CHARACTERIZATION OF SUCROSE METABOLIZING ENZYMES IN

CHARACTERIZATION OF SUCROSE METABOLIZING

ENZYMES IN SUGARCANE UNDER HEAT STRESS

FAISAL MEHDI

DEPARTMENT OF AGRICULTURE AND AGRIBUSINESS

MANAGEMENT, UNIVERSITY OF KARACHI,

KARACHI, PAKISTAN

2019

ii



By

FAISAL MEHDI

A thesis submitted in

fulfilment of the requirements for the degree of

Doctor of Philosophy (Ph.D.) in Agriculture

DEPARTMENT OF AGRICULTURE AND AGRIBUSINESS

MANAGEMENT, UNIVERSITY OF KARACHI,

KARACHI, PAKISTAN

2019

iii

Dedication

I dedicate this dissertation to my beloved parents

Ghulam Mehdi Masroor and Amina Masroor

for their love, endless support, encouragement and sacrifices

without whom none of my success would be possible.

iv



Thesis Approved

Supervisor

Dr. Saddia Galani Associate Prof.

Dr. A.Q Khan Institute of Biotechnology and Genetic Engineering

University of Karachi, Karachi, Pakistan

Supervisor’s Signature:

Examiner’s Signature:

v

Acknowledgements

All Praise to ALLAH, for giving me the blessing, the strength, the chance and endurance

to complete this dissertation. My special gratitude and appreciation to my parents, my

brothers and sisters for encouraging, supporting and always believing me to follow my

dreams throughout the study. I gratefully acknowledge to My Supervisor Dr. Saddia

Galani Associate Professor in the Karachi Institute of Biotechnology and Genetic

Engineering (KIBGE), University of Karachi she has been a tremendous mentor for me.

Without her continuous guidance, advice, effort, support, suggestions and constant

feedback throughout the research, this Ph.D. would not have been achievable. My

utmost gratitude to Prof. Dr. Abid Azhar Director General Meritorious Professor in the

Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi

for providing me the lab facilities to carry out my research successfully. My deepest

gratitude goes to Prof. Dr. Saleem Shahzad Registrar University of Karachi and Ex-

chairperson department of agriculture and agribusiness management university of

Karachi. I am indebted to Dr. Saboohe Raza chairperson department of agriculture and

agribusiness management university of Karachi. I also acknowledge to Mr. Touqeer Mirza

Cane Procurement and Development Officer Mehran Sugar Mills Ltd and Mr. Sharif Khan

Deputy General Manager Mirpurkhas Sugar Mills Ltd. Last but not least, my particular

appreciation goes to Dr. Afsheen Aman, Dr. Urooj Javed, Dr. Kazim Ali, Dr. Shagufta,

Sahar, Dr. Ishrat Jameel, Riaz Ahmed, Sohail Ahmed and Abid Hussian as well as my

fellow lab mates Neshaman Huma, Zubia Rashid, Nabeela Afzal, Zunaira Riaz, Lubna

Faraz, Fatima Haider, Maleeha Akbar, Iffat Imran, Ali Muntazir Naqvi and lab assistant

Aijaz bhai and Zaheer bhai for their support and encourage during all experiments.

Faisal Mehdi

vi

TABLE OF CONTENTS

Description Page no.

Acknowledgments v

Table of Contents vi-xii

List of Tables xi-xii

List of Figures xii-xvi

List of Abbreviations xvi-xviii

Abstract xix-xx

Abstract in Urdu xxi-xxii

Section # 1 Introduction 1

1.1. Introduction 2-6

1.2. Objectives 7

Section # 2 Literature Review 8

2.1. Importance of Sugarcane 9

2.2. Phenology of Sugarcane 10

2.3. Heat Stress 11

2.4. Effect of Heat Stress on Sugarcane 11-12

2.5. Effect of High Temperature on Growth and Development 13

2.6. Physiological Indicators of Tolerance to Increased Temperatures

2.6.1 Cell Membrane Thermosability

2.6.2 Accumulation of Compatible Solutes (Proline)

2.6.3 Malondialdehyde (MDA)

2.6.4 Hydrogen peroxide (H2O2)

13-16

3.1. Molecular Indicators of Tolerance to Increased Temperatures

3.1.1. Reactive Oxygen Species (ROS)

3.1.2. Heat Shock Protein (HSPs)

17-18

4.1. Antioxidant Defense Response to Heat Induced Oxidative Stress

4.1.1. Enzymatic Antioxidants

4.1.2. Non-Enzymatic Antioxidants

19

5.1. Plant Adaptation to Heat Stress 20

vii

6.1. Mechanism of plant Adaptation to Heat stress

6.1.1. Avoidance Mechanism

6.1.2. Tolerant Mechanism

20-21

7.1. Sucrose Metabolism and Regulation in Sugarcane

7.1.1. Biosynthesis of Sucrose in Sugarcane Plant

7.1.2. Source-sink Regulation of Sucrose Accumulation in

Sugarcane

21-23

8.1. Sucrose Metalizing Enzymes Response Under Heat Stress

8.1.1. Invertases

8.1.2. Soluble Acids

8.1.2.1. Soluble Acid Invertase (Vacuolar Invertase)

8.1.2.2. Insoluble Acid Invertase (Cell Wall Invertase)

24-26

8.2. Cytoplasmic Invertase 26

8.3. Sucrose Synthase 26-28

8.4. Sucrose Phosphate Synthase 28

8.5. Sugar Recovery Rate 29

9.1. The Mitigation of Heat Stress Strategies

9.1.1. Cultural Method 30-31

9.2. Genetics and Genomics Strategies

9.2.1. Omics-Led Breeding and Marker-Assisted Selection

9.2.2. Genome Wide Association Studies for Stress Tolerance

9.2.3. Genetic Engineered Plants for Stress Tolerance

31-33

9.3. Genome Editing Strategies 33-34

Section # 3 Methodology 35

3. General Experimental Details 36

3.1. Physiochemical Properties of Soil and Water

3.1.1. Soil and Water Analysis 37

3.2. Crop Husbandry

3.2.1. Cultivation of Sugarcane 37

3.3. Heat Stress Treatments 38

3.4. Sample Collection 39

viii

3.5. Morphological Analysis 39

3.6. Physiological Analysis

3.6.1. Cell Membrane Thermostability (CMT)

3.6.2. Proline Estimation (Osmolyte accumulation)

3.6.3. H2O2 Quantification

3.6.4. Determination of Lipid Peroxidation

40-43

3.7. Biochemical Analysis

3.7.1. Sugar Extraction

3.7.2. Total Sugar Estimation

3.7.3. Reducing Sugar Estimation

3.7.4. Non-Reducing Sugar Estimation

3.7.5. Protein and Enzymes Extraction

3.7.5.1. Extraction

3.7.5.2. Total Soluble Protein Quantification

43-47

3.8. Quantification of Sugar Metabolizing Enzymes

3.8.1. Vacuolar Acid Invertase (VAI)

3.8.2. Quantitative Analysis of Cell Wall Invertase (CWI)

3.8.3. Quantification of Cytoplasmic Invertase (CyIN)

3.8.4. Quantification of Sucrose Phosphate Synthase (SPS)

3.8.5. Sucrose Synthase (SS)

47-52

3.9. Qualitative Analysis of Isozymes through Native PAGE

3.9.1. Sample Extraction

3.9.2. Native Polyacrylamide Gel Electrophoresis

3.9.2.1. Preparation of Reagents

3.9.2.2. Preparations of Buffers

3.9.2.3. Preparation of Resolving Gel (lower gel) (pH-8.8)

3.9.2.4. Preparation of Stacking Gel (Upper gel) (pH-6.8)

3.9.2.5. Preparation of Running Buffer (1X)

52-56

3.9.3. Invertase Zymography 56-57

3.9.4. SDS-PAGE Protein Profiling 57

3.9.5. Quality Parameters Analysis 57-59

ix

3.9.5.1. Pol (%) Estimation

3.9.5.2. °Brix Estimation

3.9.5.3. Moisture Content

3.9.5.4. Fiber Content (%)

3.9.5.5. Sugar Recovery Rate Estimation

3.9.6. Statistical Analysis 59

Section # 4 Results 60

4.1. Morphological Analysis

4.1.1. Shoot Length (cm)

4.1.2. Root Length (cm)

4.1.3. Number of Tillers (plant-1)

4.1.4 Number of Leaf (plant-1)

4.1.5. Leaf Length (cm)

4.1.6. Leaf Width (cm)

4.1.7. Fresh to Dry Weight Ratio (%)

4.1.8. Stem Diameter (cm)

4.1.9. Number of Nodes (plant-1)

4.2.0 Number of Internodes (plant-1)

4.2.1. Internode Distance (plant-1)

61-71

4.3. Stress Damage Indicators Quantification

4.3.1 Malondialdehyde (MDA)

4.3.2 Proline Estimation

4.3.3 Hydrogen-peroxide

4.3.4 Relative Membrane Permeability (RMP)

72-79


4.4.1. Total Sugar Quantification

4.4.2. Reducing Sugar Quantification

4.4.3. Non-reducing Sugar Quantification

4.4.4. Total Soluble Protein Analysis

80-89

4.5. Sugar Metabolizing Enzymes

4.5.1. Quantitative Analysis 90-100

x

4.5.1.1. Sucrose Synthase (SS)

4.5.1.2. Sucrose Phosphate Synthase (SPS)

4.5.1.3. Cytoplasmic Invertase (CyINV)

4.5.1.4. Cell Wall Invertase (CWIN)

4.5.1.5. Vacuolar Invertase (VIN)

4.5.2. Qualitative Analysis

4.5.2.1. Cytoplasmic Invertase

4.5.2.2. Vacuolar Invertase

4.5.2.3. Cell wall Invertase

101-110

4.6. Quality Parameters Analysis

4.6.1. °Brix Estimation

4.6.2. Fiber Content

4.6.3. Pol Estimation

4.6.4. Sugar Recovery Estimation

111-114

4.7. Protein Profiling at Different Growth Stages

4.7.1. Protein Profiling at Vegetative Stage

4.7.2. Protein Profiling at Grand Growth Stage

4.7.3. Protein Profiling at Maturity Stage

115-120

4.8. Correlation 121-122

4.9. Heat Map 123

Section # 5 Discussion 124

5.1. Morphological Analysis 125-127

5.2. Thermotolerant Indicators Analysis 127-131

5.3. Sugar Analysis 131-132

5.4. Sugar Recovery Rate Analysis 132-134

5.5. Sugar Metabolizing Enzymes Analysis


5.5.2. Quantitative Analysis of Invertase Isozymes

134-140

5.6. SDS-PAGE Protein Profiling 141-142

5.7. Correlation 142-143

Key Findings 144-145

xi

Future Directions 146

Conclusion 147

References 148-188

Oral Presentations 189

Publications 189

Poster Presentations 189

LIST OF TABLES

Table no. Title Legends Page no.

1. Five year statistics of area, can production and yield 9

2. Five year statistics of sugar crushing, production and sugar

recovery rate. 9

3. Heat stress treatment used for the estimation of plant heat

tolerant based on the common ion leakage measurement (EC). 14

4. Details of physiochemical properties of soil and water used in

experiment 37

5. Morphological analysis of both cultivars S2003-US-633 and

SPF-238 under control (30±2°C), heat shock (45±2°C) and

recovery (30±2°C) for 24, 48 and 72 h at vegetative stage.

Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at

p level p<0.05.

69



recovery (30±2°C) for 24, 48 and 72 h at grand growth stage.

Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at

p level p<0.05.

70



recovery (30±2°C) for 24, 48 and 72 h at maturity stage. Cultivar

(C), Treatments (T) and Cultivar × Treatments (C×T) at p level

p<0.05.

71

xii

8. Quality parameters estimation of both cultivars S2003-US-633

and SPF-238 under control (30±2°C), heat shock (45±2°C) and

recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand

growth and maturity stages. Cultivar (C), Treatments (T) and

Cultivar × Treatments (C×T) at p level p<0.05.

114

9. Correlation among sugar profile, sugar metabolizing enzymes

and quality parameters at all growth stages. 122

10. Status of thermotolerant sugarcane cultivars. 123

LIST OF FIGURES Figure no. Description Page no.

1. ROS accumulation in sugarcane plant cells as consequence of

heat shock. These ROS generated from different organelles

such as mitochondria, chloroplast, endoplasmic reticulum,

peroxisome and extracellular side of cell membrane.

12

2. Free radicals are known to exist in crops. The Lewis diagram of

these free radicals presented in black, with unpaired electrons

highlighted in red. The half-life time (t1/2) is given for each type

of radicals. Colour coded with highest value for H2O2 (red) and

lowest value for OH• (green). Abbreviations: (ms=milli second,

µs=micro second, and ns= Nano second).

17

3. Sucrose biosynthesis: Sucrose is synthesized from uridine

diphosphate synthase (UDP) glucose and fructose 6-

phosphate, which are synthesized from triose phosphates in

the plant cell cytosol. The sucrose 6-phosphate synthase of

most plant species is allosterically regulated by glucose 6-

phosphate and Pi.

22

4. Schematic presentation of sucrose metabolism and

transportation from source (leave) to stem (sink). 23

5. Annual mean temperature and relative humidity of sugarcane

field for the year 2016-2018. 38

xiii

6. MDA quantified of sugarcane cultivars S2003-US-633 and SPF-

238 under control (30±2°C), heat shock (45±2°C) and recovery

(30±2°C) for 24, 48 and 72 h at vegetative, grand growth and

maturity stages.

73

7. Free proline estimated of sugarcane cultivars S2003-US-633



growth and maturity stages.

75

8. H2O2 estimated of sugarcane cultivars S2003-US-633 and SPF-

238 under control (30±2°C), heat shock (45±2°C) and recovery

(30±2°C) for 24, 48 and 72 h at vegetative, grand growth and

maturity stages.

77

9. Electrolytes leakage quantified of sugarcane cultivars S2003-

US-633 and SPF-238 under control (30±2°C), heat shock

(45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at

vegetative, grand growth and maturity stages.

79

10. Total sugar estimated of sugarcane cultivars S2003-US-633 and




82

11. Reducing sugar estimated of sugarcane cultivars S2003-US-633




85

12. Nonreducing sugar estimated of sugarcane cultivars S2003-US-

633 and SPF-238 under control (30±2°C), heat shock (45±2°C)

and recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand


87

13. Total soluble protein estimated of sugarcane cultivars S2003-

US-633 and SPF-238 under control (30±2°C), heat shock 89

xiv

(45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at


14. Specific activity quantified of sucrose synthase (SS) of

sugarcane cultivars S2003-US-633 and SPF-238 under control

(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48

and 72 h at vegetative, grand growth and maturity stages.

92

15. Specific activity quantified of sucrose phosphate synthase (SPS)

of sugarcane cultivars S2003-US-633 and SPF-238 under

control (30±2°C), heat shock (45±2°C) and recovery (30±2°C)

for 24, 48 and 72 h at vegetative, grand growth and maturity

stages.

94

16. Specific activity quantified of cytoplasmic invertase (CyIN) of




96

17. Specific activity quantified of cell wall invertase (CWIN) of




98

18. Specific activity quantified of vacuolar invertase (VIN) of




100

19. Native–PAGE analysis of cytoplasmic invertase (CyIN) of

cultivar S2003-US-633 subjected to control (C) heat shock

(45±2°C) and recovery treatments for 24, 48 and 72 h at all

growth stages. (Markers used ovalbumin (45 kDa), albumin

bovine (monomer 67 kDa and dimer 134 kDa) and gama

globulin human (160 kDa).

103

20. Native–PAGE analysis of cytoplasmic invertase (CyIN) of

cultivar SPF-238 subjected to control (C) heat shock (45±2°C) 104

xv

and recovery treatments for 24, 48 and 72 h at all growth

stages. (Markers used ovalbumin (45 kDa), albumin bovine

(monomer 67 kDa and dimer 134 kDa) and gama globulin

human (160 kDa).

21. Native–PAGE analysis of vacuolar invertase (VIN) of cultivar

S2003-US-633 subjected to control (C) heat shock (45±2°C) and

recovery treatments for 24, 48 and 72 h at all growth stages.

(Markers used ovalbumin (45 kDa), albumin bovine (monomer

67 kDa and dimer 134 kDa) and gama globulin human (160

kDa).

106

22. Native–PAGE analysis of vacuolar invertase (VIN) of cultivar

SPF-238 subjected to control (C) heat shock (45±2°C) and


(Markers used ovalbumin (45 kDa), albumin bovine (monomer

67 kDa and dimer 134 kDa) and gama globulin human (160

kDa).

107

23. Native–PAGE analysis of cell wall invertase (CWIN) of cultivar

S2003-US-633 subjected to control (C) heat shock (45±2°C) and


(Markers used ovabumin (45 kDa), albumin bovine (monomer

67 kDa and dimer 134 kDa) and gama glubulin uman (160 kDa).

109

24. Native–PAGE analysis of cell wall invertase (CWIN) of cultivar

SPF-238 subjected to control (C) heat shock (45±2°C) and

recovery treatmnets for 24, 48 and 72 h at all growth stages.

(Markers used ovabumin (45 kDa), albumin bovine (monomer

67 kDa and dimer 134 kDa) and gama glubulin uman (160 kDa).

110

25. SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-

633 (B) SPF-238 at formative stage under control at (30±2°C),

heat shock (45±2°C) and recovery treatments (30±2°C) for 24,

48 and 72 h.

116

xvi


633 (B) SPF-238 at grand growth stage under control at

(30±2°C), Heat shock (45±2°C) and recovery treatments

(30±2°C) for 24, 48 and 72 h.

118


633 (B) SPF-238 at maturity stage under control at (30±2°C),

heat shock (45±2°C) and recovery treatments (30±2°C) for 24,

48 and 72 h.

120

LIST OF ABBREVIATIONS / ACRONYMS ANOVA Analysis of Variance

AOS Active Oxygen Species

APS Ammonium Per Sulfate

AS Amount of Sample

°Bx °Brix

BSA Bovine Serum Albumin

C Control Treatment

°C Degree(s) Celsius

CBB Commassie Brilliant Blue

CMT Membrane Thermostability

Conc Std Concentration of Standard

CWIN Cell Wall Invertase

CyIN Cytoplasmic Invertase

DNA Deoxyribonucleic Acid

DNSA Dinitro Salicylic Acid

EC Electrical Conductivity

E.C Enzyme Commission

Ext pol Extraction of pol

EDTA Ethylene Diamine Tetra Acetic Acid

FAO Food and Agriculture Organization

FW Fresh Weight

FYM Farm Yard Manure

xvii

G Gram

GDP Gross Domestic Product

H Hour

HCl Hydrochloric Acid

H2O2 Hydrogen-peroxide

HSPs Heat Shock Proteins

HST Heat Shock Treatment

IPCC Intergovernmental Panel on Climate Change

KDa Kilo Dalton

KI Potassium Iodide

KOH Potassium Hydroxide

M Mole

Min Minute

MY Marketing Year

MNFSR Ministry of National Food Security and Research

MDA Malondialdehyde

MOPS Morpholino Propane Sulfonic Acid

mg ml-1 Milligram Per Milli Liter

MgCl2 Magnesium Chloride

MW Molecular Weight

N Normality

Nm Nano Meter

NaCl Sodium Chloride

NASA The National Aeronautics and Space Administration

NPK Nitrogen, Phosphorous and Potassium

OD Std Standard of Optical Density

ODT Optical Density of Test Sample

1O2 Singlet Oxygen

O.-2 Super Oxide

OH Hydroxyl Radical

PAGE Polyacryl Amide Gel Electrophoresis

xviii

PAR Photo Synthetically Active Radiation

Pb Plumbum (Lead)

PCD Programmed Cell Death

pH Power of Hydrogen Ions

PSMA Pakistan Sugar Mills Association

R Recovery

ROS Reactive Oxygen Species

RMP Relative Membrane Permeability

RPM Revolution Per Minute

RT Reaction Time

SR Sugar Recovery

SPS Sucrose Phosphate Synthase

SPSS Statistical Package for the Social Sciences

SS Sucrose Synthase

SSA Sulfosalicylic Acid

T Temperature

TBA Thiobarbituric Acid

TCA Trichloroacetic Acid

TEMED Tetramethylethylenediamine

U Unites

UDPG Uridine Diphosphate Glucose

USDA The United States Department of Agriculture

µg ml-1 Microgram Per Milli Liter

VIN Vacuolar Invertase

xix

Abstract Sugarcane is a valuable cash crop in Pakistan. It is cultivated in tropical and

subtropical areas worldwide and produced 80 % sugar globally. It requires optimum

temperature 30°C-32°C for economic production but shift in temperature declined

sugarcane production. Heat stress causes serious yield reduction in sugarcane by

affecting its morphology, physiology, biochemical, molecular levels which leading to

poor yield production and low sugar recovery rate. In this study, two local sugarcane

cultivars, high sucrose accumulation (S2003-US-633) and low sucrose accumulation

(SPF-238) were analyzed for morphological, physiological and biochemical analysis as

well as sugar quality parameters to heat stress. With particular reference to sucrose

metabolizing enzymes such as sucrose phosphate synthase (SPS), sucrose synthase

(SS) and invertase isozymes (cytoplasmic CyIN, vacuolar (VIN) and cell wall bound

(CWIN) were investigated under heat stress and recovery treatments. For this both

sugarcane cultivars subjected to extreme heat shocked treatments at (45±2°C) for

different episodes (T24, T48 and T72 h) and recovery treatments at (30±2°C) for

different episodes (R24, R48 and R72 h). The samples were collected at different

growth stages such as vegetative, grand growth and maturity stages. Results revealed

that after exposure of heat stress for different episodes 24 (T24), 48 (T48) and 72

(T72) h altered most of the morphological attributes due to the differences in the

thermotolerant potential. Both the cultivars indicated the differential expression of

proteins and sucrose metabolizing enzymes at all growth stages. Efficient activity of

sucrose metabolizing enzymes and maximum proline content, highest sucrose

accumulation, maximum sugar recovery rate, reduced electrolytes leakage (EC),

xx

declined malondialdehyde (MDA) and hydrogen peroxide (H2O2) improved

thermotolerance in cultivar S2003-US-633. While lower content of free proline,

sugars, poor performance of ROS scavenger, lower or irregular expression of enzymes

and proteins failed to protect cellular damages caused by extreme temperature,

subsequently hampered growth and development along with reduced sugar recovery

rate in cultivar SPF-238. Present study revealed that high and low molecular mass

protein bands ranging from 15 kDa to 150 kDa proteins in cultivar S2003-US-633. It is

expected that heat shock proteins (HSPs) might be play significant role in

development of thermotolerant without inhibiting the expression or activity of

sucrose metabolizing enzymes. In additions, maximum accumulation of free proline

that performance as a computable solute or osmolytes and oxidative markers,

responsible to stabilize the activity of sucrose metabolizing enzymes activity under

heat stress conditions. It can be concluded that cultivars S2003-US-633 showed more

thermotolerant cultivar with great sucrose accumulation as well as high recovery rate

under different regimes of heat shock treatment conditions. Consequently, these

biochemical attributes can index the degree of thermotolerant of sugarcane crop to

exhibit under heat stress conditions given that insight to molecular breeders to

recognize the thermotolerant sugarcane cultivars with improved recovery rate of

sugarcane.

xxi

خلاصہ

گن

پاکستتتتماںایکقیمی نم کاشتتتتر دنی اک سق فصتتتت۔ قدا دں مگرمعلایقونگرمک

ق عالن ستتتپر ر سق -حاصتتت۔ک جات چین گن ستتت ف صتتتد(08ستتت ایکک جات قون

قچ شتتتتتتتتتتتترقون کا بمیسستتتتتتتتتتتتت ن گ ک ستتتتتتتتتتتتتیس دقوقنک لئ نجہحرقنتشتتتتتتتتتتتتت اعاک

گل کن نجہحرقنتایک-ضتتتتتتتتتتترونت دت ک تبدیل ک وجہستتتتتتتتتتتت کاف کن ن ایک دقوقن

کا-آئ لدج ،اانفدلدج ،ک د ےتناؤگرا کدںستتتتتتتتتتتتپ دستتتتتتتتتتتتالنات قونبائیدک ن ک۔فز ا

ایکک کرماثرا ک دقوقن ستتتتتتابےما کن شتتتتتدیدگن دقوقنکگن جسک وجہستتتتت -کا

چ ک ن قون ین جتتات کدنی کتتاف کن آ ک -ک شتتتتتتتتتتتتتتریایک گن قساپتتالع ایک، واقتتاا

کر وقل ی قک ا کر قون322-یدق س3882-ستتتتمق سقیستتتتام،د ا کستتتتدکرود قک ا کمستتتتتدکرود

گ ا320- قیفستتتمق سی وقل ک ا ک و دںقیستتتام-کدقستتتمعنای حرقنت نجہکد،د ا کگن

ییر نجتہ قون لدج ،ک بتاؤ فدلدج ،فز ا اان وقل ایکنکتککر قونک ن ک۔بائیدحرقنتک بتاؤ

ت ا چین ک گ ا ک ا تجز ہ دگرکساتساتکق ک اع انک یرقای ردکا جیساکہخاارےوقل بنا ش

زقیندنٹیسآسددقئنقونق س( ق سےم یسسدسدکرو ق س(، ق سسےم یسفاستی ستدکرود

قونستتتتتتتتائ لاستتتتتتتتنم، یعن گ ا(ستتتتتتتت ۔وقیو کیدلر ک ا ب تجز ہ ک -کا گن و دں قسک لئ

ک د وںکد،قی نجہک ڈگریستتتتتتتت ن گ (54±3 نمالیس یعن شتتتتتتتتدیدگرا ایکستتتتتتتتام

ک قڑتتتتتتالیسقون،حرقنت رچ بیس گ ن د ک ییرہجبکتتتتویی بہمر بتتتتتاؤوقل نجتتتتتہحرقنت

ک لئ نجہ چ بیسڈگری(28±3حرقنتتیس د وں بہمرگ ن دںقڑتالیسقون،س ن گ ر

-ک ویی رنک گئ ک ارقحتت کی ند اخملفنشتتتتتتتتتتتتتتدو نتتا جنع ونقںقست قیقک-گئ ۔ ر

گ ا ک ا اخملفویی ر نجہحرقنت رک (ڈگریستتت ن گ 54±3کہ نمالیس یہاشتتتا دک

گئ چ بیس،قڑتالیسقون نک ثرخصتتتتتتتتتتتتتدصتتتتتتتتتتتتتت ات رق د وںک ظا ریبہمرگ ن دںک ویی ر

xxii

ک صتتتتتتتتلاح ر و دںقیستتتتتتتتامایکقل قں-ق دقد دئ اخملف- تقل حرقنتبر قشتتتتتتتترکر

بنا وقل خااروں دگر شتت ک ارحل ایک و دںقیستتتامایکل ن اتقون ک قیکستتتشر شتتتننشتتدو نا

د ا ک د ا،ل ن ات- دئ کاف فرقظا رب ایک کا بنا وقل خااروںک قیکستتشر شتتن دگر شتت

قک اد ا ک د ا،د ا کک قجزق دگر د ا،ستتتتتدکرود

قیستتتتت ،قون د ا،د ا کن کدنیک شتتتتتریشتتتتت

ایک322-ق سید3882-ق سکہرتا کثابریہ د اکماکقےڈیقیمآکستتتتتتتتتتتتتائ ،ر ای نوجن

ک صتتتتتتتتتتلاح ربر قشتتتتتتتتتترحرقنت دگرکا د ا،کماقدقنل ن اتک جبکہ-ت بہمرکر

کمشتتتتتتتتتت

قو خااروںک ظا رک ایصکانکر گ ،ب یائدکل ن اتق سقستتتتتتتتتتتتکدقینجرد د ا،آن د ا،قون

ن دگر

ک ستتتتتتتتتتتتاتکستتتتتتتتتتتتتاتکشتتتتتتتتتتتتت قس-کاوٹ دئ ب ن کدنیایکخلیدںک ت یظایک اکامنہن

یمستتد ندنکستت قایکبہرستتانےستتالنات ودںوقل 322-یدق س-3882ق س ونقںت قیقک

ستتکم -ل ن اتق کشتتاہ دئ ک ک ڈیقےپچاس تدیعک جا قیچ-قسستت یہ یشتتن دئ یا

بنا وقل خااروںک دکہق س یس(ج دگر

کاوٹکئ بغیر د وںایکد ا ککدن ستتتتتتتترگرایدںشتتتتتتت

ک صتتتلاح رکدبہمرکر ایکق کرگرا بر قشتتترکر ق ق ا کعلاوکقد ند- یکستتتکم مکر قن

قک ا د ا،جد رولین ایتک باؤک حگرا جدکہداانکرقونآکست ی دستندییسسبپدنآکہکا

کاااہ قن ایک ک خصتتتتتدصتتتتت ات ن ائ قسک تیج ایکح ات ات ک-قستتتتتم کام کد۔فصتتتتتگن

ک گرا گرا بر قشتترد-ستتکم کراشتتتا دکحایتایکک باؤ ک صتتتلاح روقلکر ا ک

دگرک شتتتتتتتتناخرک یستتتتتتتتمک گن

-بہمریت شتتتتتتتتریایکب ک ن کدنیستتتتتتتتاتکستتتتتتتتاتکشتتتتتتتت

1

SECTION # 1 INTRODUCTION

2

1.1 Introduction

Sugarcane (Saccharum Officinarum) is a crop of immense agronomical value and due

to high sucrose content in it, that is cultivated in more than hundred countries and

it contributes almost 65 % of world sugar production (Carson and Botha, 2002).

According to Food and Agriculture Organization and United state department of

Agriculture stated that Asian countries contribute 37 % of the world’s total sugar

production while around 46 % of sugar consumption, confirming its role as a key

player in the global sugar market (FAO 2015; USDA 2015). In Pakistan, cane is a

valuable cash crop and plays an imperative role in the economy of the country and

it provides raw material to 81 sugarcane mills and thus employment to over 4 million

people with sugarcane. It is also reported that Pakistan Sugar Mills Association

(PSMA) and Ministry of National Food Security and Research (MNFSR), Marketing

year (MY 2017-2018), sugarcane produced 82 million tons with 9 % increase as

compared to 2016/2017.

Sugarcane yield and sugar recovery are the primary objectives of famers and sugar

industrialists. Unfortunately, Pakistan has low sugar yield and recovery rate as

compared to the international market. The sugar production depends mainly on

cane yield and percentage of recovery. Despite extension in sugarcane production

in Pakistan, average sugar recovery rate is reported 8.1 % only which is far beyond

from other developed countries (PSMA Report, 2019). On the other hand, altering

climate conditions along with low sugar recovery potential of sugarcane poses a

challenge to sugarcane industry.

3

The increasing threat of climate shift is already having an extensive effect on

agricultural production all over the world as heat wave causes substantial yield

decline with great risk for upcoming global food security (Christensen and

Christensen, 2007). However, there are so many biotic and abiotic factors such as

pests, diseases and high temperature are the major constrain to achieve high cane

production and sugar yield (Khalid et al., 2005). NASA, 2017 reported that increase in

temperature up to 0.99°C makes agricultural crops most susceptible and projected

to have negative affect on plant growth and development, resulting in wide spread

famine or food security will encounter in near future. It is projected that increase in

temperature due to climate change will have effect production (Neumeister, 2010)

negatively affect growth and development of sugarcane (Rasheed et al., 2011)

because Pakistan having wide range of temperature range and varied environmental

condition (Hameed et al 2013).

Heat stress effects the crop physiology, biochemical and morphological alterations

such as decrease in internode distance, shoot and root growth inhibition

(Hasanuzzaman et al., 2013) which disturb plant growth and development and may

lead to drop the crop yield, while temperature threshold for sugarcane growth and

development is 32-33°C (Wahid et al., 2007). Heat stress also affects the sugarcane

crop by reduced concentration of sucrose content, decline in shoot dry mass,

increased number of tillers and smaller internodes at greater than 40°C (Azevedo et

al., 2011). While, cultivars were usually indexed on basis of these agronomically

important traits for improvement of sugarcane yield and sugar content and

susceptible to environmental constrains (Abbas et al., 2013).

4

This poses a serious problem for achieving high yield and sugar recovery for industrial

sector and there is more demand for more potential for sucrose accumulation in cane

tissues (Datir and Joshi, 2016; Batta et al., 2011). It is reported that limited number

of cultivars with the low capability to store high sucrose contents are major cause for

low sugar recovery and it also depends on the ratio of sucrose synthesis to sucrose

cleavage (Office of the Cane and Sugar Board, 2013) regulated by sugar metabolizing

enzymes. Sucrose metabolism enzymes such as acid invertase, cytoplasmic invertase,

sucrose synthase and sucrose phosphate synthase play important role in primary

metabolism and plant development in parenchyma cells of sugarcane plant (Schaffer

and Quick, 2017; Batta et al., 2008; Botha and Black, 2000; Batta et al., 1995).

Invertase (E.C 3.2.1.26) involves in sucrose hydrolyses (Zhu et al., 1997), while

sucrose-phosphate synthase (E.C 2.4.1.14) synthesizes sucrose and sucrose synthase

(E.C 2.4.1.13) can either degrade or synthesize sucrose (Geigenberger and Stitt,

1993). Regarding localization, invertases are divided in to three subgroups, viz cell

wall, cytoplasmic and vacuolar (Ruan et al., 2010). Sucrose and its hydrolysis product

glucose and fructose not only provide energy to growing tissues but also function as

signals in regulation of gene expression and thus invertase activity at wrong time and

place can drastically affects plant viability and development (Xu et al., 1996). Various

physiological functions have been suggested for invertases, that is, to provide

growing tissue with reducing sugar (glucose and fructose ) as a source of energy, to

produce a sucrose concentration gradient and to partition sucrose between source

(leaf) and sink tissues (cane stalk), as well as to aid sucrose transport (Chandra et al.,

2012). In sugarcane, sugar (sucrose) is translocated through the phloem to the sinks,

where it is used for cell growth, metabolism, respiration or storage (Ayre, 2011; Wang

5

et al., 2013). These sucrose metabolizing enzymes in sugarcane genotypes are

diversely affected during different growth stages (Tana et al., 2014). It has been

investigated that accumulation of sucrose in sugarcane tissue is mainly dependent on

soluble acid invertase and sucrose phosphate synthase activities (Ansari et al., 2013),

which are adversely affected by climatic alterations. It has been reported that there

are substantial yield reductions observed at temperature more than 45°C, also

affecting the sugar recovery rate in sugarcane leading to huge economic losses (Mali

et al., 2014; Bonnett et al., 2006). This sugar reduction is due to the down regulation

of specific genes in carbohydrate metabolism which may lead to the altered activities

of sugar enzymes with compromised sucrose accumulation and sucrose synthesis

under heat stress (Ruan et al., 2010). There is contradictory opinion regarding the

association between the sucrose level and the activities of enzymes contributing to

sucrose accumulation in the culm (Botta et al., 2011). The growth of internodes

decrease along with reduced sucrose in sugarcane due to significant rise in

temperature (Bohnert et al., 2006). Heat stress inhibits the processes of

photosynthesis and respiration mainly by limiting electron transport and rubisco

activase activity (Sage and Kubien, 2007). Due to small difference in the temperature

requirement for cyclic reactions may result in large changes in the net amount of

sucrose, the temperature dependence of the overall process of sucrose accumulation

is unpredictable (Ebrahim et al., 1998). On the other hand, heat stress results in the

production of reactive oxygen species (ROS) such as, hydrogen peroxide (H2O2)

superoxide anion (O-2) and hydroxyl radical (OH)-, which are highly reactive and can

change the metabolism of plant through oxidative damage to membrane, denaturing

of protein and nucleic acids leading to cell death (Pastore et al., 2007). Moreover,

6

H2O2 is involved in disruption of various metabolic activities like calvin cycle (Akram

et al., 2012).

Although it is known that sugarcane is severely affected by heat stress, cellular,

biochemical, physiological and molecular mechanisms of the response of sugarcane

to heat stress is needed to be elucidate. In present scenario of global warming, the

major challenges for crop scientists is to produce new thermotolerant varieties to

fetch the growing population with improved sugarcane productivity and sugar

recovery. These sugarcane varieties development programmes are dependent on

biochemical, molecular and biotechnological strategies in order to develop new

varieties having important economic quality characteristics (Khanum et al., 2006;

Moore, 2005).

Due to present scenario, development of thermotolerant cultivars are the significant

approach in adaptation of climate change. In this regard, biochemical

characterization is fundamental step to identify cultivars with desirable agronomic

characters or traits to meet sugar industry requirements. The current study was

designed to determine the biochemical, physiological, molecular and quality

parameters analysis as well as expression of sucrose metabolizing enzymes in

sugarcane at formative, grand growth and maturity stages under heat stress

conditions. Thus, these findings can be helpful in providing information for

engineering sugar improvement along with thermotolerance in sugarcane varieties

and providing new avenues towards the economic development of the country.

7

1.2 Objectives This study was planned with the following objectives:

• To study the temporal expression of sucrose metabolizing enzymes and their

characteristic role in sucrose accumulation in different varieties of sugarcane under

heat stress.

• To elucidate the biochemical mechanism involved in sucrose production

underlying heat stress.

• To find out a genotype that produces maximum sucrose under heat stress.

• To evaluate the characteristic role of sucrose metabolizing enzymes isoforms

responsible for sugar metabolism under heat stress.

8

SECTION # 2 LITERATURE REVIEW

9

Literature Review

2.1 Importance of Sugarcane

Globally sugarcane production for the marketing year 2019-2020 is forecast 181

million metric tons (USDA, 2019). Among sugarcane producing countries, Australia,

India, China, Brazil and Pakistan are the top sugarcane producing countries. In

Pakistan, the sugar industry is the 2nd largest agro-base industry after textiles and it

is being cultivated on 1343 m hectors with production 83,333 tons and its production

accounts for 2.9 % value addition in agriculture sector and 0.5 % in overall Gross

Domestic Production (GDP) (Pakistan Bureau of Statistics 2018-2019). Sugar industry

plays a vital role in the national economy of the country. Five year statistic of

sugarcane production, sugar recovery rate, and yield presented in Table 1-2 (PSMA

annual report, 2018-2019).

Table 1. Five Year Statistics of Area, Can Production and Yield.

Year Area Hectares Production Tons Yield Tons/Hectare

2013-2014 1,171,687 67,427,975 57.55

2014-2015 1,113,161 62,794,827 56.41

2015-2016 1,130,820 65,450,704 57.88

2016-2017 1,216,894 75,450,620 62.00

2017-2018 1,340,926 83,289,340 62.11

Table 2. Five Year Statistics of Sugar Crushing, Production and Sugar Recovery Rate.

Year Cane crushing Tons Sugar Production Tons Recovery %

2013-2014 56,460,524 5,587,568 9.90

2014-2015 50,795,218 5,139,566 10.12

2015-2016 50,024,249 5,082,110 10.16

2016-2017 70,989,946 7,005,678 9.87

2017-2018 65,615,550 6,576,534 10.02

10

2.2. Phenology of Sugarcane

Plant development phases of sugarcane are germination (seedling), vegetative

(tillering), grand growth (elongation) and maturity (ripening) stages (Silva et al.,

2008). Optimum temperature for germination of sugarcane is about 28°C to 30°C

which begins from 10 to 35 days after planting in field conditions. The germination of

bud is influenced by biotic and abiotic factors especially high temperature (Farooq et

al., 2009) and affected early seedling growth in sugarcane and other plants (Wahid et

al., 2007). Vegetative stage (tillering formation) is a physiological process of repeated

branches of cane and it provides maximum stalk for higher yield commencing from

around 40 days and may last up to 180 days after planting. The optimum temperature

for this stage is 26.2 °C which is the most important for crop growth and

development. Grand growth stage is considered from 120 to 250 days after planting

of sugarcane, where cane formation and elongation take place. Under favorable

conditions stem grow swiftly almost 5 to 6 internodes per month. Its optimal

temperature from (26.2°C to 35.5°C) also plays imperative role during the active

growth stage (Samui et al., 2003). At maturity or ripening stage starts from 9 to 12

months with declined vegetative growth with maximum sugar accumulation in

sugarcane stem. Sugarcane ripening is very important in improving quality of

sugarcane but evaluated temperature due to climate change affect the sugarcane

natural ripening and quality (Gawander, 2007).

11

2.3. Heat Stress

Heat stress is defined as the increase in temperature away from a threshold level for a

period of time sufficient to cause irreparable loss to plant growth and development.

According to NASA the surface temperature increased by 0.8 °C worldwide (NASA'S

Goddard Institute for Space Studies, 2018). While the world metrological organization

reported that global mean temperature has increased by 1.1°C (WMO, 2019). Climate

changes partially and completely damaged regional crop production (Abdelrahman et al.,

2017; Lobell et al., 2011). A single degree increase from the threshold level is consider

heat stress (Hasanuzzaman et al., 2013) and may cause 2.5 % and 10 % in crop yield

reduction (Hatfield et al., 2011). As manifested by massive yield decline in many crops,

the increasing extreme impact of heat stress are putting global food security at high risk.

Global food productions must rise by 70 % to meet the demand of a projected increasing

in population growth to nine billion by 2050 (Stratonovitch and Semenov, 2015). High

population growth rate countries are working more aggressively to improving crop yields.

To mitigate high temperature stress and for the development of thermotolerant crop

varieties, it is very important to understand heat stress mechanisms in plant.

2.4. Effect of Heat Stress on Sugarcane

Climate related event such as carbon dioxide, temperature, precipitation are key factors

for sugarcane production (D. Zhao and Li, 2015). Heat stress may cause alteration in

physiology, morphology, as well as other biomolecules during the various growth stage

of crop. Mostly sugarcane propagated by setts, germination of can sett is badly affected

by high temperature. It is reported that reduced growth and water relation were evident

in sugarcane at temperature greater than 36°C (Wahid et al., 2007).

12

Optimum temperature of the sugarcane is between 8°C to 34°C but low temperature

declined the photosynthesis and leaf growth rate ultimately reducing yield (Gawander,

2007). High temperature results increased number of nodes, short internodes, higher

fiber content in stem and lower sucrose content (Bonnett et al., 2006). High temperature

also triggers the production of reactive oxygen species (ROS) which are strong oxidizers

and react with a large variety of biological molecules in plant cells. When these ROS and

antioxidants are in equilibrium, plant shows normal activities. However, any

environmental imbalance between ROS and antioxidants that causes oxidative stress to

plant cells. This oxidative stress may lead to molecular damages, such as improper

functioning of membranes and eventually plant cell death (Fig 1).

Fig 1: ROS accumulation in sugarcane plant cells as consequence of heat shock: These ROS

generated from different organelles such as mitochondria, chloroplast, endoplasmic reticulum,

peroxisome and extracellular side of cell membrane.

13

2.5. Effect of High Temperature on Growth and Development

Temperature plays a vital role in dry matter, transpiration, partitioning (Crawford et

al., 2012; Zhao et al., 2013) respiration and photosynthetic activity (Sage and

Kocacinar, 2012; Atkin and Tjoelker, 2003). Plants survive at optimal temperature

while high and low temperatures decline the growth rate (Sanchez et al., 2015; Ciais

et al., 2005). It has been investigated that the rise in temperature to optimal

thresholds effect biochemical mechanisms, causing altered slow growth and

development rates (Thornton et al., 2014; Cleland et al., 2007) imposing devastating

impact on crop yield (Chmielewski et al., 2004). The reproductive stage is crucial,

mostly effected by fluctuation of high temperature such as plant reproductive male

and female organs and seeds subsequently declining the pollen viability and yield

(Yang et al., 2018; Chao et al., 2017; Tashiro and Wardlaw, 1990). Crop yield is

negatively affected by high temperature due to biochemical changes, physical

damages and physiological distribution. For instance, yield some major crops are

declined such as, wheat (6.0 %), rice (3.2 %), maize (7.4 %), and soybean (3.1 %) (Zhao

et al., 2017), sorghum (44 %) (Tack et al., 2017) and sunflower (10 %) (Debaeke et al.,

2017), tobacco plants (Yang et al., 2018), oil palm, rapeseeds, barely, cassava and

sugarcane (Ray et al., 2019).

2.6. Physiological Indicators of Tolerance to Increased Temperatures

2.6.1. Cell Membrane Thermostability

Electrolyte leakage (EC) is a stress induce marker of membrane injury, has been used

successfully to quantity cell membrane thermostability due to various environmental

stresses (Demidchik et al., 2014; J. Lui et al., 2006). Cytoplasmic membranes are

14

considered the most sensitive components of all plant cells as they are the primary

sites for damage (Abraham Blum, 2018). During heat stress, the plants undergo

transition phase from solid-gel structure to flexible liquid-crystalline structure. This

denaturization of protein and rise in unsaturated fatty acids consequence in

enhanced fluidity of the cell membrane (Savchenko et al., 2002). The unsaturated

fatty acids are less rigidly packed with membrane due to non-linearity of fatty acid

chains (Horvath et al., 2012; Cyril et al., 2002) along with initiation of lipids base

signaling cascades, calcium2+influx and cytoskeleton reorganization (Bita and Gerats,

2013). Under stress conditions, organic and inorganic ion leakage from cells occurred

due to membrane damage. Overexpression of Ppexp1 gene in tobacco observed a

minimum EC and membrane lipid peroxidation damage than wild type plant (Xu et

al., 2014). Cell membrane thermo-stability has been successfully employed to

measure heat resistance in various crops (ElBasyoni et al., 2017; Khaushal et al., 2016)

(Table 3).

Table 3. Heat stress treatment used for the estimation of plant heat tolerant based

on the common ion leakage measurement (EC).

Plant Samples Heat Shock Treatments References

Cicer arietinum

Wheat Triticum aestivum

Zea mays

Arabidopsis thaliana

Oryza sativa

Nicotiana Tabacum

Agrostis capillaris

Cynodon transvaallensis

Festuca arundinacea

40°C /30°C d/n

30°C /40°C d/n

42°C /26°C d/n

44°C /22°C d/n for 2d

42°C for 2d

42°C for 10d

38°C /33°C d/n for 28d

42°C for 6h

35°C /30°C d/n for 1d

Kumar et al., 2013

Kumar et al., 2016

Naveed et al., 2016

Lin et al., 2015

Feng et al., 2015

Liu et al., 2016

Jespersen et al., 2016

Wang et al., 2016

Bi et al., 2016

SECTION 2 LITERATURE REVIEW

15

2.6.2. Accumulation of Compatible Solutes (Proline)

Proline is compatible solute that maintain the cell’s water environment and helps the

organism to sustain under severe environmental stresses during its life cycle (Singh

et al., 2015) and is correlated with degree of stress tolerance (Carline and Santos,

2009). Plants defense through various osmolytes such as trehalose, free proline and

glycine betaine and that contribute to maintain equilibrium in cellular structures like

protein, enzymes and cell membrane via hydrophilic interaction and hydrogen

bonding (Ahanger et al., 2014). Under stress conditions plants accumulate proline

content hundred times more than favorable conditions (Verbruggen and Hermans,

2008). There are various studies on proline accumulation in plants under different

stresses (Das et al., 2014) such as in drought (Anjum et al., 2017; Ajithkumar et al.,

2014; Anjum et al., 2017), salinity (Wang et al., 2004), osmotic (Conde et al., 2011),

heavy metal (Sharma and Dietz, 2006), temperature (Munns and Tester, 2008), light

and pesticides stresses (Ningthoujam et al., 2013). It has many functions such as it

acts as stabilizer for subcellular structures energy, signaling molecules and play role

in homeostasis of metabolic pathways under various stress conditions (Sharma et al.,

2011; Szabados and Savoure, 2010).

2.6.3. Malondialdehyde (MDA)

When ROS level increases due to heat stress, may lead to lipid peroxidation in

biological membranes, altering the fundamental properties of the membrane, such

as fluidity, loss of activity of enzymes and denaturing consequently cell death (Zafar

et al., 2018; Sharma et al., 2012; Gill et al., 2010). Among the oxidative damaging

indicators malondialdehyde (MDA) is one of them and it is a by-product of lipid

16

peroxidation under biotic and abiotic stresses (Hameed and Iqbal 2014; Aly et al.,

2012; Dallagnol et al., 2011). Maximum malondialdehyde (MDA) content was

exhibited in rice and wheat under high temperature stress respectively (Sanchez-

Reinoso et al., 2014; Savicka and Skute, 2010), also reported in rape seed oil (Kong et

al., 2016), Algerian Plants (Bentahar et al., 2016) and sugarcane (Abbas et al., 2014).

2.6.4. Hydrogen peroxide (H2O2)

Hydrogen peroxide (H2O2) is a chemical compound discovered in 1818 by Louis

Jacques and also a by-product of aerobic metabolism in plants (Mittler, 2002).

Reactive oxygen species (ROS) produced in plants and animal cells due to various

biotic and abiotic stresses (Sieas et al., 2017). In plants it produced in different

organelles such as peroxisome, chloroplast and mitochondria. Hydrogen peroxide

play imperative roles in plant physiological and developmental processes including

programmed cell death (Cheng et al., 2015), root development (Hernandez et al.,

2015), stomatal aperture regulation (Ge et al., 2015). H2O2 can be produced either by

enzymatically or non-enzymatically. There are different routes of hydrogen peroxide

production in plant cell, such as redox reaction and photorespiration. H2O2 is a

signaling molecules in normal condition in the signaling pathway which relates to

abiotic stress response. Many studies revealed that hydrogen peroxide could respond

to abiotic stresses such as cold (Orabi et al., 2015), high temperatures (Wu et al.,

2015). Heat stress which cause oxidative stress in both plants and animals (Kotak et

al., 2007).

17

3.1. Molecular Indicators of Tolerance to Increased Temperatures

3.1.1. Reactive Oxygen Species (ROS)

Reactive Oxygen Species (ROS) commenced in 20th century, initially it was defined as

intermediate organic and inorganic compounds. ROS are derivatives of oxygen, more

reactive than oxygen molecules under stress conditions (Mittler et al., 2017) and

mainly produced in different compartments of plant cell such as chloroplast,

mitochondria, peroxisomes, apoplast, cell wall, cell membrane and cytosol (Noctor

and Foyer, 2016). Among various forms of reactive oxygen species, singlet oxygen

(half life time 1 to 4 µ second) can oxidize lipids and proteins while superoxide with

same half life time of 1O2 and react with Fe-S proteins and hydroxyl radicals (half life

time 1 nano second) are extremely reactive and unstable (Waszczak et al., 2018) (Fig

2). While hydrogen peroxide are fair stable signaling molecules and treated by various

enzymes like catalases (CATs) and ascorbate peroxidases (APXs) in plant cell.

Fig 2: Free radicals are known to exist in crops: The Lewis diagram of these free radicals

presented in black, with unpaired electrons highlighted in red. The half-life time (t1/2) is

given for each type of radicals. Colour coded with highest value for H2O2 (red) and lowest

value for OH· (green). Abbreviations: (ms=milli second, µs=micro second, and ns= Nano

second).

18

3.1.2. Heat Shock Proteins (HSPs)

Heat shock protein was discovered by Italian scientist R. Ritossa in Drosophila

melanogaster and named as heat shock proteins (Tissieres et al., 1974). Under stress

conditions, plants produce cluster of proteins called heat shock or heat induced

proteins. Overall, many heat inducible genes are expressed that is called as heat

shock genes which encode heat shock proteins, essential for existence plant in heat

stress conditions (Charng et al., 2007). Several kinds of HSPs have been recognized

in many organisms (Bharti and Nover, 2002). In plants, according to their molecular

mass and their activities, five classes of heat shock proteins were characterized as:

HSPs60, HSPs70, HSPs90, HSPs100 and small heat shock proteins (Kotak et al., 2007).

The HSPs60 and HSPs70 normally conserved protein in nature and play vital role in

heat stress conditions (Kultz, 2003). HSPs70 present in different organelles such as

nuclear, chloroplast, cytosol, mitochondria and endoplasmic reticulum (Usman et

al., 2017) in barley crop (Landi et al., 2019) while HSPs90 in Arabidopsis thaliana

(Toumi et al., 2019). Heat shock protein play important role in the stabilization of

protein, avoiding the aggregation of polypeptide and facilitate the protein

maturation (Hartl et al., 2011). It is reported that HSP90 play vital role in plant

defense mechanisms (Bao et al., 2014), cellular homeostasis, growth and

reproductive and flowering development (Margaritopoulou et al., 2016).

19

4.1. Antioxidant Defense Response to Heat Induced Oxidative Stress

Thermotolerant plants have ability to protect against the damaging effects of reactive

oxygen species during stress conditions, which produces different enzymatic and non-

enzymatic biomolecules for detoxification of ROS (Apel and Hirt, 2004). These enzymatic

and non-enzymatic antioxidant defense system are as follows:

4.1.1. Enzymatic Antioxidants

In higher plants, ROS scavenging enzymes are catalase (CAT), ascorbate peroxidase

(APX), glutathione peroxidase (GPX), peroxiredoxins (Prx) and thioredoxins (Trx).

These antioxidant enzymes have various temperature ranges but the activity of

these enzymes increases at high temperature. It is reported that the activities of CAT,

APX and SOD were increased in high temperature while POX and GR declined their

activities in temperature ranges from 20°C to 50°C (Chakraborty and Pradhan, 2011).

This thermotolerance and susceptibility depend on crop varieties, growth phase and

growing season (Almeselmani et al., 2006).

4.1.2. Non-Enzymatic Antioxidants

Non-enzymatic antioxidants including ascorbic acid, tocopherol glutathione and

carotenes work with antioxidant enzymes against intracellular ROS, which may help

plant growth and development as well as strengthen the responses against harsh

environmental circumstances. Non-enzymatic antioxidants such as carotene,

tocopherol, ascorbic acid and glutathione defends against oxidative stress (Sairam

et al., 2000). Heat stress increased the glutathione level that enhanced the tolerance

of wheat crop under high temperature (Chauhan, 2005).

20

5.1. Plant Adaptation to Heat Stress

On the basis of temperature tolerance plant can be divided into fallowing groups:

(i) Psychrophilic: Plant grow at very low temperature range from 0°C to 10°C.

(ii) Mesophylls: Its moderate temperature between 10°C to 30°C for growing plant.

(iii) Thermophiles: Those plant can be cultivated between 30°C to 65°C (Zrobek, 2012).

Different plant species have different responses and resistance to heat stress. Another

scientist grouped pant species into three categories: (i) heat sensitive species (ii) relative

heat resistant species and (iii) heat tolerant species (Larcher, 1995).

6.1. Mechanism of Plant Adaptation to Heat Stress

6.1.1 Avoidance Mechanism

Plant as sessile organism which cannot move from one place to another, under high

temperature. Plants have developed different mechanisms to avoid any environmental

stress such as short term and long-term avoidance mechanisms. First long-term

mechanism which includes evolutionary phonological and morphological adaptations

and secondly, short term avoidance mechanism in which plants change leaf direction,

change lipid compositions and close stomata to prevent water losses (Srivastava et al.,

2012). Plants can also reduce heat intensity by protective waxy covering, leaf rolling.

Under heat stress physiological leaf rolling was evident in wheat plant (Sarieva et al.,

2010). During grand growth stage crops are extremely sensitive to heat stress. Good

agriculture practices like selecting proper sowing method (late or early sowing), crop

varieties can also be avoided against high temperature stress (Hall, 2011).

21

6.1.2. Tolerant Mechanism

Plants have various responses against different environmental stresses, which depend on

stress types, intensity and duration (Queitsch et al., 2000). Plants fight against any biotic

and abiotic stresses by through many tolerance mechanisms that includes expression of

heat shock proteins, accumulation of osmolytes, ion transporters and antioxidant

defense systems (Rodriguez and Borras, 2005; Wang et al., 2004). This stress responsive

mechanisms maintain the homeostasis and protect enzymes, protein and membrane

damages (Vinocur and Altman, 2005).

7.1. Sucrose Metabolism and Regulation in Sugarcane

The word sucrose was coined by William Miller in 1857. It is non-reducing sugar

(disaccharide) which synthesized by hexose sugar (glucose and fructose) that naturally

occurs in many plants such as sugarcane and sugar beets.

7.1.1. Biosynthesis of Sucrose in Sugarcane Plant

During photosynthesis, sugarcane leaves produce more carbohydrate (as

triphosphate) then it is converted to sucrose and transported to other compartment

of plant cells such as stalk or stem. Sucrose is the primary form of stored sugar in

sugarcane and sugar beet. Sucrose is synthesized at different compartment such as

plastid and cytosol. After condensation of two triphosphates to form fructose 1, 6

biphosphate), hydrolysis by fructose 1, 6 bisphosphates yields fructose 6-phosphate.

Sucrose 6-phosphate synthase then catalysis the reaction of fructose 6-phosphate

with UDP-glucose to form sucrose 6-phosphate. Mostly, the triose phosphate

generated by carbon dioxide fixation process is converted to sucrose as following

reaction:

22

Fig 3: Sucrose biosynthesis: Sucrose is synthesized from uridine diphosphate synthase

(UDP) glucose and fructose 6-phosphate, which are synthesized from triose phosphates in

the plant cell cytosol. The sucrose 6-phosphate synthase of most plant species is

allosterically regulated by glucose 6-phosphate and Pi.

7.1.2. Source-sink Regulation of Sucrose Accumulation in Sugarcane

Sucrose is accumulated in stem according to their capacity, supply of sources (leaves)

and sink demand. When source supply is not fulfilled for sink demand that is called

source limited plant. In contrast, when source supply exceeds sink demand, it is

called sink limited plant. Most of the sugar plants are in last category (sink limited)

(W. Patrick et al., 2013). Furthermore, high sucrose accumulating cultivars of

sugarcane had lower photosynthesis activity than low sucrose accumulating cultivars

(McCormick et al., 2008). Sugarcane has a typical source to sink system, sucrose

transported parenchyma and apoplastic pathway with concentration ranged from

400 to 700 nm (Welbaum and Meinzer, 1990; Moore and Cosgrove, 1991). Suberized

cell walls barrier prevent the apoplastic sucrose to reverse into phloem (Welbaum

et al., 1992). So, sucrose transporting from vacuole to apoplast is a controlling step

in sucrose accumulation. In juvenile internodes, 66 % of carbon utilizes for

respiration and synthesis of protein and fibers while 34 % of carbon stores as

sucrose, but reverse the case in mature internodes (Bindon and Botha, 2002). The

23

sucrose content depends on many enzymatic activities which play important role in

sucrose metabolism in sugarcane. In sugarcane, sucrose metabolizing enzymes are

sucrose phosphate synthase (SPS), sucrose synthase (SS) and invertases are

expressed in leaves and stem (Kalwade and Devarumath, 2014). Sucrose synthase

(SS) is key enzyme for sucrose synthesis playing dual role of hydrolysis or synthesis

but invertases only cleaves the sucrose into hexose sugar (glucose and fructose). The

sucrose phosphate synthase activity is higher in high sucrose content cultivars and

mature internode than low sucrose cultivars and immature internodes (Verma et al.,

2011). Sucrose synthesized in photosynthetic leave (source) is translocated through

phloem to stalk (Fig 4).

Fig 4: Schematic presentation of sucrose metabolism and transportation from source

(leave) to stem (sink).

24

8.1. Sucrose Metalizing Enzymes Response Under Heat Stress

In the past thirty years, quick development in understanding the dynamic of sucrose

metabolizing enzymes during leaf growth (Ruan, 2014). Sucrose metabolizing enzymes

are divided into two groups first group is hydrolyzed enzymes including sucrose synthase

and invertases which hydrolyzed sucrose irreversibly into glucose and fructose while

second is synthesis enzyme group such as sucrose phosphate synthase which sucrose

reversibly in the presence of uridine diphosphate (UDP) into uridine diphosphate-glucose

and fructose (UDPG-F), SPS is also responsible for sucrose synthesis in leaves (Wang,

2013).

8.1.1. Invertases

Invertase also called beta-fructofuranosidase (E.C.3.2.1.26). It is found commonly in

plants such as in pear, pea, grapes, oats and microorganisms like S.cerevisia, Candida

utilus and niger ect. In sugarcane, invertases are important for growth and

development (Moore, 1995; Zhu et al., 1997). There are different types of invertases

terms as isoform or isozymes codded by the different gene or one enzyme more than

one locus gene duplication. Sucrose transported from source (Leaf) to stem (sink)

through different cellular compartments cell wall, cytoplasm and vacuole (Ma et al.,

2000). Different invertase isozymes present in plant with different functions,

properties and beneficial role to the crop (Lahiri et al., 2012; Kim et al., 2011). Based

on subcellular localization, solubility, isoelectric point and pH plant invertase can be

divided into following groups.

8.1.2. Soluble Acids

In sugarcane two types of acid invertase have been reported based on solubility and

cellular localization.

25

8.1.2.1. Soluble Acid Invertase (Vacuolar Invertase)

Vacuolar acid invertase present on vacuole, has an acidic pH-4.5 to 5.0. It is believed

to be important in the regulation of hexose level in certain tissues, rate of return

sugar from storage, sucrose import, sugar signaling and remobilization of stored

sucrose from the vacuole (Sturm et al., 1999). High accumulation of sucrose directly

proportional to soluble acid invertase (SAI) in sugarcane plant during rapid growth

phase. The substrate of this enzyme is sucrose while metal ions such as mercury and

silver are inhibitors having molecular weight of is about 70 kDa, 80 kDa and 86 kDa

respectively (Hashizume et al., 2003). Vacuolar invertase also have more than two

isoforms, these isozymes can be characterized and purified from many plants such

as barley, pear, tobacco etc.

8.1.2.2. Insoluble Invertase (Cell Wall Invertase)

Acid invertase, glycosylated protein belongs to family GH32 and play significant role

in sucrose partitioning, growth and development (Roitsch and Gozale, 2004). It is

true member of β-fructofuranosidase, sucrose and raffinose as substrate (Belcarz et

al., 2002). Cell wall invertase is confined to the cell wall (ionically bond) and

exhibiting optimum activity at pH-3.2 to 3.6 and temperature 45°C with different

molecular weight ranges from 28 kDa to 64 kDa. The first cloned acid invertase was

found in carrot and tobacco (63 kDa), in tomato (68 kDa) (Klann et al., 1992; Konno

et al., 1993), Potato 58 kDa (Bracho and Whitaker, 1990) (Weil and Rausch, 1990). In

mung bean 70 kDa protein and subunit heterodimer 30 kDa and 38 kDa were

exhibited. Acid invertase dimer in barley 64 kDa (Avigad and Dey, 1997), rice 46 kDa

(Isla et al., 1995) proteins were observed. Cell wall invertase also act as gateway for

26

the entry of sucrose into the cell in juvenile tissues. It is found in maize seed kernel

and also found in seed coat of parenchyma cell.

8.2. Cytoplasmic Invertase (Alkaline/Neutral)

Cytoplasmic invertase is a non-glycosylated polypeptide but belong to the GH100

family (lammens et al., 2009) expressed at low level ranging from 54 kDa to 65 kDa

molecular weight at different growth and development stages. Cytoplasmic

invertase was localized in cytosol, mitochondria chloroplast and nucleus (Vargas and

Salerno, 2010) and involved in flowering, seed germination, growth and

development (Jia et al., 2008; Barratt et al., 2009). It is reported that cytoplasmic

invertase activities negatively correlates with sucrose content (Chandra and

Solomon, 2012). While, in inter nodal tissues it exhibited positive correlation with

sucrose concentration and isozymes of molecular weight (60, 120 and 240 kDa) were

reported (Vorster and Botha, 1998). It is antioxidant against reactive oxygen species

homeostasis (Xiang et al., 2011).

8.3. Sucrose Synthase (pH 7.5)

Sucrose synthase (SS) (EC 2.4.1.13) is very important sucrose metabolizing enzyme

belongs to subfamily glycosyltransferases and its protein are characteristically

considered homotetramers (Schmolzer et al., 2016). Sucrose synthase (SS)

expressed in vasculature of many plant species in phloem (Goren et al., 2017). SS

plays important role in high temperature in plants and may play signaling role in the

development of flowers (Cho et al., 2018). It is reported that SUS3 allele that is highly

expressed during seed maturity stage may confer resistance to chalky grain in brown

rice caused by heat stress (Takehara et al., 2018). Additionally, heat resistant sucrose

27

synthase was purified in wheat line (WH-1Q21). It catalyses the reversible hydrolyse

of sucrose into fructose in the presence of NDP-glucose (Kolman et al., 2015) as

following:

NDP-glucose + D-fructose ⇌ NDP + sucrose

Both NDP-glucose and D-fructose are substrate of this enzyme while D-fructose and

sucrose are products. The product of sucrose hydrolyzed by sucrose synthesis

enzymes are important for energy production, primary metabolites as well as starch

synthesis. SS is present in cytosol, cell wall, vacuole and mitochondria. The optimal

SS activity is pH 5.5-7.5 and with molecular mass of approximately 90 kDa. It is also

found in other plants such as., banana with 110 kDa (Yang and Su, 1980) Arabidopsis

with 107 kDa (Baud et al., 2004) wheat with 63 kDa (Verma et al., 2018), bean with

78 kDa (Fujii et al., 2010). When SS activity was reduced, growth and development

is affected with low tolerance against stress conditions. Whereas overexpression of

SS activity had shown increased starch content and growth, making SS high possible

candidate gene for the development of economical important crops. Activity of this

enzyme is regulated by two phosphorylation positions such as, serine

phosphorylation site at position 11 to 15 which is assumed to play important role in

membrane association and other site also serine, at about position 170 which is

supposed to regulate protein degradation (Hardin et al., 2003). In rice this

phosphorylation protein Rsus1-3 could promote sucrose synthases activity (Takeda

et al., 2017). Sucrose synthase isozymes have also been noticed in cell wall in various

plants for example, rice, carrot, tomato, sugarcane and cotton (Salnikov et al., 2003)

in tobacco pollen tubes (Persia et al., 2008). However, it is demonstrated that

sucrose synthase localized to the cell wall remains unclear (Brill et al., 2011). Under

28

stress conditions, sucrose synthase play important role in metabolism. Sucrose

synthase genes were found in various plants species, in rice 6 genes (Hirose et al.,

2008) in grapes and sugarcane 5,5 genes are characterized (Zhang et al., 2013; Zhu

et al., 2017). Sucrose synthase genes exhibited in apples (Tong et al., 2018) while 30

in Chinese-pear (Abdullah et al., 2018). It activity was noticed in in delayed ripening

in straw berry fruits (Zhao et al., 2017) and in immature internodes in sugarcane stalk

(Schafer et al., 2004).

8.4. Sucrose Phosphate Synthase (pH 7.5)

Sucrose phosphate synthase (SPS) (EC 2.4.1.14) is the most important enzyme

among sucrose metabolizing enzymes. Sucrose phosphate synthase (SPS) and

Sucrose synthase (SS) activity declines at increasing temperature of 42°C in

sugarcane (Gomathi et al., 2013). Sucrose phosphate synthase found in both

photosynthetic and heterotrophic tissues such as leave, stem, roots and nodules

(Aleman et al., 2010; Haigler et al., 2007). Sucrose phosphate synthase has very

important role in growth and development for many plants such as maize plant

(Causse et al., 1995a) and its activity associated with dry mass yield (Causse et al.,

1995b). In transgenic rice its activity is directly proportional to growth rate (Ishimaru

et al., 2004), in sugarcane non reducing sugar (sucrose) accumulation in the stalk

depends on sucrose phosphate synthase (Zhu et al., 1997). SPS also present in alfalfa,

pea (Aleman et al., 2010), it is also reported that Arabidopsis has 4 genes encoding

SPS enzymes (Lunn and MacRae, 2003; Langenkämper et al., 2002). SPS crucial role

in water stress condition and low temperature in potato crop (Krause et al., 1998;

Geigenberger et al., 1999). However, it activity gradually decreased at 28°C but it

inactivated when temperature at 60°C by heat stress (Neliana et al., 2019).

29

8.6. Sugar Recovery Rate

High population growth rate countries are working more aggressively to improving

crop yields. Sugar recovery rate is the percentage (%) of sugar production in metric

ton to the sugarcane crushed in metric ton. Sugar recovery is a significant parameter

for both farmers and industrialist (Costa et al., 2014). Quality cane depends on some

characteristics such as sugar recovery rate, high °brix, pol and high sucrose content

in stem while minimum non-sugar and optimum fibers. But the other factors like

climate change adversely affects on sugar recovery rate. Recovery rate of Pakistan is

9.87 to 10.02 % (PSMA report, 2019). Which is less than other sugarcane producing

countries. Average sugar recovery rate countries are Brazil (14.6 %), Australia (13.8

%, European Union (13 %), USA (11.7 %) Mexico (11.6 %), Egypt (11.5 %), Thailand

(11.3 %) and India (10 %) (Roy et al., 2018; PSMA report, 2007). Furthermore, there

are many other factors which also contribute to decline sugar recovery rate. For

instance, late harvesting, transportation, storage, processing and improper hygienic

conditions etc. Soon after harvesting sugar recovery rate start to decline, after 24 h

is consider reduction in cane weight due to moisture loss, reduction in sucrose

content due to sucrose hydrolyse (Roy et al., 2018). This is one of the most

challenging factors for sugarcane producing countries (Suma et al., 2000). It is

suggested that due to staling of canes for 4 days, there is reduction in sugarcane

mass (7.4 % to 17.0 %) (Datir and Joshi, 2015), recovery rate 2 % (Rakkiyappan et al.,

2009). So, improvement in sugar recovery rate is very important with high sugarcane

yield against biotic and abiotic factors.

30

9.1. The Mitigation of Heat Stress Strategies

Variation in environment has long lasting impact on agriculture and food security

worldwide. Food security is threatened by global warming and severe climate conditions.

This changing global temperature is challenging for crop scientists to combat this

problem. Therefore, to cope theses changing climate, development of thermotolerance

crop varieties against high temperature stress are required.

9.1.1. Cultural Method

There are so many agronomical practices to combat the climate change such as changing

sowing and harvesting time, crop rotation, irrigation techniques and variation in cropping

scheme. Under heat stress condition these approaches are useful for crop adaptability

Deligios et al., 2019; Duku et al., 2018). So the choice of suitable sowing and harvesting

time, planting density, best irrigation practices are essential techniques to tackle any

environmental stress conditions (Battisti et al., 2018). Fertilizer also play very important

role under stress conditions, it provides to support better adoptability, to provide energy

to maintain the soil fertility and increase the crop productivity (Henderson et al., 2018).

Under high temperature stress, macronutrients (k, Ca) and micronutrients (B, Se and Mn)

regulate stomata functions and also activate the physiological and metabolic activities

contributing to maintain high water potential in tissues and reduce toxicity to ROS by

enhancing the activity of antioxidant enzymes in plant cell ( Waraich et al., 2012).

Under any environmental stress conditions, plant breeding is the best techniques for

enhancement of crop productivity. It provides potential to guarantee food security,

escape from stress through a crucial growth and development phases by developing

stress resistant cultivars (Abraham Blum, 2018). For this, genetic divergent analysis is

31

considered a significant method for the improvement of new varieties based on genetic

distance and similarities (Raza et al., 2019; Raza et al., 2018).

9.2. Genetics and Genomics Strategies

9.2.1. Omics-Led Breeding and Marker-Assisted Selection (MAS)

Omic approaches provide valuable resources to elucidate biological functions of any

genetic information for crop progress and growth (Stinchcombe and Hoekstra, 2008). The

breeding program is attached with genomic approaches to succeed great heights in

molecular breeding and to screen elite germ plasms with multiple trait assembly (Bevan

and Waugh, 2007). Genomic also allows exploration of the molecular mechanism

underlining the abiotic stresses resistance. These approaches assistance in the

improvement of climate smart crops for high yield and production under high

temperature stress (Roy et al., 2011). With the initiation of high quantity of sequencing

and genomic led breeding paved the way for recognizing various stresses that are

projected to badly affect on crop production. In addition, the data available on many

environmental stresses, DNA fingerprinting and qualitative trait loci (QTL) mapping

permits the screening of best germplasms under heat stress (Kotle et al., 2015). QTL

dissention of yield related characteristic under high temperature stress allows the

development of new varieties with better adaptability in abiotic stress (Collins et al.,

2008). Molecular plant breeding is very important approach for improving crop yield

(Gosal and Wani, 2018). For the immediate breeding progression marker assisted

selection (MAS) presents a fundamental part in the improvement of crop trait and yield.

With the development in crop genomics, DNA markers have been recognized which are

valuable for marker-assisted breeding (Da Silva, 2014).

32

9.2.2. Genome Wide Association Studies (GWAS) for Stress Tolerance

Genome wide association studies is a powerful tool for understanding the whole set of

genetic variants in different crop varieties to identify allelic variants connected with any

particular characteristic (Manolio, 2010). Genome Wide Association Studies (GWAS)

mostly highlight association among SNPs and characteristic and based on GWAS design,

genotyping tools, statistical models for examination and results interpretation (Bush and

Moore, 2012). In many crop GWAS has been carried out to exploit the genetic process

responsible for genetic resistant under heat high temperature stress conditions

(Mousavi-Derazmahalleh et al., 2019). In plants, GWAS has extensive applications

associated to environmental stresses. GWAS have been applied to describe salt tolerance

(Wan et al., 2017), drought tolerance (Thoen et al., 2017) and thermo tolerance (Lafarge

et al., 2017).

9.2.3. Genetic Engineered Plants for Stress Tolerance

The genetic alteration via biotechnology is an important tools and powerful strategy for

the improvement of plant against biotic and abiotic stresses. Encouraging data collected

from genetics which can be exploited significantly to numerous biotic and abiotic stresses

including heat stress. Identification of stress response transcription factors are influential

discoveries to improve thermotolerant crop varieties. These transcription factors can

control the phenotypes of genes in genetic engineered crops associated with different

environmental stresses (Reynolds et al., 2015). There are many transgenic plants which

have been recognized by genetic engineering to tackle the various environmental

stresses. These transgenic plants revealed important resistance under heat stress

conditions as compared to normal plants (Nejat and Mantri, 2017; Shah et al., 2016).

33

Various plant specific TFs are identified such as AP2/ERFBP group (Riechman and

Meyerowits, 1998). This family or groups of TFs is responsible for plant growth and

development pathway and has functions in different ecological stresses (Licausi et al.,

2010). The subfamilies of TFs including DREB (dehydration-responsive element-binding

protein) and ERF play vital role under biotic and abiotic tresses conditions (Phukan et al.,

2017).

9.3. Genome Editing Strategies

Genome editing (GE) is very important tool to manipulate the plant genome by means of

sequence-specific nucleases. This GE tools use for crop development has the

extraordinary capability to tackle food insecurity and advances in climate-smart

agriculture system (Liu et al., 2013). It has been using for many years due to fast and

precise manipulation in crop genomes to defend them against various environmental

stresses including heat stress (Taranto et al., 2018). This genome editing tools including

Crisper-case9, zinc-finger nucleases (ZFNs) and transcription activator like effector

nucleases (TALENs) (Zhu et al., 2017). This crisper-case9 has been used in many plant

genome editing to survive against different stresses (Manolio, 2010). Many plant

manipulated by crisper-case9 gene editing tools against different stresses, 21 KUP genes

identified against stress conditions in cassava (Ou et al., 2018), herbicides resistant was

developed in rice (Shen et al., 2017) and enhance the seed size in wheat (Wang et al.,

2018). To develop new thermotolerant crop varieties regarding global climate change,

there is urgent need to study about thermotolerance mechanisms at physiological,

biochemical and molecular levels. In present scenario, we must understand heat stress

mechanism for avoiding heat shock induced detrimental changes that play a vital role in

34

crop survival. In this situation of global warming, the major concern for plant scientist is

to develop new cultivars or varieties resistant to biotic and abiotic stresses (Zhang et al.,

2005). In the coming decade, due to high temperature, crop yield will decline but

constant growing population, will increase food demand, that create a gap between the

current crop yield achievement and yield potential (Koevoets et al., 2016). There are

many molecular techniques that have been used to develop high yielding and

thermotolerant crop varieties. Present study analysis effect of heat shock for sucrose

metabolizing enzymes that might reveal ways to develop thermotolerance cultivars that

vital in adverse environmental conditions.

35

SECTION # 3 METHODOLOGY

36

Methodology

3. General Experimental Details

This study was performed using two local sugarcane cultivars S2003-US-633 (high

sucrose accumulation) and SPF-238 (low sucrose accumulation) provided by “Cane

Testing Lab” Mehran Sugar Mills Limited. Tando Allahyar, Pakistan. All the

experiments were conducted Plant Care Unit, Agricultural Biotechnology Section, The

Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of

Karachi, Karachi, Pakistan. Experiment was conducted in Completely Randomized

Design (CRD) with three replicates per treatment group at vegetative (30 to 50 days),

grand growth (150 days) and maturity stages (250 days). All chemicals and reagents

of either analytical or optical grades were purchased from Aldrich, Bio-Rad, BDH,

Fluka, Merck, Scharlau and Sigma. Crop was cultivated in February, 2016 and

morphological analysis was measured along with determination of thermo tolerance

indicators such as proline, electrolytes leakage, lipid per oxidation malondialdehyde

(MDA), hydrogen peroxide (H2O2). For second year 2017, sugar metabolism in terms

of total sugar, reducing sugar, non-reducing sugar, total soluble protein, sucrose

metabolizing enzymes sucrose phosphate synthase (SPS), sucrose synthase (SS) and

Invertase isozymes such as cytoplasmic invertase (CyIN) cell wall invertase (CWIN)

and vacuolar invertase (VIN) as well as sugar recovery was quantified. During last year

2018 invertase isozymes were analyzed through Native-PAGE and differential staining

for proteins SDS-PAGE electrophoresis was done, in addition to confirmation of

previous year’s results. The experiments were conducted consecutively for three

years 2016 to 2018.

37

3.1. Physiochemical Properties of Soil and Water

3.1.1. Soil and Water Analysis

Table 4: Detail Physiochemical Properties of Soil and Water Used in Experiment.

Year 2016 2017 2018

Soil texture Clay (%) Silt (%) Sand (%) Soil textural class EC (dSm

-1)

pH Fertilizer Value NO

3-N

P K Water properties EC (dSm

-1)

pH

11.6 12.4 74

Sandy loamy 0.36 7.92

1.23 2.91 192

5.9 7.6

10.5 13.8 75


1.20 2.82 190

6.2 7.9

12.2 11.3 78


1.30 3.10 193

6.6 8.0

3.2. Crop Husbandry

3.2.1. Cultivation of Sugarcane

Before sowing, the sugarcane sets were kept in hot water at 50 C temperature for

two hours (h) to control red-rot, grassy shoots and other virus diseases and to

improve germination. Sugarcane were cultivated in pots filled with 20 kg loamy soil

and 5 kg farm yard manure (FYM). All agronomic practices were done such as

application of fertilizers (NPK), weeding and regularly application of supplement with

half strength nutrients (Hoagland and Arnon, 1950). During experiment, temperature

and relative humidity were recorded on daily bases, at vegetative (50 days), grand

growth (150 days) and maturity stages (250 days) (Fig 5).

38

Figure 5: Annual mean temperature and relative humidity of sugarcane field for the year

2016-2018.

3.3. Heat Stress Treatments

All sugarcane plants were shifted to growth room for heat shock treatments. Where,

white fluorescent tube lights/mercury lamps were used for maintaining

photosynthesis active radiation (PAR) ranging from 650 to 700 µmol m-2 s-1 in long

day conditions (16 h light/8 h dark). Temperature was set at 45±2°C and 34±2°C for

day and night respectively while humidity was maintained at 60% to 70 %. For air

circulation, fans were adjusted. Heat shock treatments at 45±2°C for 24 h (T24), 48h

39

(T48) and 72 h (T72) along with control (30±2°C) at different time intervals were

imposed at vegetative (50 days) grand growth (150 days) and maturity stage (250

days) after planting. The plants were then allowed to recover. For recovery

experiment, pots were again shifted from growth room to field at 30±2°C for 24 h

(R24), 48 h (R48) and 72 h (R72).

3.4. Sample Collection

Samples were collected for control, heat stress and recovery treatments at all

growth phases (vegetative, grand growth and maturity) and stored at -80°C for

further analysis.


The morphological observations were recorded in replicates from each treatment as

shoot length (cm), root length (cm), leaf length (cm), leaf width (cm), stem diameter

(cm), number of leaves, number of tillers, number of nodes, number of internodes

and internode distance (cm).

Fresh weight of shoot was determined on electronic weighing balance immediately

after harvesting while dry weight was taken after drying shoot from oven at 60 °C

for a week. Measurement for shoot, root, leaf, stem diameters and internode

distances were taken using meter scales.

40

3.6. Physiological Analysis

Following physiological parameters were analyzed for assessing heat stress imposed

damages in sugarcane cultivars at different growth stages.

3.6.1. Cell Membrane Thermosability (CMT)

Relative membrane permeability (RMP) in terms of % measured by assessing

electrolytes leakage (EC) using the method by Yang et al., (1996), with the help of

electrical conductivity meter. Fresh leaves (500 mg) cut into small pieces and soaked

in 25 ml distilled water then vortexed for 20 to 30 seconds. Initial electrical

conductivity (EC₀) was measured within 10 min while (EC1) measured after

incubation of tubes for overnight at 4°C then tubes were autoclaved for 20 min, all

tubs were placed at room temperature till it cooled down and (EC2) was measured,

same steps were repeated three times.

Calculation

The electrolyte leakage was calculated as following formulae;

RMP(%) =EC1 − EC₀

EC2 − EC₀ × 100

3.6.2. Proline Quantification

Reagents

Ninhydrin: 1.25 g ninhydrin was dissolved in 30 ml glacial acetic acid and 20 ml 6 M

ortho phosphoric acid.

3 % Aqueous Sulphosalicylic Acid (SSA): 3 g of sulphosalicylic acid was dissolved in

100 ml deionized water.

Proline (Osmolyte accumulation) was determined in sugarcane using method by Bate

41

et al., (1973). Leaf tissues (100 mg) were extracted in 2 ml sulphosalicylic acid with

mortar and pestle. The residue was removed by centrifugation at 12000 rpm for 10

min. Supernatant was used for estimation of proline. In 1 ml aliquot, 1 ml ninhydrin

reagent, 1 ml acetic acid were added, the mixture then heated at 100 °C for 1 h and

transferred on ice bath for termination of reaction. The reaction mixture was

extracted with 4 ml toluene then vortexed for 20 to 30 seconds and kept for 30 min

at room temperature, upper phase (toluene layer) was separated in a dry glass tube

then the absorbance or intensity at 520 nm using spectrophotometer was measured,

while toluene was used as a blank. Standard curve of proline ranging from 10 µg to

50 µg / 2 ml was constructed and slope value was used in formula for calculation.

Calculation

The Proline was calculated as following formulae;

Proline mg ml −1 =Slope × Absorbance

Extract Used (ml)

Proline (uMg−1FW) =µg Proline × 115.5 × Volume of Extract

Toluene (ml) × Sample(g)

Note: (Where 115.5 is the molecular weight of proline)

3.6.3. H2O2 Quantification

Reagents

0.1 % w/v TCA: 0.01 g trichloroacetic acid (TCA) dissolved in 10ml water.

10 mM phosphate buffer (pH 7.0): Disodium phosphate (0.141g) and monosodium

phosphate (0.119 g) dissolved in 100ml distilled water. Mixed both solution and

adjusted pH at 7.0.

42

1 M Potassium iodide (KI): 16.6 g Potassium iodide dissolved in 100ml distilled water.

Hydrogen peroxide (H2O2) content was assayed by the method of (Jessup et al., 1994).

For this 0.1 g sugarcane leaf was homogenized in 2 ml 0.1 % trichloro acetic acid in

mortar and pestle. The extract was centrifuged at 12000 rpm for 15 min. From

supernatant, an aliquot of 0.5 ml is added to 1 ml of phosphate buffer and 1.5 ml of

potassium iodide. The mixture was vortexed for 15 to 20 seconds then its absorbance

was measured at 390 nm. 2ml of potassium iodide and 1ml potassium buffer were

used as blanks in the absence of leaf extract. Standard curve of H2O2 was constructed

using different concentration of H2O2 standards. Results were expressed as μmol

H2O2 g-1 fresh weight.

3.6.4. Determination of lipid peroxidation

Reagents

5 % Tetracholoroacitic acid (TCA): 0.5 g of TCA was dissolved in 10 ml distilled water.

0.5 % Thiobarbituric acid (TBA): 0.05 g of TBA was dissolved in 10 ml distilled water.

To estimate the malondialdehyde (MDA) content, byproduct of membrane lipid

peroxidation was quantified as described by (Heath and Packer, 1968). Fresh leaf of

sugarcane (100 mg) was homogenized in pestle and mortar in 2 ml tetracholoroacitic

acid solution. After centrifugation at 12000 rpm for 15 min, 1 ml supernatant was

mixed with 1 ml thiobarbituric acid then kept in water bath for 30 min at 95 C. The

mixture was allowed to cool rapidly on an ice bath and again centrifuged at 10,000

rpm for 10 min then absorbance of MDA was measured at 532 nm. The value of non-

specific absorbance at 600 nm were subtracted by using spectrophotometer, 5 % TCA

used as blank.

43

Calculation

Lipid peroxidation was expressed as nmole g-1 FW using the formula;

MDA =A532 − A600

15500 × 106


Sugar analysis in terms of total, reducing and non-reducing sugars were carried out

from both sugarcane varieties from heat shock, recovery along with control

treatments at all growth stages.

3.7.1. Sugar Extraction

For extraction of reducing and total sugar, 100 mg of sugarcane leaves were

homogenized in 5 ml of 80 % ethanol.


Reagents

Standard sucrose stock—10 mg sucrose dissolved in 10 ml distilled water.

Working standard: 1 ml stock solution was added into 3 ml water (1:3) for working

standard and diluted as (25 to 250 µg ml-1).

Anthrone reagent: 100 mg anthrone was dissolved in 50 ml of ice-cold 95 %

hydrochloric acid (H2SO4).

Total sugars were determined by using Anthrone reagent method (Hedge and

Hofreiter, 1962). The homogenate was centrifuged at 1000 rpm for 10 min and used

for the estimation of total sugars. Volume was made up 975 µl distilled water added

in 25 µl supernatant of ethanol extraction. 5ml of anthrone reagent was added and

the reaction mixture was heated for 15 min in a boiling water bath, cooled rapidly

44

and vortexed. The absorbance of the green colour solution was measured at 620 nm

by using spectrophotometer. Sucrose was used as standard. The total sugar content

was expressed in terms of percentage on fresh weight basis.

Calculation

Total sugar was calculated as µg ml-1 using the formula:

Total sugar (µg ml−1 ) =ODT × Conc Std (µg)

OD Std × AS

whereas,

ODT= Optical Density of Test (Sample)

Conc std= Concentration of Standard

OD std= Optical Density of Standard

AS= Amount of Sample


Reagents

0.5 gm DNS: dissolved in 25 ml distill water on stirring. After solubilizing 15 gm

potassium sodium tartrate was added after completed solubalization milky yellow

solution was obtained.

0.8 gm NaOH: dissolved in 10 ml of distill water and added NaOH solution in to DNS

solution it was turned into clear orange solution made volume up to 50 ml then kept

in refrigerator in dark bottle.

Standard Stock: 0.05 gm glucose dissolved in 50 ml distilled water and diluted (0.1

to 1.0 mg ml-1).

Reducing sugar was estimated by using DNSA (dinitro salicylic acid) reagent method

45

(Miller, 1959). The homogenate was centrifuged at 1000 rpm for 10 min and used

for the estimation of reducing sugar. 1 ml DNS was added with 1ml reaction mixture

and kept in a boiling water bath for 5 min. After cooling 9 ml distilled water was

added and then vortexed. The absorbance of the orange colored solution was

measured at 546 nm using spectrophotometer. Glucose was used as standard. The

reducing sugar content was expressed in terms of percentage on fresh weight basis.

Calculation

Reducing sugars was calculated a mg ml-1 using the formula:

Reducing sugar (mg ml−1 ) =ODT × Conc Std (mg)

OD Std × AS

3.7.4. Non-Reducing Sugar Estimation:

Non-reducing sugar was estimated by following formula;

Non reducing sugar ( mg ml−1) = Total sugar − Reducing sugar

3.7.5. Total Soluble Proteins and Sugar Metabolizing Enzymes

Extraction:

Reagents

500 mM MOPS: 10.465 gm MOPS dissolved in 100 ml deionized water.

15 mM MgCl2: 0.914 gm MgCl2 dissolved in 300 ml deionized water.

10 mM EDTA: 0.371 gm EDTA dissolved in 100 ml deionized water.

10 mM DTT: 0.462 gm DTT dissolved in 300 ml deionized water.

Bovine serum albumin (BSA): 0.25 gm BSA dissolved in 50 ml deionized water (freshly

prepared).

0.05 % (v/v) Triton X-100

46

Working solution:

50 ml MOPS, 16 ml MgCl2, 50 ml EDTA, 125 ml DTT, 50 ml BSA and 0.25 ml Triton X-

100 were added and adjusted pH through NaOH and stored at 4°C.

3.7.5.1. Extraction

Sucrose metabolizing enzymes were extracted by buffer containing, MOPS-NaOH

(pH -7.5), MgCl2, EDTA, DTT, Triton X-100 and Bovine serum albumin (BSA). For this

100 mg tissues were homogenized in 2 ml buffer and then centrifuged at 12000 rpm

for 30 min. Supernatant were separated into other tubes and then stored at -20 for

further biochemical analysis.

3.7.5.2. Total Soluble Protein Quantification

Reagents

Coomassie Brilliant Blue (CBB) G-250 dye: 0.01 g CBB (G-250) was dissolved it in 5

ml 95 % ethanol. 10 ml of orthophosphoric acid was added to make the volume up

to 20 ml with deionized water. Reagent was filtered with Whatman paper # 01 and

stored in dark bottle at 4°C.

0.15N NaCl: 0.87 g NaCl was dissolved in 100 ml deionized water.

Bovine Serum Albumin (BSA): 0.01 g of bovine serum albumin was dissolved in 10

ml water.

Total soluble proteins were calculated through Bradford Assay (Bradford, 1976). The

3 ml reaction mixture containing, 50 µl protein sample, Bradford dye 150 µl and 0.15

N NaCl 2800 µl vortexed for 5 to 10 seconds then incubated at room temperature

15-20 min and read at 595 nm by spectrophotometer. Protein standard curve was

constructed by using known concentration of BSA (10 to 100 µg ml-1).

47

Calculation

Total soluble protein was calculated as mg ml-1 using the formula;

Protein (mg ml−1 ) =ODT × Conc Std (mg)

OD Std × AS

3.8. Quantification of Sugar Metabolizing Enzymes

3.8.1. Vacuolar Acid Invertase (VAI) pH 5.0

Unit Definition:

One unit is defined as that quantity of vacuolar invertase that will convert or

generate sucrose to 1.0 µmol of glucose per minute at pH 5.0 at 37 °C under assay

conditions.

Principle

Sucrose + water → Glucose + Fructose

Reagents

0.05 M potassium-acetate buffer (pH 5.0): 4 g sucrose was dissolved in 100 ml

acetate buffer (freshly prepared).

DNSA reagent (1.6 % NaOH): (As mentioned in reducing sugar protocols).

Soluble acid invertase activity was assayed using modified method of (Hatch et al.,

1963) and (Voster and Botha, 1999). The assay medium consisted of 0.05 M

potassium-acetate buffer (pH 5.0), 250 µl 4 % sucrose, 500 µl plant extraction and

250 µl deionized water. All control reactions tubes were stopped at 0 minute with 1

ml of (1.6 % NaOH, DNSA reagent) before incubation. The assay mixture was

incubated at 37 °C for 1 h. After that reaction mixture was neutralized with 1 ml of

(1.6 % NaOH, DNSA reagent) and heated at 100 °C for 30 min in a boiling water bath.

After cooling, 9 ml of water added in each tub then absorbance was measured at

48

540 nm using spectrophotometer. The blank tube contained all mixture excluding

enzyme. The enzyme activity was measured as glucose mg ml-1 protein mint-1 .

Calculation

Vacuolar acid invertase was calculated µmole ml−1 using the formula;

Vacuolar invertase (units ml−1min−1) =ODT × Conc Std (mg)

OD Std × AS × MW × RT × TSP

whereas,

ODT=Optical Density of Test (Sample)

Conc std=Concentration of Standard

OD std=Optical Density of Standard

AS=Amount of Sample

MW=Molecular Weight

RT=Reaction Time

TSP=Total Soluble Protein

3.8.2. Quantitative Analysis of Cell Wall Invertase (CWI) pH 3.5

Reagents

0.05 M potassium-acetate buffer (pH 3.0): 4 g sucrose was dissolved in 100 ml

acetate buffer (freshly prepared).

Unit Definition:

One unit is defined as that quantity of cell wall invertase that will convert or generate

sucrose to 1.0 µmol of glucose per min at pH 3.0 at 37 °C under assay conditions.

Principle


49

The activities of cell wall bond invertase using with modified procedure by (Hatch et

al., 1963). The assay medium consisted of (250 μl) 0.05 M potassium-acetate buffer

(pH 5.0) with 4 % sucrose, (250 μl) supernatants and (500 μl) deionized water. The

pellet was washed with extraction buffer and re-suspended in 2000 μl of MOPS-

NaOH (pH-7.5), kept for overnight at 4°C. Then aliquot was used for activity assay in

1 ml containing acetate buffer (pH-3.5) with 4 % sucrose as a substrate (250 µl), for

control and blank (excluding 4 % sucrose buffer) were used. The reaction mixture

was incubated at 37 °C for one hour. All reactions were terminated by added 1 ml of

(1.6 % NaOH, DNSA reagent) after incubation, boiling at 100°C in water bath for 5

min. After cooling at room temperature, samples were re-centrifuged at 14000 rpm

for 5 min and absorbance was taken at 540 nm using known glucose standard curve.

Calculation

Cell wall invertase was calculated µmole ml-1 using the formula;

Cell wall invertase (units ml−1min−1) =ODT × Conc Std (mg)


3.8.3. Quantification of Cytoplasmic Invertase (CyIN) pH- 7.0

Unit Definition:

One unit is defined as that quantity of neutral invertase that will convert or generate

sucrose to 1.0 µmol of glucose per minute at pH 7.0 at 37 °C under assay conditions.

Principle


Reagents

0.1 M Potassium-phosphate buffer (pH 5.0): Solution # A: 1.74 g K2HPO4 was

dissolved in 100 ml deionized water and solution # B: 1.36 g KH2PO4 sucrose was

50

dissolved in 100 ml deionized water and both solution mixed and adjusted the pH 7.0.

DNSA reagent (1.6 % NaOH): (As mentioned in reducing sugar protocols)

Procedure

The assay medium consisted of 0.1 M potassium-phosphate buffer (pH 7.0), 250 μl

4 % sucrose, 500 μl plant extraction and 250 μl deionized water. All control reactions

tubes were stopped at 0 min with 1 ml of (1.6 % NaOH, DNSA reagent) before

incubation. The assay mixture was incubated at 37 °C for 60 min. After that reaction

mixture was neutralized with 1 ml of (1.6 % NaOH, DNSA reagent) and heated at 100

°C for 30 min in a boiling water bath. After cooling, 9 ml of water added in each tub

then absorbance was measured at 540 nm using spectrophotometer. The blank tub

contained all mixture excluding enzyme.

Calculation

Cytoplasmic was calculated µmole ml-1 using the formula;

Cytoplasmic invertase (units ml−1min−1) =ODT × Conc Std (mg)


3.8.4. Quantification of Sucrose Phosphate Synthase (SPS) pH-7.5

Principle

Higher plants synthesize sucrose through two processes. Suc 6'-phosphate is then

dephosphorylated by Suc-P phosphatase (E.C 2.3.1.14; SPS) to produce sucrose as

the final product. The sucrose phosphate synthase a soluble enzyme located in the

cytoplasm that catalysis the reaction:

UDPGlc + Fru6-phosphate ⇌ UDP + Suc6'-phosphate

51

Reagents

50 mM MOPS-NaOH (pH-7.5)

2 mM UDGP (Substrate)

5 mM MgCl2

1 mM EDTA

4 mM Frutose-6-p

20 mM Glucose-6-p

30 % KOH

0.14 % (w/v) anthrone reagent in 95 % sulphuric acid (H2SO4)

Procedure

Sucrose phosphate synthase activities were quantified according to the method

described by (Huber et al., 1989). The reaction mixture containing 10 μl enzymes, 25

μl (UDPG) substrate, 70 μl KoH, MOPS-NaOH (pH -7.5) buffer consisted of MgCl2 and

EDTA, Fru 6-P, Glu 6-P. The mixture was incubated at 37°C for 20 min before adding

70 µl 30 % (w/v) KOH and heating for 10 min at 100°C. To this 5 ml anthrone reagent

was then added. The mixture was incubated at 100°C for 20 min and A620 was

measured.

Calculation

Sucrose phosphate synthase (SPS) was calculated µmole ml-1 using the formula;

SPS (units ml−1min−1) =ODT × Conc Std (mg)


52

3.8.5. Sucrose Synthase (SS) pH-7.5

Principle

Sucrose synthase (E.C 2.4.1.13) which catalysis a reversible reaction:

UDP - glucose + fructose ⇌ sucrose + UDP

Reagents

50 mM MOPS-NaOH (pH-7.5)

2 mM UDGP (Substrate)

5 mM MgCl2

1 mM EDTA

4 mM fructose

30 % KOH

0.14 % (w/v) anthrone reagent in 95 % sulphuric acid (H2SO4)

Procedure

The procedure followed for the assay of sucrose synthase (SS) was similar to that of

sucrose phosphate synthase (SPS), except that 4 mM fructose was used instead of

fructose 6-p and glucose 6p in the reaction mixture.

Calculation

Calculation also mentioned in sucrose phosphate synthase (SPS) protocol.

3.9. Qualitative Analysis of Isozymes through Native PAGE

Native or non-denaturing gel electrophoresis is technique for analysis and

separation of macromolecules such as nucleic acids and proteins in their native state

separation of proteins in the absence of SDS.

53

3.9.1. Sample Extraction

Invertase isozymes (CWIN, VIN and CyIN) were extracted with MOPS-NaOH (pH-7.0).

For this, 10 g of leaf tissues were crushed into liquid nitrogen and homogenized with

buffer and left for 30 min, then centrifuged at 12000 rpm for 30 min. Supernatant

was filtered by syringe filter. To concentrate the sample, centricon device was used.

For this 3 ml supernatant was collected in centricon centrifugal filter device and

centrifuged at 2000 rpm till concentrated and stored -20°C for gel electrophoreses.

3.9.2. Native Polyacrylamide Gel Electrophoresis

3.9.2.1. Prepration of Reagents

a. Acrylamde bisacrylamide (solution A)

Acryle amide 30 g, bisacryle amide 0.8 g, both polymer and cross linkers were

dissolved in 80ml double deionized filrtrate water and the volume was filtred via

whatman filter paper # 01 and stored at 4°C for further use.

b. 1.5 M Tris-Hcl (pH-8.8)( solution B)

36.3 g trizma base was dissolved in 200 ml double deionized water to achive 1.5 M

concerntrantion and pH-8.8 was maintained by gradually incorporated 1 N HCL.

c. 0.5 M Tris-Hcl (pH-6.8)( solution C)

6.05 g trizma base was dissoved in 100 ml double deionized water to get 0.5 M

concerntration and the pH was adjusted upto 6.8 by gradually incorporated in HCl.

The buffer was stored at 4°C.

d. Ammonium Per Sulphate (APS 10 % solution D)

Fresh solution of ammonium per sulphate was prepared. For this, 10 g APS salt was

dissolved in 100 ml double deionized water and placed on ice bath before use.

54

e. N,N,N,N-Tetramethyleethylenediamine (TEMED)

TEMED was stored at 4°C.

f. 0.025 M Tris-glycine Buffer pH 8.4-8.6 (Reservior buffer)

First 14.4 g of glycine was dissolved in 1000 ml double deionized water. After

complete dissolving then 3.025 g of trizma base was added and pH was adjusted at

8.4 and stored at 4°C.

g. Sample Diluting Buffer (SDB):

For this, glycerol (2 ml) was mixed with 0.5 M Tris-HCL ( 2.5 ml) and few crsytals of

bromophenole blue were added. The volume was made upto 10 ml using double

deionized water. Aliquotes (1 ml) were prepared and stored at 4°C.

3.9.2.2 Preprations of Buffers:

a. 0.05 M Sodium Acetate Buffer pH-3.5 and pH-5.0

Solution # A: 0.4 g sodium acetate dissolved in 100 ml double deinized water.

Solution # B: 0.14 ml acetic acid was added in 50 ml double deionized water.

Gradually acid (solution # A) incorporated into base(solution # B) to achieve upto

pH-3.5 and pH-5.0.

b. 0.1 M Potasium Phosphate Buffer pH-7.0

Solution # A: 1.36 g of KH2PO4 dissolved 100 ml double deinized water

Solution # B: 1.74 g of K2HPO4 dissolved 100 ml double deinized water.

Both solutions (A and B) were mixed to get pH-7.0

c. 0.3 %- 2,3,5 Triphenyle Tetrazolium Chloride (TTC) in 4 %- NaOH

Tetrazolium chloride (TTZ) solution was prepared fresh.

For this, 2, 3, 5 triphenyle tetrazolium chliride (TTZ) (0.1 g) was dissolved in 4 %

NaOH.

55

The solution was stored at 4°C.

d. Prepration of Staining Solution

Coomassie Briliant Blue G-250

Coomassie briliant blue G-250 ( 0.05 g ) was mixed with 50 ml 2-propanol and 30 ml

gacail acitic acid. The final volume was made upto 200 ml with double deionized

water. The prepared dye was filtered through whatman filter paper # 01 and stored

at 4°C.

e. Prepration of Destaining Solution

For this, 25 ml ethanol and 75 ml glacail acitic acid were mixed. The final volume was

raised upto 1000 ml with double deionized water. The prepared solution was stored

at 4°C.

3.9.2.3. Preparation of Resolving Gel (pH-8.8)

3.9.2.4. Preparation of Stacking Gel (pH-6.8)

56

3.9.2.5. Preparation of Running Buffer (1X)

Extracted proteins were resolved on NATIVE-PAGE gel (Laemmli, 1970). Spacer

plates (1.0 mm) were used to cast the gel. Resolving (12 %) and stacking (4 %) gels

were prepared by combination of acrylamide and bis-acrylamide solution, resolving

(pH 8.8), stacking buffers (pH 6.8) fresh APS (10 %), TEMED and double distilled

water. For invertase isozymes sample preparation, ultra-filtration or centricon (10

kda) were used. Concentrated protein samples (100 µg to 140 µg) mixed with sample

diluting buffer SDB (20 µl): Sample (80 µl) ratio, placed in ice prior to loading. Sample

(50 µl) was loaded in each well and gel was run at 120 voltage for 2 h. The gel was

incubated with substrate solution at 37°C for overnight with slightly shaking. Next

day the gel was stained by 2,3,5,-tripbenyltetrazolium chloride in NaOH solution.

3.9.3. Invertase Zymography

Principle

Alkaline 2,3,5,-tripbenyltetrazolium chloride (TTC) forms a red insoluble formazan in

reaction with glucose and fructose. For Invertase isozymes gel was stained with

some modification by the method discribed by (Cairns and Ashton, 1991). Samples

were prepared 1:4 (Dye : sample) mixed 4 part of samples with 1 part of craking

solution or (SDB). Sample (60 ul) was loaded in each well. Two gels were run parallel;

one for commassie staining while second for (TTC) satining. The gel was run at 120

voltage for 2 h.

57

At the end of gel electrophoresis, the gel was washed thrice with double distilled

deinized water followed by K-acetate buffer pH-5.0 for vacoular invertase while K-

Phospahte buffer pH-7.0 for alkaline or cytoplasmic invertase at 37°C for 2 h. And

the buffer was replaced by substrate buffer (20 % sucrose) and further incubated at

37°C for overnight with slight shaking. Next day substrate was changed and again

incubated at 37°C for 2 h. Then the gel was washed tree time with buffer. The hexose

released was visualized by soaking the gel in 50 ml of 0.1 % (TTC) in 4 %-NaOH

solution and heated in microvave oven for 30 seconds. Alkaline TTC formed a red

insoluble formazan is reacted with glucose and fructose and the red band was

appeared with light pick background. The second gel was stained with commasie G-

250 solution for overnight shaking. Then the gel was destained for visualizaton of

bands. Both gels were scaned and documented.

3.9.4. SDS-PAGE

Protein qualitative analysis was carried out by Laemmli method (Laemmli, 1970).

Method of gel preparation and buffer composition detailed already given in Native-

PAGE.

All procedures are same as in Native-PAGE but the differences are 10 % SDS (250 µl)

was added in resolving, stacking and sample diluting buffers. Blue protein marker pre-

stained (16 to 270 kDa, Cat No PM-PM-002-500, size 500 µl) was used for each gel

electrophoresis.

3.9.5. Quality Parameters Analysis

3.9.5.1. Pol (%) Estimation

Pol % was estimation the protocol used (Tahir et al., 2014a). For this, 4000 ml water

58

was mixed with 400 g crushed sample then disintegrated for 30 min. Lead (pb)

powder 1-2 g was added in 150 ml of sieved cane extraction and filtered, 20 ml of

filtered sample was measured using polari meter, this procedure was repeated three

times. And pol cane % was calculated by following formula;

𝐏𝐨𝐥 𝐜𝐚𝐧𝐞 (%) =Ext Pol × 0.26 (W + C − (0.0125F × C)

Specific Gravity × C

whereas,

Ext pol=Extraction of Pol

W=water and C=Sugarcane

3.9.5.2. °Brix Estimation

Degree brix (symbol °Bx) is the sugar concentration in water solution. One degree

brix is one gram of sugar in hundred gram of solution and represents the strength of

the solution as percentage by mass.

Brix was measured using brix meter and calculated by this formula:

°𝐁𝐫𝐢𝐱 𝐜𝐚𝐧𝐞 =Ext B × W − (0.25C) − (0.0125C × M)

C × 1 − (0.0125 × ExtB)

whereas,

ExtB=Extraction °Brix

W=Water

C=Cane

M=Moister

3.9.5.3. Moisture Content

For moisture calculation. 50 g of crushed sample was placed in oven at 40-60 min at

150°C then dry weight was measured following formula;

59

Moister Content = Fresh Weight-Dry Weight-Tare weight

3.9.5.4. Fiber Content (%)

Fiber content was calculated using this formula;

Fiber Content (%) = 100-moisture-calculated °brix

3.9.5.5. Recovery Estimation

Sugar recovery was estimated according to Foster (Foster, 1955). For this, 1 kg

sugarcane stems were crushed in crusher machine and weighted with the weighing

balance. Sample was divided into two portions one (400 g) for pol % and other (50

g) for moisture.

Recovery (%) =Pol %-2.5 %

Note: (2.5 % is operational loss in sugar mills during processing which is differ

according to different mills).

3.9.6. Statistical Analysis:

Data was statistically analyzed for analysis of variance (ANOVA), Pearson Correlation

and statistical significance was determined at p < 0.05 level using the LSD test. All

calculation and data analysis were done using the SPSS package program, version

17.

60

SECTION # 4 RESULTS

61

Results


Morphological characterization is very important role in identification of a cultivar

and source of variability. Morphological analysis was carried out on two sugarcane

cultivars S2003-US-633 (high sucrose accumulation) and SPF-238 (low sucrose

accumulation) under different temperature regimes (45±2°C) for 24, 48 and 72 h at

different growth stages.

4.1.1. Shoot Length (cm)

Statistical analysis revealed that there were significant differences (p<0.05) between

cultivars (C), treatments (T) and their interactions (C×T) at formative stage for shoot

length. Under control conditions, maximum shoot length was observed in cultivar

S2003-US-633 (90 cm).While, under heat stress conditions, both cultivars declined in

shoot length but the drastic reduction in shoot length was exhibited in cultivar SPF-

238. Upon recovery, swift improvement was observed in cultivar S2003-US-633 while

slow progress in shoot length was observed in cultivars SPF-238 at vegetative stage

(Table 2). At grand growth stage, only the cultivars were significantly influenced

(p<0.05) while heat stress treatments and interactions were not significantly different

(p>0.05). After 48 and 72 h of heat exposure, both cultivars exhibited similar shoot

lengths. However, highest shoot length was depicted in cultivar S2003-US-633 under

recovery treatments (Table 3). At maturity stage, only significant changes in shoot

length was noted upon heat stress treatments, overall (Table 4). With consistently

decline it is evident from the results that varietal differences were also observed

among cultivars.

62

4.1.2. Root Length (cm)

Root length is an important morphological parameter, result revealed that root

length was statistically significant (p<0.05) affected by heat stress treatments while

no significant differences (p<0.05) were observed for their intersections (C×T) at

formative and grand growth stages. During formative stage, under control conditions,

root length were showed in both cultivars S2003-US-633 (19.7cm) and SFP-238 (18.3

cm) respectively. Moreover, both cultivars manifested the same trend for root length

under heat stress but the cultivars SPF-238 (17) was decreased root length after

exposure of heat stress as compared to S2003-US-633 (18) while recovery treatments

cultivar SPF-238 slowly improvement was observed upon recovery conditions (Table

2). At grand growth stage, when heat stress applied for different episodes root length

was gradually decreased in both varieties, maximum root length declined was

showed in cultivar S2003-US-633. Upon recovery treatments root length was

increased, comparatively, quick improvement was observed in cultivar SPF-238 as

compared to S2003-US-633 (Table 3).At maturity stage, results revealed that S2003-

US-633 (82 cm) and SFP-238 (78 cm) under control conditions. However, root length

was constantly declined under high temperature stress conditions. While

improvement of root length was found under recovery treatments in both cultivars

(Table 4).

4.1.3. Number of Tillers (plant-1)

It is manifest from results that number of tillers per sugarcane plant was not

significantly affected (p>0.05) by heat stress treatments at both formative and

maturity stages. At formative stage means of number of tillers exhibited that under

63

control conditions, maximum number of tillers were found in cultivar S2003-US-633

(5) as compared to cultivar SPF-238 (3). Inconsistent trend was observed for number

of tillers for heat shock and recovery treatments for both varieties but no drastic

reduction in number of tiller was observed (Table 2).At grand growth stage, no

remarkable differences were observed at heat stress and recovery treatments for the

number of tillers per plant (Table 3). Number of tillers were significantly affected by

heat stress for cultivars (C) and their interactions (C×T) but not significantly (p>0.05)

affected for treatments (T) at maturity stage. Although among all stages, highest

number of tillers were evident at maturity stage in both cultivars (Table 4).

4.1.4. Number of Leaf (plant-1)

It is evident that no significant difference (p>0.05) for number of leaf count per plant

at formative and grand growth stages were noted. At formative stage, results

indicated that maximum number of leaves (18) were found in cultivar S2003-US-633

as compared to SPF-238. Upon heat stress conditions, number of leaves were reduced

in both cultivars ranging from 18 to 15 showing reduction from control (Table 2).At

grand growth stage, similar number of leaves were recorded in both cultivars under

control, heat stress and recovery treatments on both cultivars (Table 3).While at

maturity stage, maximum number of leaves were observed under control conditions.

Significant differences (p<0.05) were observed between treatments (T) but not for

their interactions (C×T) and cultivars (C). Although heat stress treatments should

reduce the number of leaves from control in both varieties (Table 4).

64

4.1.5. Leaf Length (cm)

Leaf length was measured to explore the effect of high temperature treatment.

Statistical analysis for treatments, cultivars and their intersections showed non-

significant differences (p>0.05) at vegetative, grand growth and maturity stages.

Under control condition leaf length exhibited in S2003-US6-33 (65 cm) and SPF-238

(63 cm) respectively at formative stage. Under heat stress, no differences were

observed in leaf length while upon recovery conditions in both cultivar SPF-238 (Table

2). At grand growth stage, similar pattern was found in both cultivars (Table 3). At

maturity stage, highest leave length (127 cm) was recorded in cultivar S2003-US-633,

although there is a varietal difference but no notable difference was recorded

between both cultivars at all treatment (Table 4).

4.1.6. Leaf Width (cm)

Leaf width was measured of sugarcane at all growth stages. No significant difference

(p>0.05) was found in the leaf width among treatments (T), cultivars (C) and their

interactions (C×T) at vegetative stages. Under control conditions, the leaf width was

observed in both cultivars S2003-US-633 (1.67 cm) and SFP-238 (1.5 cm).While it was

reduced after 48 and 72 h heat treatments in both cultivars. However, upon recovery

treatments, leaf width was continuously reduced in cultivar SFP-3238 (Table 2). Only

significant differences were observed between cultivars at grand growth stage. Under

control conditions, both cultivars S2003-US-633 (2.67cm) and SFP-238 (2.5 cm) were

exhibited respectively. Under high temperature stress and recovery treatments, the

leaf width did not consistently increased or decreased in both cultivars (Table 3).

While at maturity stage, leaf width was significantly different (p<0.05) between

65

treatments (T) but no difference was found in cultivars and interactions. Leaf width

showing declined after heat stress compared to control but the reduction was

remained consistent from 24 to 72 h (T24 to T72) in cultivar S2003-US-633 (Table 4).

4.1.7. Fresh to Dry Weight Ratio (%)

At vegetative stage analysis of data for fresh to dry weight ratio (%) revealed,

significant differences (p<0.05) between treatments (T) and non-significant

differences (p>0.05) among cultivars (C) and their interactions (C×T). Under control

conditions, fresh to dry weight ratio was observed 27.9 % in cultivar S2003-US-633

and 26.49 % in cultivar SPF-238. During the heat treatments, initially the fresh weight

to dry weight ratio was slightly declined at 24 h (T24) but drastic reduction were

observed at after 72 h (T72) in both cultivars. Upon recovery, rapid improvement was

observed in cultivar SPF-238 (Table 2). While at grand growth stage, there were

significant differences for treatments and their interactions except cultivars (C). At

control conditions fresh to dry weight ratio was 43 % in cultivar SPF-238. But under

heat shock conditions, fresh to dry weight ratio was consistently reduced in cultivar

SPF-238.However, after 72h of recovery conditions, similar results were found in

both cultivars (Table 3). At maturity, there were significantly affected (p<0.05) for

cultivars and treatments but non-significant (p>0.05) for their interactions (C×T).

Fresh to dry weight ratio were found in both cultivars, S2003-US-633 (44 %) and SPF-

238 (40 %) at control conditions. Fresh to dry weight ratio of sugarcane significantly

reduced due to heat shock in both cultivars. Initially both cultivars slowly declined

fresh to dry weight ratio but at T72 h drastic reduction was observed in both cultivars.

Upon recovery conditions, similar pattern of improvement was exhibited in both

66

cultivars. Overall, cultivar S2003-US-633 had better performance under high

temperature and earlier recovery than other cultivar.

4.1.8. Stem Diameter (cm)

At grand growth stage, significant differences (p<0.05) were evident for stem

diameter among cultivars, treatments but not for their interactions (cultivars ×

treatments). Under control conditions, maximum stem diameter was observed in

cultivar S2003-US-633 (2.2 cm) and SPF-238 (1.8 cm) respectively. Under heat stress

treatments, minimum stem diameter was showed in both cultivars. However, upon

recovery treatments, stem diameter was found in both cultivars S2003-US-633 (1.9

cm) and SPF-238 (1.7 cm) respectively (Table 3). While at maturity phase, there were

non-significant (p>0.05) for all parameters. Stem diameter were increased as

compared previous stages in both varieties both varieties S2003-US-633 and SPF-238.

Although, there varietal differences but maximum stem diameter was found in

cultivar S2003-US-633 (Table 4). This result indicated that cultivar S2003-US-633

showed higher growth rate and better performance at various environmental

conditions than cultivar SPF-238.

4.1.9. Number of Nodes (plant-1)

There was no significant (p>0.05) difference between cultivars (C) and treatments

(C×T) at grand growth and maturity stages. At grand growth stage, number of nodes

per plant was noted in cultivars S2003-US-633 and SPF-238 in all treatments. Same

number of nodes of sugarcane were found in both cultivars S2003-US-633 (23) and

SPF-238 (23) under control conditions. No changes were found of number of nodes

under stress and recovery conditions (Table 3). The above mentioned results were

67

also noticed at maturity stage (Table 4). Although there were no differences (p>0.05)

between cultivars, treatments and their interactions at maturity stage but the

number of nodes were increased as compared to grand growth stage.

4.2.0. Number of Internodes (plant-1)

At grand growth, number of internodes per plant was also counted after each

treatment. Data revealed that there was no significant difference (p>0.05) among

cultivars, treatments and their interactions (C×T). At control conditions same number

of internodes (22, 22) were observed in both cultivars. Upon the heat stress

treatments, number of internodes reduced at T48 h after heat exposure in both

cultivars. However, upon recovery conditions, no changes occurred in number of

internodes in both cultivars (Table 6). Again no difference was exhibited at maturity

stage for all parameters (Table 7). Similar trend of number of internodes was shown

in both cultivars at all treatments, control and recovery conditions. At both stages,

the study revealed that no remarkable change was observed upon heat stress and

recovery treatments in both cultivars.

4.2.1. Internode Distance (plant-1)

Internode distance is very important morphological parameters because sucrose

content depends on internode distance. Internode distance is directly proportional

to sucrose content in sugarcane stalk. Analysis of data for internode distance per

plant revealed non-significant differences (p>0.05) between treatments (T), cultivars

(C) and their interactions (C×T) at grand growth stage. In control conditions,

internode distance was recorded in both cultivars S2003-US-633 (8.3 cm) and SPF-

238 (8 cm) respectively. Upon heat shock and recovery treatments internode distance

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were at T48, R24 and R72 in cultivar S2003-US-633 (7.7 cm) while equal internode

distance (7.7 cm) were observed at R48 in recovery conditions (Table 6).

At maturity stage, no statistically significant affect was observed by heat shock

treatment for internode distance. Mostly the highest internode distance (9 cm) was

recorded in cultivar S2003-US-633 as against SPF-238 (Table 7). However, the length

of internode were slightly increased in cultivar S2003-US-633 at grand growth stage

as compared to maturity stage. This results indicated that higher length of internodes

may increase sucrose content in sugarcane stalk.

69

Table 5: Morphological parameters of both cultivars S2003-US-633 and SPF-238 under control at (30±2°C), heat shock (45±2°C) and recovery (30±2°C)

for 24, 48 and 48 h at vegetative stage. Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at p level p<0.05.

Vegetative Stage

Parameters Varieties

Mean ± SEM P Value

Control Heat shock Recovery Cultivar Treatment Interaction

C T24 T48 T72 R24 R48 R72 C T C×T

Shoot length

(cm)

S2003-US-633 90.00±0.57 89.66±0.33 89.33±0.88 88.33±0.88 88.00±0.66 87.66±0.33 88.66±0.33 * * *

SPF-238 88.00±0.88 87.00±0.57 84.00±0.57 84.00±0.57 84.00±0.57 84.33±0.33 84.33±0.66

Root length

(cm)

S2003-US-633 19.0±0.30 19.3±0.30 19.3±0.70 18.3±0.90 18.7±0.30 19.0±0.06 19.3±0.30 * * ns

SPF-238 18.3±0.30 17.3±0.30 17.0±0.00 16.0±0.00 16.3±0.30 16.3±0.30 17.7±0.30

Number of

tiller

S2003-US-633 5.00±0.58 2.67±0.67 3.67±0.67 4.00±0.58 3.67±0.67 5.00±0.58 4.00±0.58 ns ns ns

SPF-238 3.67±0.67 4.00±0.58 4.33±0.88 6.00±0.58 4.00±0.58 5.00±0.58 4.33±0.88

Number of

leaf

S2003-US-633 18.00±0.58 18.00±0.58 17.00±0.88 16.00±0.33 16.00±0.58 17.00±0.33 18.00±0.33 ns ns ns

SPF-238 17.00±0.58 16.00±0.33 16.00±0.33 15.00±0.33 15.00±0.33 16.00±0.33 16.00±0.58

Leaf length

(cm)

S2003-US-633 65.00±1.0 66.00±0.6 66.00±1.5 65.00±2.61 64.00±1.20 64.00±0.00 63.00±1.3 ns ns ns

SPF-238 63.00±0.6 63.00±1.8 63.00±1.5 63.00±2.6 63.00±2.4 65.00±0.6 64.00±2.00

Leaf width

(cm)

S2003-US-633 1.67±0.29 1.83±0.29 1.17±0.17 1.50±0.29 1.50±0.29 1.83±0.33 1.67±0.33 ns ns ns

SPF-238 1.50±0.33 1.50±0.17 1.67±0.17 1.50±0.29 1.50±0.29 1.33±0.17 1.33±0.33

Fresh to dry

wt ratio (%)

S2003-US-633 27.94±0.32 27.30±0.02 26.01±0.72 24.89±0.45 25.27±0.22 25.32±0.71 25.79±0.48 ns * ns

SPF-238 26.49±0.59 25.12±1.11 25.02±1.35 24.79±0.33 25.45±0.13 25.97±0.60 26.03±0.43

Significant difference p<0.05 (*) and non-significant differences p<0.05 (ns)

70

Table 6: Morphological parameters of both cultivars S2003-US-633 and SPF-238 under control at (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at grand growth stage. Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at p level p<0.05.

Significant difference p<0.05 (*) and non-significant differences p<0.05 (ns)

Grand Growth Stage


Mean ± SEM P Value


C T24 T48 T72 R24 R48 R72 C T C×T

Shoot length (cm)

S2003-US-633 167.3±0.33 166.7±0.30 167.3±0.47 167.3±0.33 166.3±0.33 166.3±0.33 166.6±0.88 * ns ns

SPF-238 165.6±0.51 166.3±1.06 165.3±0.33 165.3±0.33 165.6±1.35 166.1±0.88 164.5±0.88

Root length (cm)

S2003-US-633 41.0±0.58 36.5±1.80 35.67±1.77 34.33±0.33 35.0±0.58 33.67±0.88 31.33±0.67 * * ns

SPF-238 38.18±1.09 38.0±0.58 36.33±0.33 36.00±0.58 36.67±0.88 37.0±1.00 37.33±0.33

Number of tiller

S2003-US-633 5.0±0.58 5.0±1.00 5.0±0.58 4.0±0.58 5.0±0.00 5.0±1.00 5.0±1.16 ns ns ns

SPF-238 4.0±1.00 4.0±0.58 5.0±0.58 5.0±0.58 5.0±0.58 6.0±0.58 5.0±1.16

Number of leaf S2003-US-633 24.0±0.58 24.0±0.58 25.0±0.67 25.0±0.00 24.0±0.33 26.0±0.33 24.0±0.58

ns ns ns SPF-238 24.0±1.16 25.0±0.00 25.0±0.67 26.0±1.00 26.0±0.33 26.0±0.67 25.0±1.33

Leaf length (cm)

S2003-US-633 125±0.29 125±0.88 125±0.88 124±0.99 124±0.80 125±0.99 126±1.31 ns ns ns

SPF-238 125±0.59 124±0.59 124±0.40 123±0.68 126±0.00 125±0.00 125±0.33

Leaf width (cm)

S2003-US-633 2.67±0.17 2.63±0.13 2.50±0.29 2.70±0.47 2.57±0.07 2.47±0.03 2.80±0.06 * ns ns

SPF-238 2.50±0.29 2.37±0.37 2.27±0.15 2.50±0.06 2.13±0.13 2.33±0.09 2.44±0.06

Fresh to dry wt ratio (%)

S2003-US-633 39.47±0.48 38.0±0.54 37.6±1.19 38.8±0.75 39.8±0.85 41.8±2.21 34.2±0.94 ns * *

SPF-238 43.0±1.44 42.3±1.13 39.6±0.81 38.9±0.69 30.6±2.55 34.0±1.57 34.2±3.77

Stem diameter (cm)

S2003-US-633 2.23±0.10 1.77±0.12 1.67±0.17 1.57±0.03 1.70±0.06 1.67±0.09 1.90±0.06 * * ns

SPF-238 1.83±0.09 1.60±0.06 1.53±0.03 1.53±0.03 1.60±0.06 1.60±0.15 1.70±0.06

Number of nodes

S2003-US-633 23.0±0.00 22.7±0.33 22.7±0.33 23.0±0.00 22.7±0.33 22.7±0.33 22.7±0.33 ns ns ns

SPF-238 23.0±0.00 23.0±0.00 22.7±0.33 22.7±0.33 23.0±0.00 23.0±0.00 23.0±0.00

Number of internodes

S2003-US-633 22.0±0.00 21.7±0.33 21.7±0.33 22.0±0.00 21.7±0.33 21.7±0.33 21.7±0.33 ns ns ns

SPF-238 22.0±0.00 22.0±0.00 21.7±0.33 21.7±0.33 22.0±0.00 22.0±0.00 22.0±0.00

Internodes distance (cm)

S2003-US-633 8.33±0.33 8.00±0.00 7.70±0.33 8.00±0.00 7.70±0.33 8.3±0.33 7.70±0.33 ns ns ns

SPF-238 8.00±0.00 8.33±0.33 8.33±0.33 8.33±0.33 8.00±0.00 7.7±0.33 8.33±0.33

71

Table 7: Morphological parameters of both cultivars S2003-US-633 and SPF-238 under control at (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at maturity stage. Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at p level p<0.05.

Significant difference p<0.05 (*) and non-significant differences p<0.05

Maturity Stage


Mean ± SEM P Value


C T24 T48 T72 R24 R48 R72 C T C×T

Shoot length (cm)

S2003-US-633 317.3±1.33 316.0±0.58 315.6±0.88 314±1.58 314±0.33 315.0±0.58 315.33±0.88 ns * ns

SPF-238 316.33±0.33 315.33±0.33 314.33±0.33 313.6±0.88 314.6±0.33 315.3±0.33 315.67±0.67

Root length (cm)

S2003-US-633 82.00±1.70 81.00±0.60 78.00±0.90 76.00±0.90 77.00±0.90 78.00±0.90 79.00±1.50 * * *

SPF-238 78.00±0.30 77.00±0.90 77.00±0.60 76.00±0.30 76.00±.0.90 77.00±0.70 77.00±0.30

Number of tiller

S2003-US-633 6.67±0.33 6.33±0.33 6.33±0.17 6.17±0.17 6.00±0.00 6.23±0.23 6.17±0.17 * ns *

SPF-238 6.33±0.33 6.00±0.00 5.83±0.17 5.67±0.33 5.83±0.17 5.67±0.17 5.87±0.13

Number of leaf

S2003-US-633 30.00±0.58 27.67±0.33 27.00±0.58 27.33±0.33 27.33±0.33 27.67±0.33 28.67±0.33 ns * ns

SPF-238 29.33±0.33 28.00±0.58 27.67±0.33 27.33±0.33 27.67±0.33 26.33±0.33 27.33±0.33

Leaf length (cm)

S2003-US-633 127.7±1.20 127.3±2.91 127.3±2.85 126.3±0.33 127.7±1.20 127.7±1.86 127.3±0.67 ns ns ns

SPF-238 123.7±0.88 127.7±1.20 127.7±0.88 127.7±1.67 126.0±0.58 126.3±2.41 126.3±0.88

Leaf width (cm)

S2003-US-633 2.93±0.07 2.77±0.03 2.70±0.06 2.70±0.00 2.57±0.03 2.63±0.09 2.77±0.03 ns * ns

SPF-238 2.70±0.06 2.80±0.09 2.70±0.10 2.70±0.10 2.73±0.09 2.57±0.07 2.83±0.07

Fresh to dry wt

ratio (%)

S2003-US-633 44.0± 1.29 40.99±0.37 39.29±1.73 37.91±0.08 38.16±0.35 38.73±0.50 40.29±0.55 * * ns

SPF-238 40.47±0.33 39.84±0.57 38.57±0.49 37.06±0.42 37.57±0.39 37.87±2.22 38.25±0.21

Stem diameter

S2003-US-633 2.50±0.06 2.53±0.03 2.47±0.03 2.33±0.18 2.43±0.07 2.57±0.03 2.53±0.03 ns ns ns

SPF-238 2.33±0.17 2.30±0.15 2.33±0.17 2.17±0.17 2.23±0.15 2.33±0.17 2.37±0.19

Number of nodes

S2003-US-633 26.00±0.00 26.00±0.58 25.00±0.33 26.00±0.58 25.26±0.00 26.00±0.33 26.00±0.58 ns ns ns

SPF-238 25.00±0.33 26.00±0.33 25.00±0.33 26.00±0.33 26.00±0.33 25.00±0.00 26.00±0.00

Number of internodes

S2003-US-633 25.00±0.00 25.00±0.58 24.00±0.33 25.00±0.58 24.26±0.00 25.00±0.33 25.00±0.58 ns ns ns

SPF-238 24.00±0.33 25.00±0.33 24.00±0.33 25.00±0.33 25.00±0.33 24.00±0.00 25.00±0.00

Internodes distance

(cm)

S2003-US-633 9.00±0.00 9.00±0.00 9.00±0.00 8.83±0.17 8.33±0.33 8.67±0.33 9.00±0.00 ns ns ns

SPF-238 8.33±0.58 8.67±0.33 8.33±0.33 8.77±0.33 8.33±0.33 8.83±0.20 9.00±0.00

72

4.3. Stress Damage Indicators Quantification

Stress damage indicators such as malondialdehyde, hydrogen per oxide and relative

membrane permeability and proline were quantified as following.

4.3.1. Malondialdehyde (MDA)

MDA is the indicator of lipid peroxidation, it was quantified in sugarcane plant and

is expressed in terms of nano-mole per ml. It is a vital resistant physiological index

of plant in any environmental stress conditions. As shown in the below (Figure 6).

MDA content was statistically different (p<0.05) for treatments (T) and cultivars (C)

and their interactions (C×T) at all stages. Under control conditions, the amount of

MDA content was observed for cultivar, SPF-238 (12.74 n mole ml-1) and S2003-US-

633 (11.78 n mole ml-1) at vegetative stage. Exposure of heat stress for 72 h trigger

the highest MDA content in cultivar SPF-238 (42.4 n mole ml-1) as compared to

S2003-US-633 (38.36 n mole ml-1). While under recovery treatments, both cultivars

depicted similar pattern of MDA accumulation. However, at grand growth stage,

maximum MDA content was also exhibited under heat stress but upon recovery

conditions both cultivars slowly declined the MDA content. At maturity stage, the

amount of MDA content was significantly lower than the MDA quantifies under

control, heat stress and recovery conditions at grand growth stage. However, under

control conditions, cultivar S2003-US-633 indicated lowest MDA accumulation than

SPF-238 at maturity stage. Heat stressed plants slightly increased MDA content over

no heat stressed plants. But between cultivar S2003-US-633 was observed quick and

better recovery of MDA than cultivar SPF-238.

73

Fig 6: MDA quantified of sugarcane cultivars S2003-US-633 and SPF-238 under control

(30±2oC), heat shock (45±2oC) and recovery (30±2oC) for 24, 48 and 72 h at vegetative,

grand growth and maturity stages.

74

4.3.2. Proline Estimation

Higher accumulation of osmolyte (proline) is an important physiological response

in sugarcane plant exposed to different environmental stresses (biotic and abiotic).

Free proline was quantified in terms of µmole gm-1 FW. The amount of proline was

significantly different (p<0.05) between cultivars (C) and treatments at all stages

except their interactions (C×T) at grand growth stage. While proline content was not

significantly different (p>0.05) for their interactions (C×T) at vegetative and maturity

stages. Under control conditions, both cultivars did not show any differences for the

accumulation of free proline at vegetative stage (Fig 7). Under heat shock conditions,

the amount of free proline was significantly higher than control and recovery

conditions in both cultivars. However, during recovery conditions, proline content

was slightly declined in both cultivars S2003-US-633 and SPF-238 but SPF-238

showed a drastic reduction than S2003-US-633. While at grand growth stage,

significant differences for the free proline content among all factors. Under heat

shock conditions, both cultivars indicated an enhancement in the free proline

contents however proline accumulation was noticeably greater in cultivar S2003-US-

633 than SPF-238. Upon recovery, proline content dropped sharply in both cultivars.

At maturity stage, proline content was accumulated in cultivar S2003-US-633 (61.4

µM g-1 FW) and SPF-238 (58.3 µM g-1 FW). After exposure to heat stress for 24 h

(T24) the amount of proline content slightly increased and after 48 h (T48) much

more increased. While, after 72 h (T72) it increased many folds in both cultivars

S2003-US-633 (190 µM g-1 FW and SPF-238 (173.8 µM g-1 FW) respectively.

75

Fig 7: Free proline estimated of sugarcane cultivars S2003-US-633 and SPF-238 under

control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at


76

4.3.3. Hydrogen peroxide

Hydrogen peroxide is produced in plant cell and it plays vital role as signaling

molecules in different physiological processes. In plant cell hydrogen peroxide

content increases under biotic and abiotic stresses. Hydrogen peroxide was

estimated in terms of µmole g-1 FW. The amount of hydrogen peroxide exhibited

significant differences (p<0.05) between treatments (T) and cultivars (C). While non-

significant differences (p>0.05) were observed between their interactions (C×T) at

all stages. At formative stage, the amount of hydrogen peroxide was (64.39 µM g-1

FW) S2003-US-633 and (92.42 µM g-1 FW) SPF-238 under control conditions. Under

heat stress treatments, its content gradually increased with the increasing

temperature. Maximum hydrogen peroxide content was noted in SPF-238 (208.2 µM

g-1 FW) than S2003-US-633 (187.8 µM g-1 FW). Under recovery conditions, rapid

improvement was depicted in cultivar S2003-US-633. At grand growth phase, the

amount of hydrogen peroxide exhibited in S2003-US-633 (66 µM g-1 FW) and SPF-

238 (86 µM g-1 FW) respectively. But after 72 h (T72) exposure to heat stress, the

content of hydrogen peroxide was increased in both cultivars S2003-US-633 (154.9

µM g-1 FW) and cultivar SPF-238 (173.35 µM g-1 FW), respectively. However, upon

recovery conditions, lower accumulation of hydrogen peroxide was depicted in both

cultivars. Same results were also found at maturity stage in both cultivars. Figure 8

indicates cultivar S2003-US-633 showing tolerance under heat stress conditions.

77

Fig 8: H2O2 estimated of sugarcane cultivars S2003-US-633 and SPF-238 under control

(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at vegetative,

grand growth and maturity stages.

78

4.3.4. Relative Membrane Permeability (RMP)

Membrane thermo stability is a significant stress damage indicator in various

physiological processes. It is quantified by measuring electrical conductivity (EC) of

sugarcane plant. It is evident from the results EC was significantly different (p<0.05)

between treatments, cultivars and their interactions but non-significant difference

(p>0.05) was observed among their interactions (C×T) at vegetative and grand

growth stages. At formative stage, electrolyte leakage (EC) was exhibited in S2003-

US-633 (14.7 %) and SPF-238 (16.6 %) respectively. After 24 h heat stress treatment

(T24) initially maximum EC content was observed in both cultivars S2003-US-633 (20

%) and SPF-238 (27.7 %) and at 72 h (T72), EC content was continuously increased in

both cultivars S2003-US-633 (24.3 %) and SPF-238 (28.71 %) respectively. Recovery

treatments for 72 h (R72) showed maximum EC in cultivar S2003-US-633 (19.2 %)

while minimum RMP manifested by SPF-238 (18.5 %). At grand growth stage, data

stated that EC was observed in both cultivars S2003-US-633 (15.24 %) and SPF-238

(18.71 %) of untreated plants. But after heat treatments EC was increased in both

cultivars with increasing heat for different episodes (T24, T48 and T72). Upon

recovery treatments, EC was declined in both cultivars with same pattern (Fig 9). At

maturity stage, data revealed that significant difference between cultivars and

treatments except their interactions (C×T). Minimum RMP was observed in

untreated plants in both cultivars. Under heat stress conditions, EC increased

gradually after 72 h heat treatments in both cultivars. Upon recovery both cultivars

recovered EC content after 72 h (R72). However, minimum EC was displayed in

cultivar S2003-US-633 that shows tolerance under heat stress conditions.

79

Fig 9: Electrolytes lekeage quantified of sugarcane cultivars S2003-US-633 and SPF-238

under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at


80


The biochemical analysis of two sugarcane cultivars S2003-US-633 (high sucrose

accumulation) and SPF-238 (low sucrose accumulation) under different temperature

regimes (45±2°C) for 24, 48 and 72 h at vegetative, grand growth and maturity stages.


Total sugar was quantified in terms of (µg ml-1). Results revealed that total sugar

showed statistically significant differences (p<0.05) between cultivars (C) and

treatments (T) at all stages. While non-significant differences (p>0.05) were

exhibited between their interactions (C×T) at both vegetative and maturity stages

but only significant differences (p<0.05) were found between their interactions (C×T)

at grand growth stage. Under control conditions, maximum total sugar content was

exhibited in cultivar S2003-US-633 (712.6) as compared to cultivar SPF-238 (573).

Upon the application of thermal stress, total sugar content gradually decreased at

24, 48 h (T24-T48) and reached maximum reduction at 72 h (T72) in both cultivars

S2003-US-633 (263.15) and SPF-238 (228). However, upon recovery treatments after

24, 48 and 72 h (R24, R48 and R72) both cultivars recovered sugar loss with same

pattern at vegetative stage. At grand growth stage, maximum total sugar content

was showed in S2003-US-633 (1641). Incontrast minimum sugar content was found

SPF-238 (1581.1) under control conditions. But during exposure to heat stress both

cultivars showed gradual declined from 24 to 72 h (T24, T48 and T72) in cultivar

S2003-US-633 (1623.9), (1367.37) and (0.1213.67) while in cultivar SPF-238

(1581.19), (1402.2) and (1241.37) respectively. However, during recovery

treatments, total sugar content progressively increased from 24 to 72 h (R24, R48

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and R72) in cultivar S2003-US-633 (1230.76), (1316.23) and (1461.53) and SPF-238

(1213.675), (1307.69) and (1410.25) against heat stress conditions. At maturity

stage, the amount of total sugar was found in cultivar S2003-US-633 (10818.3) and

SPF-238 (8573.31) under control conditions. But under high temperature stress

conditions, total sugar content was declined in cultivar S2003-US-633 at 24

(9471.84), at 48 (9044.49) and at 72 h (8663.16) and SPF-238 at 24 (8573.31), at 48

(7020.93) and at 72 h (7027.62) respectively. However, during recovery treatments

from (24, 48 and 72 h) regained total sugar loss in cultivar S2003-US-633 at 24

(9020.38) at 48 (9298.78) and at 72 h (9714.22) and SPF-238 at 24 (7392.92), at 48

(7131.78) and at 72 h (7388.57) respectively. Although, there is varietal difference,

but it was revealed that the amount of total sugar gradually increased from

vegetative stage to grand growth stage and maturity stage (Fig 10).

82

Fig 10: Total sugar estimated of sugarcane cultivars S2003-US-633 and SPF-238 under

control (30±2°C),heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at


83


Reducing sugar of both varieties during this study estimated in terms of (mg ml-1) at

all three growth stages. Data revealed significant difference between cultivars and

treatments except their interactions at vegetative and grand growth stages. Under

control conditions, reducing sugar content was observed in S2003-US-633 (0.25) and

in SPF-238 (0.19) respectively (Fig 11). Under high temperature stress treatments

the amount of reducing sugar was significantly reduced in cultivar S2003-US-633 at

24, 48 and 72 h (0.15), (0.13) and (0.12) while SPF-238 (0.15), (0.12) and (0.11)

respectively. During recovery conditions, both varieties showed positive adaptation

with regaining of reducing sugar loss. In addition, the content of reducing sugar was

recovered in cultivar S2003-US-633 after 24, 48 and 72 h (0.19), (0.20) and (0.21) and

cultivar SPF-238 (0.18 ), (0.182) and (0.187) at vegetative stage respectively. At

grand growth stage, under control conditions, the amount of reducing sugars were

in both cultivars S2003-US-633 (0.52) and SPF-238 (0.61) but significant variation was

noticed depending upon the duration of high temperature and recovery treatment.

Reducing sugars were declined in SPF-238 after 24, 48 and 72 h (0.58), (0.58) and

(0.44) and S2003-US-633 (0.48), (0.38) and (0.31) during stress treatments. The

amount of reducing sugar was maximum in SPF-238 as compared to S2003-US-633.

However, both cultivars improved sugar loss in recovery treatment with same

pattern. At maturity stage, only significant differences (p<0.05) were observed

between cultivars (C) but non-significant (p>0.05) differences were found among

their interactions (C×T). Plant under control conditions, reducing sugars content

found in S2003-US-633 (0.23) and SPF-238 (0.35) but under thermal stress

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conditions, the amount of sugar slightly declined in both cultivars while

improvements were exhibited during recovery treatments. These responses were

noticed in both varieties. Collectively, heat stress caused reduction in sugar profile.

The amount of reducing sugar was maximum in SPF-238 as compared to S2003-US-

633 in all three stages but highest level of reducing sugar was noticed at grand

growth stage than the other two stages. In addition, the amount of reducing sugar

was declined in formative stage and maturity stage but increased in grand growth

stage.

85

Fig 11: Reducing estimated sugar of sugarcane cultivars S2003-US-633 and SPF-238 under

control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at


86

4.4.3. Non-reducing Sugar Estimation

Non-reducing sugar also quantified in terms of (mg ml-1). Statistical analysis results

were found same as total sugar at all growth stages.

At formative stage, the amount of non-reducing sugar was observed in S2003-US-

633 (0.45) while in cultivar SPF-238 (0.37) under control condition. While after 72 h

of heat exposure the amount of non-reducing sugar was declined in cultivar S2003-

US-633 (0.14) and in SPF-238 (0.11). However, after72 h recovery treatments both

varieties showed same pattern of improvement in both cultivars S2003-US-633

(0.39) and SPF-238 (0.39) respectively. At grand growth stage, reduction of non-

reducing sugars content was noticed from 24, 48 and 72 h after heat shock

treatments as compared to under control condition S2003-US-633 (0.30) and SPF-

238 (0.14) and similarly recovered sugar losses in both varieties. At maturity stage,

under control conditions, SPF-238 had less amount of non-reducing sugars (8.23)

than S2003-US-633 had (10.59 ) while during different episode of heat stress at 24,

48 and 72 h (9.24), (8.8 ) and (8.44) in S2003-US-633 but in SPF-238 (7.34), (6.6) and

(6.71) showed substantial reduction respectively. Cultivar S2003-US-633 got better

recovery than SPF-238. Despite of different cultivars, maximum amount of non-

reducing sugar was revealed at vegetative stage and maturity stage while minimum

contents of non-reducing sugars were found in grand growth stage in both cultivars.

(Fig 12).

87

Fig 12: Non reducing sugar estimated of sugarcane cultivars S2003-US-633 and SPF-238



88

4.4.4. Total Soluble Protein Analysis

Total soluble protein (TSP) was quantified in terms of (mg ml-1) in both cultivars.

Significant differences (p<0.05) were observed among all cultivars (C), treatments

(T) and their interactions (C×T) at all stages. At formative stage, TSP content was

noted in cultivar S2003-US-633 (1.187) and cultivar SPF-238 (1) under control

conditions. While TSP concentration declined under heat stress conditions in both

cultivars. Comparatively, minimum accumulation of TSP content was exhibited in

SPF-238. After exposure of heat shock treatments at different time intervals (24, 48

and 72 h), TSP content showed in cultivar S2003-US-633 (1.11), (0.938) and (0.819)

while in SPF-238 (0.879), (0.652) and (0.634) respectively. Upon recovery

treatments, the amount of protein in cultivar SPF-238 minimum recovered than

cultivar S2003-US 633. At grand growth stage, under normal conditions, protein

concentration was exhibited in both cultivars, S2003-US-633 (1.8) and cultivar, SPF-

238 (1.7) respectively. But during the heat shock treatments both cultivar was

exhibited less amount of TSP. In addition under stress conditions, the amount of

protein observed in S2003-US-633 (1.6) (1.5) and (1.4) and in SPF-238 (1.5), (1.4) and

(1.2) respectively. However, after 24 and 48 h of recovery treatments both cultivars

had same amount of protein contents, except 72 h (1.6) and (1.5). At maturity stage,

cultivar S2003-US-633 (3) while other variety, SPF-238 (2.8) were noted under

control conditions. However, similar results were exhibited under high temperature

stress conditions as at both vegetative and grand growth stages. Comparatively,

cultivar S2003-US-633 showed best resistance in heat stress conditions as well as

swift recovery in recovery treatments than SPF-238 (Fig 13).

89

Fig 13: Total soluble protein estimated of sugarcane cultivars S2003-US-633 and SPF-238



90

4.5. Sugar Metabolizing Enzymes

Sugar metabolizing enzymes such as sucrose synthase (SS), sucrose phosphate

synthase (SPS) and invertase isozymes such as cytoplasmic invertase (neutral), cell

wall invertase (acidic) and vacuolar invertase (acidic) of two sugarcane cultivars

S2003-US-633 and SPF-238 were quantified at vegetative, grand growth and

maturity stages. Characterization of enzymes activities were carried out through

quantitative and qualitative analysis as follows:

4.5.1. Quantitative Analysis

4.5.1.1. Sucrose Synthase (SS)

Sucrose synthase (SS) activities exhibited significant differences (p<0.05) among

cultivars (C), treatments (T) and their interactions (C x T) at maturity stage. However,

non-significant differences (p>0.05) variation were observed for cultivars (C) and

their interactions (C x T) at vegetative and grand growth stage. The exposure of heat

stress declined the enzymes activities in both varieties as compared to the control

(Fig 14). Drastic reduction in SS activity was noted after 72 h of heat stress in S2003-

US-633 (92.14 U ml-1min-1) and SPF-238 (69.68 U ml-1min-1) while increased in

recovery treatments after (139 and 142 U ml-1min-1) respectively. While, at grand

growth stage, sucrose synthase (SS) activity exhibited significant differences (p>0.05)

only for treatments (T). The activity of sucrose synthase was found in both cultivars

S2003-US-633 (386.5 U ml-1min-1) and SPF-238 (388.3 U ml-1min-1) at normal growth

condition. But after heat shock treatments for 24, 48 and 72 h the SS activities were

observed in S2003-US-633 (382.251 and 259.2 U ml-1min-1) and SPF-238 (382.8,

91

254.7 and 245.4 U ml-1min-1) respectively. Furthermore, cultivar S2003-US-633 was

found to quickly recover the activity of SS after heat shock. At maturity stage, it is

evident from the results that SS activity was sequentially decreased as episodes of

heat stress gradually increased, maximum reduction (189.4 U ml-1min-1) was

observed at heat shock treatment (T72) in SPF-238. While both cultivars recovered

the maximum SS activity after 72 h of recovery treatment (R72).

92

Fig 14: Specific activity quantified of sucrose synthase (SS) of sugarcane cultivars S2003-

US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C)

for 24, 48 and 72 h at vegetative, grand growth and maturity stages.

93

4.5.1.2. Sucrose Phosphate Synthase (SPS)

For sucrose phosphate synthase (SPS) activity, significant differences (p<0.05) were

found for cultivars (C) and treatments (T) while their interactions (C x T) at all growth

stages but non-significant (p>0.05) difference between cultivars at grand growth

stage. At vegetative stage, heat shock treatments for 24 and 48 h slightly decreased

the SPS activity in both cultivars but depicted maximum declined in activity at T72 in

S2003-US-633 (2257.95 U ml-1min-1). While for recovery treatment after 72 h both

varieties recorded the enzymes activity. At grand growth stage, SPS activity exhibited

the same pattern in heat shock and recovery conditions as at vegetative stage. At

maturity stage, SPS activity under control condition was slightly high in S2003-US-

633 than SPF-238 (Fig 15). However, under heat stress conditions (T24, T48 and T72)

S2003-US-633 exhibited reduction in this attribute but rapid recovery from heat

stress was observed through improved SPS activity as compared to heat shock

conditions. Comparatively among different growth phases, maximum SPS activity

was exhibited at maturity stage as compared with previous two stages (vegetative

and grand growth) in both cultivars but S2003-US-633 had better performance under

heat stress conditions. This results indicated that cultivar S2003-US-633 has better

sink strength to accumulate sucrose in stem.

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Fig 15: Specific activity quantified of sucrose phosphate synthase (SPS) of sugarcane

cultivars S2003-US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and

recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand growth and maturity stages.

95


In plant different invertase isoform are presented at different cellular localization

which are subcategorized based on pH which play various different important roles.

These invertase isozymes are cell wall invertase (acidic), vacuolar invertase (acidic)

and cytoplasmic invertase (neutral). Data suggest significant difference (p<0.05) for

temperature treatments, cultivars and their interactions (C x T) both vegetative and

grand growth phase while non-significant differences (p>0.05) were observed

among all treatments at maturity stage. At vegetative stage, the exposure of heat

stress at 24 and 48 h slightly declined the enzymes activities but after 72 h drastic

reduction was noted in both varieties as compared to the control. Under recovery

treatments both cultivars depicted varietal differences for cytoplasmic invertase

activity. As SPF-238 showed fast recovery of enzyme activity S2003-US-633. At grand

growth stage, maximum enzyme activity was noted in SPF-238 than S2003-US-633

under normal growth conditions. Although SPF-238 showed maximum activity but

heat shock exposure drastically affected the enzyme activity in both cultivars.

However, in maturity stage, there were no significant differences (p>0.05) for

cultivars (C), treatments (T) and their interactions (C x T). At maturity stage,

furthermore, maximum cytoplasmic invertase specific activity was noted in cultivar

SPF-238 than S2003-US-633 at all growth stages (Fig 16).

96

Fig 16: Specific activity quantified of cytoplasmic invertase (CyIN) of sugarcane cultivars

S2003-US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery

(30±2°C) for 24, 48 and 72 h at vegetative, grand growth and maturity stages.

97


For cell wall invertase activity, significant differences (p<0.05) were found for

treatments (T) while cultivar and their interaction (C x T) were non-significant (p>

0.05) at formative and grand growth stages. However, at maturity stage significant

differences (p<0.05) were noted for cultivar and their interaction (C x T). At

vegetative stage, with increasing episodes of heat shock for 24, 48 and 72 h

decreased activity of cell wall invertase activity was observed. After 72 h of heat

stress, cell wall invertase activity declined drastically in both cultivars S2003-US-633

(0.233 U ml-1 min-1) and SPF-238 (0.211 U ml-1 min-1). Moreover, quick recovery was

observed only in SPF-238 at 72 h (0.30 U ml-1 min-1) as compared to S2003-US-633

(0.33 U ml-1 min-1). Whereas, at grand growth stage, no significant differences

(p>0.05) were evident. Only temperature treatment manifested the significant

difference (p<0.05) for cell wall invertase activity. However, maximum cell wall

enzyme activity was observed in cultivar SPF-238 (0.371 U ml-1 min-1) than cultivar

S2003-US-633 (0.301 U ml-1 min-1) control condition at grand growth stage. At

maturity stage, under heat stress conditions, both cultivars indicated reduction in

the activity of cell wall invertase enzyme, but this activity was substantially greater

in SPF-238 than S2003-US-633. During the recovery enzyme activity improved same

way in both cultivars. Collectively, SPF-238 had more invertase than S2003-US-633

in all treatments at all stages (Fig 17).

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Fig 17: Specific activity quantified of cell wall invertase (CWIN) of sugarcane cultivars S2003-

US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for

24, 48 and 72 h at vegetative, grand growth and maturity stages.

99


At vegetative stage, data revealed significant differences (p<0.05) for treatments (T)

and their interaction (C×T) while non-significant differences (p>0.05) were observed

for cultivars (C) (Fig 18). Under control conditions, vacuolar invertase activity was

(10.1 U ml-1 min-1) in S2003-US-633 and (8.2 U ml-1 min-1) in SPF-238 but heat stress

significantly reduced the activity of vacuolar invertase in both cultivars while S2003-

US-633 exhibited slow recovery of the activity of this enzyme under recovery

conditions. Furthermore, at grand growth stage, vacuolar invertase activity was

declined in all treatments as compared to control but not significant differences

(p>0.05) were found for cultivar (C), treatment (T) and their interaction (C×T).

Maximum vacuolar enzyme activity was observed in SPF-238 under control, heat

shock and recovery treatments at maturity stage. There was decline in enzyme

activity with increasing episode of temperature in both cultivars. Statistical analysis

revealed significant differences (p<0.05) for cultivar and treatment but non-

significant differences (p>0.05) were noted for their interaction (C×T). Moreover, the

activity of vacuolar enzyme activity was more in vegetative stage than grand growth

and maturity stage in both cultivars.

100

Fig 18: Specific activity quantified of vacuolar invertase (VIN) of sugarcane cultivars S2003-

US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for

24, 48 and 72 h at vegetative, grand growth and maturity stages.

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Invertase isozymes are present in plant at different location, based on location plant

invertase divided into three classes cytoplasmic, cell wall and vacuolar invertases.

Among sucrose metabolizing enzymes invertase is the most important sucrose

cleavage enzymes which was studied through Native-PAGE electrophoresis in both

cultivars S2003-US-633 and SPF-238 at different growth stages. Qualitative analysis

or differential staining of invertase isozymes (cell wall, vacuolar and cytoplasmic)

were carried out on NATIVE-PAGE at all growth stages.


Results revealed that at formative stage, cultivar S2003-US-633 expressed

cytoplasmic or neutral invertase of 160 kDa. During heat stress treatment for T24 low

expression of cytoplasmic invertase while upon recovery conditions at (R24)

maximum intensity was exhibited. At grand growth stage, three types of cytoplasmic

invertases molecular weight (134, 150 and 160 kDa) were exhibited. Band intensity

of 134 kDa was decreased at T72 but expression was observed under recovery

treatments. Intense banding pattern of cytoplasmic invertases (134 and 160 kDa) was

observed than (150 kDa) at all treatments. Bands of cytoplasmic invertase (150 kDa)

were visible under control condition but its intensity decreased at (T72). However, its

expression was increased at R24, R48 and R72 upon recovery. At maturity stage,

expression of cytoplasmic invertase different at all treatments as compared to other

growths stages. Invertase type one (160 kDa) expressed only under recovery

condition after R48 and R72 h. Cultivar S2003-US-633 explored multiple type of

invertases at grand growth but only one type of invertase was exhibited at vegetative

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and maturity stages (Fig 19). Qualitative analysis of cytoplasmic invertase of cultivar

SPF-238 at different growth stages (formative, grand growth and maturity) is

presented in (Fig 20). At vegetative stage, in SPF-238, one types of invertases

molecular weight (160 kDa) was observed. Under control conditions, band intensity

was high but diminished as temperature increased while presence of cytoplasmic

invertase was evident under recovery conditions. At grand growth stage, cultivar SPF-

238, multiple bands (160, 150 and 134 kDa) were detected. Expression pattern of this

invertase isoform 160 kDa was visible at control, T24 h and T48 h, but it was

diminished at T72 h than again reappeared at all recovery conditions. However, other

invertase isoforms (150 and 134 kDa) exhibited weak signal only but the sharp

intensity of band was evident under recovery condition. At maturity stage, cultivar

SPF-238 revealed the presence of invertase isoform molecular weight (160 kDa) at all

heat shock and recovery treatments with varying band intensity. Sharp bands were

observed only in control and heat shock condition (T72).

103

Fig 19: Native–PAGE analysis of cytoplasmic invertase (CyIN) of cultivar S2003-US-633

subjected to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at

all growth stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa

and dimer 134 kDa) and gama globulin human (160 kDa).

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Fig 20: Native–PAGE analysis of cytoplasmic invertase (CyIN) of cultivar SPF-238 subjected

to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at all growth

stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa and dimer

134 kDa) and gama globulin human (160 kDa).

105


Quantitative analysis of vacuolar invertase at formative stage revealed that explored

one type of isozyme (160 kDa) with uniform but at heat shock treatments band

intensity was declined at T72 h. After recovery treatment maximum enzymes

expression was exhibited at R24, R48 and R72 h. At grand growth stage, only 134 kDa

molecular weight isozyme was observed, their expression was confirmed at heat

shock treatments T24, T48 and T72 h, band intensity of vacuolar invertase was

reduced. Furthermore, two types of vacuolar invertases (150 and 160 kDa) were

observed in S2003-US-633 variety, at control condition the band intensity was low

but then exposure to high temperature its expression was increased at maturity stage

while during recovery conditions only, isoforms (150 kDa) was expressed under heat

shock treatment (Fig 21). In cultivar SPF-238, only molecular weight of 160 kDa

isozyme was exhibited, the expression pattern initially increased, but declined with

increased in heat stress application (T72) and recovery conditions (R24) at formative

stage (Fig 22). At grand growth stage, band at 134 kDa was evident at control

conditions and recovery treatment (R72) but band not visible at any heat stress

treatment. While under control condition vacuolar invertase band intensity (160 kDa)

was maximum but the band intensity gradually declined at T24, T48 and T72 h after

heat exposure. However, under recovery treatments, the band intensity gradually

increased at R24 and R72 h at maturity stage.

106

Fig 21: Native–PAGE analysis of vacuolar invertase (VIN) of cultivar S2003-US-633 subjected

to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at all growth



107

Fig 22: Native–PAGE analysis of vacuolar invertase (VIN) of cultivar SPF-238 subjected to

control (C) heat shock (45±2°C) and recovery treatmnents for 24, 48 and 72 h at all growth



108


Multiple bands of molecular weight (140,150 and16 kDa) were expressed in cultivar

US-633 under recovery treatments at vegetative stage. Heat stress severely affected

on enzymes activity, especially after 72 h of heat exposure (T72). On the other hand,

upon recovery, maximum enzymes expression was observed at R24 h. Sharp bands

of 150 kDa and 140 kDa molecular weight isozymes were exhibited after heat shock

treatment after 48 h (T48) at grand growth stage maximum involvement of cell wall

invertase (Fig 23). At maturity stage, only 160 kDa isozyme was evident among all the

heat shock and recovery treatments except R72 (recovery treatment after 72 h). Only

band intensity varied. In SPF-238 cultivar, only 160 kDa molecular weight band was

displayed at vegetative stage. Band of cell wall invertase was sharp under control

conditions but at heat stress treatments (T24, T48 and T72 h) expression of the cell

wall invertase was affected (Fig 24). Upon recovery treatment, only its expression was

confirmed at R24 but at R48 and R72 h the band intensity was diminished. While at

grand growth stage, their expression was not visible at T24 and T48 h after heat shock

treatment. At maturity phase, only 160 kDa isozyme band was displayed same

pattern of band intensity as at vegetative stage.

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Fig 23: Native–PAGE analysis of cell wall invertase (CWIN) of cultivar S2003-US-633

subjected to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at

all growth stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa

and dimer 134 kDa) and gama globulin human (160 kDa).

110

Fig 24: Native–PAGE analysis of cell wall invertase (CWIN) of cultivar SPF-238 subjected to

control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at all growth



111

4.6. Quality Parameters Analysis

Quality traits or parameters such as °brix, polarity, fiber content and sugar recovery

rates are the quality parameters of sugarcane (Table 8).

4.6.1. °Brix Estimation

°Brix is the sucrose content of aqueous solution. It indicates the percentage of cane

sugar (sucrose) by weight (1 g / 100 ml of water). ° Brix is conventionally used in wine

and sugarcane and fruit industries. Results indicates that significant differences

(p<0.05) were observed between treatments (T) and their interactions (C×T) except

cultivar (C) at grand growth stage. Under control conditions, greater °brix content

was depicted in cultivar S2003-US-633. Although heat shock treatment maximum

reduction affected the °brix content by declining in both cultivars. After 72 h of heat

stress in °brix content was evident in cultivar SPF-238 as compare to S2003-US-633.

Upon recovery conditions, both cultivars gradually improved after 48 h and 72 h of

recovery. Comparatively, rapid recovery was exhibited in cultivar S2003-US-633 than

SPF-238. However, at maturity stage, non-significant differences (p>0.05) were

exhibited for °brix content between treatments and their interactions (C×T) except

cultivar (C). Moreover, increased °brix content was observed in both cultivars at

maturity stage than grand growth stage. Temperature episodes also affected °brix

contents in both cultivars, after 72 h (T72) °brix content was drastically declined in

both cultivars. While cultivar SPF-238 showed a greater reduction in °brix content

than S2003-US-633. However, during recovery conditions, both cultivars gradually

improved in °brix contents while quick recovery was observed in S2003-US-633 after

72 h of recovery.

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4.6.2. Fiber Content

Regarding fiber content, it is defined as the dry fibrous insoluble materials of

sugarcane plant. Significant differences (p<0.05) were observed among cultivars (C),

treatments (T) and their interactions at grand growth stage. Under control

conditions, maximum fiber contents were noted in SPF-238 than S2003-US-633.

When temperature episodes were increased gradually, fiber content was decreased

in both cultivars while minimum fiber content was observed in cultivar S2003-US-

633 than SPF-238. But, the percentage of fiber content was progressively increased

after recovery treatments in both cultivars S2003-US-633 and SPF-238 respectively.

At maturity stage, significant differences (p<0.05) for cultivars (C), treatments (T) but

not their interactions (C×T) were observed. Overall, fiber content was slightly

increased at all treatments at maturity stage as compared to grand growth stage.

4.6.3. Pol Estimation

Pol % was investigated in both sugarcane cultivars at both growth and maturity

stages. Statistical analysis revealed that significant differences (p<0.05) were

exhibited for pol among cultivars (C) and their interactions (C×T) at growth stage.

Pol percentage at grand growth stage was gradually declined after applying heat

shock for 7 h than control conditions. In addition, in cultivar S2003-US-633 higher

pol content was noted under heat shock treatments after at (24 h T24) and (72 h

T72). Exhibiting that high temperature affected the pol of sugarcane at grand growth

stage. Moreover, upon recovery pol rate was improved in both cultivars with similar

pattern. While maximum pol percentage was found in cultivar S2003-US-633 than

SPF-238. At maturity, pol content of sugarcane reduced significantly due to heat

113

stress. There were non-significant differences (p>0.05) between cultivars (C) and

treatments (T) together with a non-significant (p>0.05) interactions (C×T) of these

factors. Under heat shock conditions, pol % was gradually declined as compared to

control conditions. But both varieties exhibited same pattern under recovery

treatments. Comparatively, current results designated cultivar S2003-US-633 had

higher percentage of pol than other at both stages.

4.6.4. Sugar Recovery Rate Estimation

Data revealed that statistically significant differences were evident among all factors

for sugar recovery rate (%) at grand growth stage. High sugar recovery rate was

depicted in cultivar S2003-US-633 under control conditions. However, under heat

stress conditions both cultivars exhibited a relatively lesser reduction in this

attribute. In addition, rapid recovery was showed from stress in both cultivars. This

trend also was observed in heat stress and recovery treatments in cultivars SPF-238

(Table 8). At maturity, both cultivars and treatments indicated significant (p<0.05)

differences but not their interactions (C×T). In control conditions, S2003-US-633 (13

%) showed greater sugar recovery rate than SPF-238. Under the stress conditions,

SPF-238 depicted more declined in sugar recovery rate while under recovery cultivar

S2003-US-633 recovered readily more than SPF-238. Above mentioned results

showed that both cultivars losses sugar recovery at harsh environmental conditions

and after recovery treatments for different time durations (24-72 h) both cultivars

observed improvement in sugar recovery rate. At maturity stage both varieties

showed higher percentage of sugar recovery rate than previous both vegetative and

grand growth stage.

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Table 8: Quality parameters of both cultivars S2003-US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48

and 48 h at vegetative, grand growth and maturity stages. Cultivar (C), Treatments (T) and Cultivar ×Treatments (C×T) at p level p<0.05.

Grand Growth Stage


Mean ± SEM P Value


C T24 T48 T72 R24 R48 R72 C T C×T

°Brix

S2003-US-633 13.78±0.12 12.85±0.37 12.82±0.06 12.41±0.13 12.14±0.11 12.52±0.13 12.74±0.05

ns * * SPF-238 13.44±0.19 13.12±0.06 12.25±0.10 11.88±0.07 12.44±0.15 12.46±0.12 12.56±0.12

Fiber content

(%)

S2003-US-633 17.55±0.40 15.48±0.70 14.51±0.38 13.92±0.29 15.19±0.39 16.81±0.33 16.92±0.33

* * * SPF-238 18.89±0.35 17.22±0.29 17.08±0.58 16.45±0.39 14.56±0.15 17.87±0.98 18.44±0.60

Polarization

S2003-US-633 12.13±0.66 10.76±0.25 10.31±0.24 9.47±0.245 9.36±0.250 10.28±0.18 9.81±0.325

* * * SPF-238 10.25±0.19 10.05±0.27 9.31±0.130 9.00±0.329 9.36±0.004 9.39±0.011 10.35±0.01

Recovery (%)

S2003-US-633 9.63±0.66 8.26±0.253 7.81±0.245 6.97±0.246 6.86±0.251 7.78±0.187 7.31±0.325

* * * SPF-238 7.75±0.18 7.55±0.272 6.81±0.130 6.50±0.329 6.86±0.001 6.89±0.015 7.58±0.007

Maturity Stage

°Brix

S2003-US-633 18.13±0.48 17.41±0.28 17.40±0.35 16.65±0.62 16.40±0.65 17.20±0.66 17.64±0.58

* ns ns SPF-238 16.69±0.48 16.41±0.67 16.16±0.59 15.48±0.71 15.54±0.33 16.18±1.03 16.14±1.03

Fiber content

(%)

S2003-US-633 14.40±1.71 13.39±0.28 11.60±0.36 11.35±0.62 11.60±0.66 11.39±0.67 11.86±0.59

* * ns SPF-238 18.68±0.73 17.71±0.67 15.17±0.31 14.52±0.13 14.79±0.58 15.82±1.00 15.59±1.03

Polarization

S2003-US-633 16.99±0.25 16.14±0.06 15.60±0.06 15.43±0.20 15.53±0.36 15.99±0.50 16.08±0.40

* * ns SPF-238 16.18±0.08 15.70±0.18 14.83±0.15 14.68±0.08 15.18±0.30 15.70±0.76 16.00±0.88

Recovery (%)

S2003-US-633 14.49±0.25 13.64±0.06 13.10±0.87 12.93±0.25 13.03±0.36 13.49±0.49 13.58±0.39

* * ns SPF-238 13.68±0.08 13.20±0.18 12.33±0.15 12.18±0.08 12.37±0.24 31.21±0.75 13.50±0.87

Significant difference p<0.05 (*) and non-significant differences p>0.05 (ns)

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4.7. Protein Profiling at Different Growth Stages

4.7.1. Protein Profiling at Vegetative Stage

At vegetative stage, protein expression was analyzed through SDS-PAGE for both

varieties. Proteins of different molecular weights were differentially expressed

during heat stress conditions. The highest molecular weight protein approximately

150 kDa (black) was clearly expressed upon heat stress and recovery treatments. The

band intensity of the protein remained stable during all the episodes of heat stress

24, 48 and 72 h (T24, T48 and T72) but during the recovery phase it was diminished

after 24 and 48 h (R24, R48) and then reappeared at 72 h (R72). Similarly, among

high molecular weight proteins 90 kDa (white), 70 kDa (yellow) and 60 kDa (red)

protein bands were consistently expressed during the heat shock treatments but at

the initial stages of recovery (R24-R48) these were not visible (Fig 25A). Thus, in both

varieties same pattern of protein expression was observed in high molecular weight

protein but in case of low molecular weight proteins there was a sharp high intensity

band observed at approx. 15 kDa (green) in cultivar S2003-US-633 which was not

present in cultivar SPF-238. During the SDS PAGE analysis 15 kDa protein might be

differentially expressed in S2003-US-633 as compared to SPF-238 during the heat

shock and recovery phases. However, protein band nearly 35 kDa (blue) evident at

heat shock as well as after 72 h recovery treatments (R72) while it was absent in

control conditions in cultivar SPF-238 (Fig 25B).

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Fig 25: SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-633 (B) SPF-238 at

formative stage under control at (30±2°C), heat shock (45±2°C ) and recovery treatments

(30±2°C) for 24, 48 and 72 h.

117

4.7.2. Protein Profiling at Grand Growth Stage

At grand growth stage, analysis of protein profiling observed induction of different

proteins molecular mass bands approximately 100 kDa, 66 kDa, 30 kDa and 35 kDa.

The 70 kDa protein band (Red) was prominent after 72 h exposure to heat shock

(T72) while under control conditions, the band intensity was slightly observed but

not prominently. Upon recovery, 70 kDa protein band was disappeared at 24 h (R24)

then after 48 and 72 h recovery treatment the bands, again reappeared at R48 and

R72. Same pattern of results was exhibited of 100 kDa protein (white). However, in

cultivar S20003-US633, the expression of 30 kDa and 35 kDa protein bands were not

higher at all treatments (Fig 26A). In cultivar SPF-238 both 66 kDa and 100 kDa

showed expression after 72 h (T72) heat treatments as compared to other

treatments. Another new protein 52 kDa (green) also observed under control

conditions and after 24 h (T24) heat shock conditions (Fig 26B). And also 30 kDa band

(yellow) molecular mass protein was expressed only under control and under heat

stress conditions at 24 h (T24) but rest of the samples showed no bands.

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grand growth stage under control at (30±2°C), heat shock (45±2°C) and recovery treatments

(30±2°C) for 24, 48 and 72 h.

119

4.7.3. Protein Profiling at Maturity Stage

At maturity stage, in both cultivars (S2003-US-633 and SPF-238) the expression

pattern was quite different than vegetative stage. In cultivar S2003-US-633, protein

band approximately 66 kDa molecular mass (red), was observed very clearly with

higher intensity under control conditions, heat shock after 24-48 h (T24 and T48) as

well as after 48 h (R48) recovery conditions while 30 kDa protein band intensity was

very low after 72 h (T72) exposure of heat treatments. However, induction of 100

kDa molecular weight protein was not visible under heat shock T72, recovery R24 and

R72 but the band was slightly visible in other treatments (Fig 27A). In cultivar SPF-

238, 66 kDa (red) and 100 kDa (white) proteins bands were dominantly expressed in

all treatment except after heat exposure at 48 h (T48). Both proteins (66 kDa and 100

kDa) were not expressed at T72 h after heat shock treatments. However, another

differential expression exhibited as 30 kDa band molecular mass (yellow) protein was

under heat shock treatment and recovery but the expression level was very low in

control conditions (Fig 27B).

120


maturity stage under control at (30±2°C), heat shock (45±2°C ) and recovery treatments

(30±2°C) for 24, 48 and 72 h.

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4.8. Correlation

Sugar metabolizing enzymes play vital role in sugarcane plant for synthesis and

hydrolyze sugar at different growth stages. Furthermore, theses enzymes also help

sugar loading and unloading at different compartment or location in plant cells to

balance the cellular mechanisms of different biomolecules. Present study was

performed to investigate correlation among sugar profile, sugar metabolizing

enzymes such as sucrose synthase, sucrose phosphate synthase, cell wall invertase,

cytoplasmic or neutral invertase and vacuolar invertase and quality parameters like

pol, fiber content, °brix and sugar recovery rate at vegetative, grand growth and

maturity stages. The correlation results of the all parameters at different growth

phases were presented in (Table 9). Data was tested at a significant p<0.01 (**) and

p<0.05 (*). Results revealed that the relationship among total sugar with sucrose

synthase (SS) and sucrose phosphate synthase (SPS) were strongly positively

correlated at all growth stages. While its relationship with reducing sugar at maturity

stage was negative at maturity stage but positive to relation was exhibited at

vegetative and maturity stage. The association of quality parameters such as °brix,

recovery rate and pol with total sugar was positive but negative association was

observed with reducing sugar at maturity stage. Sugar recovery relationship among

cell wall invertase (CWIN) and vacuolar invertase (VIN) were negatively correlated at

both vegetative and maturity stages. This result indicates that the °brix, pol and total

sugar were inversely proportional to reducing sugar in both cultivars. However, the

association of invertase isozymes such as vacuolar and cell wall invertases with total

sugar and non-reducing sugar as well as sugar recovery rate, pol and °brix were

negatively correlated at maturity stage.

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**Significant correlation at p<0.01 and *Significant correlation at p<0.01

Table 9: Correlation among sugar profile, sugar metabolizing enzymes and quality parameters at all growth stages.

Parameters Stages TS RS NRS SS SPS CyIN VIN CWIN °Bx Fb Pol RC

Total Sugar (TS)

V 1 .732** .972** .694** .707** .501** .114 .315* - - - -

G 1 .448** .390* .771** .755** .540** .378* .532** .746** .378* .739** .739**

M 1 -.701** 1.000** .629** .667** .037 -.250 -.310* .473** -.430** .451** .451**

Reducing Sugar (RS)

V .732** 1 .551** .572** .579** .245 -.150 .221 - - - -

G .448** 1 -.648** .464** .514** .627** .372* .383* .387* .721** .200 .200

M -.701** 1 -.723** -.294 -.298 .273 .513** .573** -.425** .668** -.296 -.296

Non-reducing Sugar (NRS)

V .972** .551** 1 .654** .671** .534** .193 .308* - - - -

G .390* -.648** 1 .180 .115 -.183 -.059 .065 .240 -.417** .424** .424**

M 1.000** -.723** 1 .622** .659** .023 -.264 -.326* .476** -.446** .449** .449**

Sucrose Synthase (SS)

V .694** .572** .654** 1 .715** .532** .325* .511** - - - -

G .771** .464** .180 1 .658** .534** .439** .494** .734** .414** .553** .553**

M .629** -.294 .622** 1 .822** .328* .152 .094 .503** -.009 .588** .588**

Sucrose Phosphate Synthase (SPS)

V .707** .579** .671** .715** 1 .480** .086 .326* - - - - G .755** .514** .115 .658** 1 .522** .370* .626** .750** .342* .647** .647**

M .667** -.298 .659** .822** 1 .375* .170 .130 .459** .073 .597** .597**

Cytoplasmic Invertase (CyIN)

V .501** .245 .534** .532** .480** 1 .155 .360* - - - -

G .540** .627** -.183 .534** .522** 1 .207 .408** .579** .562** .325* .325*

M .037 .273 .023 .328* .375* 1 .415** .362* .098 .385* .077 .077

Vacuolar Invertase (VIN)

V .114 -.150 .193 .325* .086 .155 1 .230 - - - -

G .378* .372* -.059 .439** .370* .207 1 .450** .443** .245 .431** .431**

M -.250 .513** -.264 .152 .170 .415** 1 .659** .024 .526** -.074 -.074

Cell Wall Invertase (CWIN)

V .315* .221 .308* .511** .326* .360* .230 1 - - - -

G .532** .383* .065 .494** .626** .408** .450** 1 .579** .293 .504** .504**

M -.310* .573** -.326* .094 .130 .362* .659** 1 -.140 .640** -.010 -.010

°Brix (°BX) G .746** .387* .240 .734** .750** .579** .443** .579** 1 .249 .703** .703**

M .473** -.425** .476** .503** .459** .098 .024 -.140 1 -.474** .308* .308*

Fiber Content (FC) G .378* .721** -.417** .414** .342* .562** .245 .293 .249 1 .204 .204

M -.430** .668** -.446** -.009 .073 .385* .526** .640** -.474** 1 .050 .050

Polarization (Pol) G .739** .200 .424** .553** .647** .325* .431** .504** .703** .204 1 1.000**

M .451** -.296 .449** .588** .597** .077 -.074 -.010 .308* .050 1 1.000**

Sugar Recovery (SR) G .739** .200 .424** .553** .647** .325* .431** .504** .703** .204 1.000** 1

M .451** -.296 .449** .588** .597** .077 -.074 -.010 .308* .050 1.000** 1

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4.9-Heat Map

Table 9: Status of Thermotolerant Sugarcane Cultivars

Growth Stages

Cultivars

Sugar Analysis Sucrose Metabolizing Enzyme Analysis

Sugar Recovery Analysis Thermotolerance Indicators Analysis

Remarks TS

RS

NRS

SS

SPS

CyIN

CWIN

VIN

°Bx

Pol

Fb

SR

EC

MDA

H2O2

Pr

Vegetative

S2003-US-633

Nil Nil Nil Nil

Least Susceptible

SPF-238

Nil Nil Nil Nil

Most Susceptible

Grand

Growth

S2003-US-633

Moderately Susceptible

SPF-238

Moderately Tolerant

Maturity

S2003-US-633

Most Tolerant

SPF-238

Least Tolerant

Most Susceptible Moderately Susceptible Least Susceptible Least Tolerant Moderately Tolerant Most Tolerant

Susceptible (SPF-238 ) Tolerant (S2003-US-633)

Total Sugar (TS), Reducing Sugar (RS), Non-reducing Sugar (NRS), Sucrose Synthase (SS), Sucrose Phosphate Synthase (SPS), Cytoplasmic Invertase (CyIN), Cell Wall Invertase (CWIN), Vacuolar Invertase (VIN),°Brix (°Bx), Sugar Recovery (SR), Polarization (Pol), Fiber (Fb), Proline (Pr), Hydrogen Peroxide (H2O2 ), Electrolytes leakage (EC) and Malondialdehyde (MDA).

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SECTION # 5 DISCUSSION

125

Discussion


Yield parameters are potential and reliable morphological indicators to select and

differentiate the thermotolerant sugarcane cultivars. In the present study, heat stress

conditions decreased the plant height and root length, fresh to dry weight ratio, stem

diameter and biomass accumulation that eventually reduced yield and related

attributes of both sugarcane cultivars at vegetative, grand growth and maturity

stages (Table 5-7).

Among all stages, vegetative stage is considered more critical, severely affecting the

shoot length by application of heat stress for different episodes but slowly

improvement was shown upon recovery conditions in SPF-238. While possible reason

for taller shoot length of sugarcane cultivar S2003-US-633 may be due to high yield

characteristics than cultivars SPF-238 exhibiting its potential for stress tolerance

(Abubakar et al., 2013; Gravois and Legendre, 2011). Varietal differences were

evident with respect to root length (Table 6) effecting root length of both sugarcane

cultivars upon heat stress. While longest root length (82 cm) was observed in S2003-

US-633 compared to SPF-238 (78 cm) indicating its deep root branching for more

water absorption to combat water loss due to evaporation (Madhav et al., 2017).

Roots play a vital role for supply of nutrients and water for growth and development

of crop and tendency to develop deep root system observed may be used as selection

criteria for drought and thermotolerant as well (Wasaya et al., 2018).

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Another important agronomic trait, number of tillering influenced by biotic and

abiotic factors because the optimal temperature for tillering formation is 30°C.

Maximum number of tillers in SPF-238 depicts its low sugar recovery potential in

stalk, delayed accumulation of sucrose in culm and stunted growth due to maximum

energy utilization during tiller formation (Misheck, 2013; Inman-Bamber et al., 2009).

No external changes were observed in sugarcane leaf length, leaf width, due to short

episode of temperature however, leaf colors were slightly changed when the plants

exposed at different heat shock episodes. High temperature resulted pre and post-

harvest damages such as, scrolling of leaf, sunburn of leaf, leaf senescence, shoot and

root growth inhibition, stem diameter and declined the yield for long exposure

(Vollenweider and Gunthardt-Goerg, 2005). Leaf structure also affected by heat

stress frequently causing of thinner leaf with higher leaf area (Poorter et al., 2009).

These morphological alterations are supported with changes in leaf anatomy. Leaf

under high temperature and drought usually have smaller cells and higher stomatal

density (Shahinnia et al., 2016) but limited data available regarding to leaf anatomy

alterations due to heat stress (Wahid et al., 2007). In this study, no changes were

observed in number of nodes and internodes per plant in both cultivars at all stages

due to heat stress. However, number of nodes was increased in maturity stage as

compared to grand growth stage in S2003-US-633. Number of nodes is important

criteria for sucrose accumulation and there is inverse relationship between number

of nodes and sucrose content in sugarcane stalk (Bonnett et al., 2006). Number of

nodes and internodal distances are also controlled by genetic factors and may also be

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affected by plantation date or other environmental conditions (Shahzad et al., 2002;

Shahzad et al., 2016).

Under high temperature stress drastic reduction was exhibited in fresh to dry weight

ratio in both cultivars. Highest fresh to dry matter ratio was found in cultivar, S2003-

US-633 upon heat stress only at vegetative stage and attributed due to increased

photosynthetic rate. Heat stress at 40 / 24 °C (day/night) significantly declined the

shoot dry weight in quinoa (Hinojosa et al., 2019), sugarcane, corn and pearl millet

(Wahid et al., 2007; Zhao et al., 2006). In this study, cultivar S2003-US-633 could be

ranked as thermotolerant on morphological basis due to high yielding characteristics,

deep rooting system and high fresh to dry matter ratio (Table 10).

5.2. Thermotolerant Indicators Analysis

Malondialdehyde (MDA) content as by-product of lipid per oxidation, was quantified

in both cultivars of sugarcane plant under heat stress. Increase in lipid peroxidation

also a marker of oxidative stress (Goel and Sheoran, 2003) for abiotic and biotic

stresses (Hameed and Iqbal, 2014) and it could be used as an important

thermotolerant indicator of physiological damages during crop growth and

development. In current study, maximum accumulation of MDA content was

revealed in both cultivars S2003-US-633 and SPF-238 under high temperature

conditions displaying substantial lipid peroxidation of biological membrane leading to

the reactive oxygen production along with losing membrane integrity (Boaretto et al.,

2014). The S2003-US-633 cultivar had minimum MDA content accumulation than SPF-

238 cultivar (Fig 6). Electrolyte leakage also closely related with the degree of cell

membrane

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injury and relative membrane thermosability under heat stress. Plasma membrane is

very sensitive to abiotic stress (high temperature) in plant cell, it is primary site for

injury (Blum, 2018). This injury can be measured by the loss of membrane integrity

reflected in ion leakage from plant cells (Liu and Huang, 2000; Jiang and Huang,

2001a; Salvucci et al., 2004) and has been used as stress induced markers in under

stress conditions. Under heat shock conditions, plant cells extremely damage and

even cell death may occur within minutes. This phenomena is extensively used as a

test for the stress induced damage of plant tissue and measure of plant. Recent study

revealed significant damage to the membrane thermal stability of SPF-238

(susceptible cultivar) under heat stress conditions, whereas S2003-US-633 (tolerant

cultivar) maintained a membrane thermal stability with minimal electrolytes leakage

(Fig 9). This may be attributed due to cell membrane damages by losing membrane

integrity with increase in unsaturated fatty acids leading to increased fluidity of the

membrane (Horvath et al., 2012) which further increase Ca2+ influx (Bita and Gerats,

2013). These outcomes are supported by previous study (Zhang et al., 2005) where

excessive permeability of membrane severely damaged the mesophyll cells and led

to the increased electrolytes leakage under heat stress conditions (Savchenko et al.,

2002; ElBasyoni et al., 2017 ; Kumar et al., 2013). It is also reported that EC content

was associated with grain yield reduction (Khan et al., 2019). Among all growth

stages, both cultivars significantly influenced under heat shock treatments at

vegetative and grand growth stages. Comparatively maximum EC content was

observed in vegetative stage than other stages. Cell membrane thermostability

depends on species, tissue, cell types and can be triggered by biotic and abiotic

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factors including pathogen attack (Maffei et al., 2007), salinity (Demidchik et al.,

2010), oxidative stress (Demidchik et al., 2003, 2010), heat (Liu and Huang, 2000) and

others. Heat stress could lead to over production of reactive oxygen species (ROS)

which deteriorate photosynthetic machineries in plants (Wang, 2004). High levels of

ROS accumulation significantly effect on physiological and biochemical functions such

as destruction of plasma membrane, lipid per oxidation, protein denaturation,

destruction of enzymes, DNA, RNA and figments (Bose et al., 2013; S Li et al., 2018)

resulted reduced crop yield and quality. (Sharma et al., 2017; You and Chan, 2015;

Gaschler and Stockwell, 2017). Among the different types of ROS hydrogen peroxide

is a lethal reactive oxygen species, which has harmful effect on plant cells (R. Sairma

and Srivasta, 2002). It is also most important signaling molecule playing vital role in

the photosynthesis (Rodrigues et al., 2017), cell wall cross linkage (Li et al., 2017)

stress acclimation (Lv et al., 2018) and antioxidant defense system (Liu et al., 2016).

In this study, increased hydrogen peroxide contents in both sugarcane cultivars were

exhibited under heat stress conditions which indicated the manifestation of oxidative

stress. But maximum hydrogen peroxide content was found in SPF-238 exhibiting

damages and low integrity of cell membrane. The level of hydrogen peroxide was

higher at vegetative stage than other growth stages (Fig 8). In general, when the level

of hydrogen peroxide level exceeds then caused damage to essential cellular

components (Mittler, 2006). Similar results were also observed in wheat (Hamurcu et

al., 2014) and canola (Akram et al., 2018) plants under stress conditions. Studies at

physiological, molecular, biochemical levels suggest that osmolytes perform

important role in mitigating heat stress in plant by decreasing reactive oxygen species

130

(ROS) level by protecting cell membrane and protein (Hayat et al., 2012). Free proline

and other compatible solutes such as glycine and soluble sugar is essential to regulate

osmotic activities and protect cellular structures, membrane stability by maintaining

the cell water balance, improve cytoplasmic acidosis and by buffering redox potential

(Khan et al., 2019; Farooq et al., 2008; Chen and Murata, 2002). As the synthesis of

proline depend on plant to plant, species to species, plant tissue under investigation

past memory and genetic factors (Ashraf and Foolad, 2007; Qaun et al., 2004).

Therefore, both cultivars S2003-US-633 and SPF-238) had ability to accumulation of

thermotolerant osmolyte (proline) under heat stress conditions at different growth

phases. Cultivar S2003-US-633 had more potential to accumulate free proline as

compared to other cultivar SPF-238 under heat stress conditions at all growth stages

(Fig 7). This higher accumulation of osmolytes such as proline maintained water

environment in the cells (Gupta et al., 2013; Giri et al., 2011; Sakamoto and Murata,

2000) and providing thermotolerance (Tanveer et al., 2019; Sharma et al., 2019) to

S2003-US-633. On the other hand, proline also serve as energy for respiration,

ammonia sources during stress conditions and directly participating in plant

metabolism after stress relief (Hussain et al., 2019) and act as electron acceptor,

osmolyte and protect the cell membrane (Bartels and Sunkar 2005; Gupta et al.,

2013). Accumulation of free proline in cultivar S2003-US-633 might contribute

minimum ROS production such as hydrogen peroxide with compromised MDA and

electrolytes leakage than cultivar SPF-238 under heat stress conditions. These

biochemical attributes can index the degree of tolerance of sugarcane to exhibit

adaptability under stressful conditions providing the insights to molecular breeders

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to identify the thermotolerant varieties with improved recovery of sugar. In this

regard, cultivar S2003-US-633 is ranked as thermotolerant variety.

5.3. Sugar Analysis

In current study, biochemical parameters such as (total sugar, reducing sugar and

nonreducing sugar) were quantified upon heat stress in both sugarcane varieties.

These biochemicals not only play imprative role in growth and development of crop

but also act as osmolytes in any environmental stress conditions such as biotic and

abiotic for protect the plant cells (Zhou et al., 2017). It is evident from results that

heat stress declined the content of total sugar in both sugarcane varieties but upon

the recovery treatment, sugar content improved only at maturity stage. These results

are in agreement with Datir et al., 2015 finding that total sugar was low at 300 day

after planting and then gradually increased at 360 day after planting (Datir et al.,

2015). There is variability in sugar content in different sugarcane varieties at different

growth stages, sucrose content was predominant at maturity stage (Tana et al.,

2014). However, higher total and non-reducing sugar content was observed in

cultivar S2003-US-633 at all stages (Fig 10 and 12). This accumulation of sucrose may

be due to the decreased invertase activity and delayed ripening processes in S2003-

US-633 (Sachdeva et al., 2003). However, reducing and nonreduing sugar analysis

depicted same pattern of decrease under high temperature in both varieties at

formative, grand growth and maturity stages. This reduction in total sugar content is

attributed due to less carbon assimilation and subsequent partitioning of carbon

derived energy products including sucrose, from source (leaves) to sink (stem),

tissues or increased respiratory demand

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(Huntingford et al., 2017; O sullivan et al., 2017; Amthor et al., 2019). Results

revealed that maximum reducing sugar was observed in cultivar SPF-238 only under

heat stress conditions at grand growth and maturity stage. But upon recovery not

much recovered reducing sugar content (Fig 11). It is assumed that enhanced

invertase activity may be responsible for increased reducing sugar (Tana et al., 2014).

High temprature stress declined the activity of key sucrose metabolizing enzymes

sucah as adenosine diphosphate glucose pyrophophorylase, SPS and invertase

affecting the sucrose accumulation (Vu et al., 2001). These alteration in sucrose

content under heat stress is fundamental for understanding the biochemical

pathways associated in the molecular response of plant (Rodziewicz et al., 2014).

5.4. Sugar Recovery Rate Analysis

Sugar industrialists and farmers required more sucrose contents, cane yield as well

as high sugar recovery rate. Quality parameters play vital role in marker assisted

selection for high cane yield and sugar recovery rate. Sucrose recovery rate analysis

comprised of different quality characteristics like sugarcane pol, brix, moisture, and

fiber content and sugar recovery rate. Present study demonstrated that S2003-US-

633 had highest sugar recovery (14.4 %) while in cultivar SPF-238 (13.6 %) were

recorded under control conditions. It is reported that pol % is directly proportional to

sugar recovery (Khan et al., 2018). It is suggested that maximum pol (18.38 %) was

exhibited in cultivar MS-92-CP-99 with highest sugar recovery (12.44 %) (Ali et al.,

2019) These high and low cane yield and sugar recovery potential are associated with

various morphological and genetical characters (Ali et al., 2019; Khan et al., 2018).

http://www.plantcell.org/content/31/2/297#ref-58

http://www.plantcell.org/content/31/2/297#ref-95

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However, it is challenging to get simultaneously cane yield and sugar recovery rate in

the same cultivar (Khan et al., 2018). Due to the highest CCS %, yield and sugar

recovery potential, cultivar S2003-US-633 was recommended for commercial cultivar

in Punjab Pakistan (Tahir et al., 2014). It is reported previously that cultivar SPF-238,

sugarcane produced 115 ton per hector and sugar recovery rate 6.02 % while cultivar

S2003-US-633 can produced 130 ton per hector and recovery rate 12.90 %

(Anonymous 2009-2010). Whereas, the cultivar S2003-US-633 was produced average

15 % commercial cane sugar (CCS) also produced excellent recovery, this variety is

fast growth rate, salt tolerant as well as highest sugar recovery potential while

cultivar, SPF-238 was banned because low yield with 43.48 to 53.40 t / hec and

recovery rate 7.96 % to 9.48 % respectively (Afzal et al., 2011). Maximum sugar

recovery was exhibited at temperature from 22°C to 26°C at maturity stage or

ripening (Srivastava et al., 1995).

However, results revealed that exposure of heat treatments declined the sugar

recovery rate in both cultivars at both grand growth and maturity stages. Upon the

recovery conditions, both cultivars recovered with same pattern. Fluctuation in

temperature and humidity adversely effect on sugar recovery and sugar

accumulation in sugarcane crop (Pathak et al., 2019) by affecting metabolizing

enzymes activities. High temperature denatures enzymes and protein ultimately

declining the sugar recovery rate (Kohila and Gomathi, 2018) and these sugar

recovery and sucrose metabolizing enzymes such as SPS and SS positively associated

with sucrose content. Cultivar SPF-238 exhibited declined SPS and SS activities at high

temperature (45 ± 2°C) but thermotolerant cultivar S2003-US-633 had highest

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sucrose metabolizing enzymes activity. Low sugar recovery rate is also attributed due

to high fiber content in sugarcane stalk (Rakkiyappan et al., 2003). Present

experiment revealed that among cultivars SPF-238 had maximum fiber content

comparison with cultivar S2003-US-633 at grand growth and maturity stages (Table

8). Regarding pol %, it content also declined due to improper harvesting season (early

or late) (Rakkiyappan et al., 2009; Kulkarni et al., 2010). Early mature cultivar of

sugarcane and less fibrous percentage are tolerant cultivar to postharvest sucrose

loss (Siddhant et al., 2009). Recent data showed that cultivar S2003-US-633 had less

fibrous percentage but high pol % than cultivar SPF-238. Sowing date also most

important factor for high sugar recovery rate, it is suggested that sugarcane crop

cultivated in September achieved high sugar recovery rate and sugarcane yield as

compared to March (Fazal-ur-Rehman, 2018). So cultivar S2003-US-633 may be the

high yielding, resistant in extreme temperature conditions and potential to high

recovery rate as compared to cultivar SPF-238.

5.5. Sucrose Metabolizing Enzymes Analysis

5.5.1. Quantitative Analysis

Sucrose metabolizing enzymes such as sucrose phosphate synthase (SPS), sucrose

synthase (SS), cell wall (CWIN), cytoplasmic (CyIN) and vacuolar (VIN) invertases, are very

important enzymes for sugar metabolism in sugarcane. There is need to understand the

characteristics of sucrose metabolizing enzymes for improvement of sugar recovery rate

against abiotic stress especially high temperature. In sugarcane, sucrose synthesis in

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leaves is carried out by photosynthesis and transported to stems which is fundamental

requirement for plant growth and development (Bihmidine, 2013; Yadav, 2015).

Moreover, this sugar synthesis and transports are affected under biotic and abiotic stress

conditions (Lemoine, 2013) and regulated by sucrose phosphate synthase (SPS). SPS

activity was higher in high sucrose accumulation cultivars in mature nodes than lower

sucrose accumulation cultivars (Verma et al., 2011). While, SPS synthesized sucrose -6-

phosphate by protein phosphorylation with glucose 6-p and resynthesize sucrose after

apoplastic hydrolysis in sugarcane (Oparka et al., 1992; Gayler and Glasziou ,1972). In this

study, sucrose phosphate synthase (SPS) activity was evaluated at different heat shock

conditions in both sugarcane cultivars (Fig 15). Quantitative analysis revealed that

significant decline in SPS enzyme was exhibited in both cultivars but cultivar S2003-US-

633 had higher SPS enzymatic activity than SPF-238 under heat stress conditions (45 ± 2

°C for 24, 48 and 72 h) at maturity stage (Gomathi et al., 2017). This swiftly inactivation

of SPS activity under heat stress was attributed due to denaturation of enzyme structure

under heat stress (Neliana et al., 2019). Another enzyme sucrose synthase, catalyzes the

reversible hydrolysis of sucrose using UDP to yield fructose and UDP-G (Granot and Stein,

2019). At vegetative stage, minimum SS activity was found in both cultivars as compared

to grand growth and maturity stages under heat stress exhibiting enzyme may be

hydrolyzed into fructose and glucose for as fuel for growth and developments at

vegetative stage (Ruan, 2014). But at maturity stage, the SS activity was declined due to

limited sink strength especially in low sucrose accumulation cultivar (SPF-238) under heat

stress (Fig 14). Temperature is very import factor for enzyme activity, sucrose synthase

showed optimal activity at 37°C (Verma et al., 2018) and at 30 °C (Schmolzer et al., 2016).

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Other studies also confirmed that SS activity can either decline or enhance at maturity or

ripening stage (Botha and Black 2000; Joshi et al., 2013). It has been reported that sucrose

metabolizing enzymes activities were found dissimilar in different cultivars at different

growth stages (Tana et al., 2014). As sucrose accumulation depends on SS and invertases

isoforms in sugarcane and sucrose synthase was negatively associated with invertases

while positive correlation with SPS enzymes activities (Gutierrez-Miceli et al., 2002;

Siswoyo et al., 2007). Now-a-days, efforts have been made for the increased sucrose

contents or sugarcane production in sugarcane crop through manipulating genes

associated with sucrose (Conradie, 2011). During ripening of rice seed, maximum SS

activity was exhibited under heat stress (Takehara et al., 2018). In addition, potential

thermotolerant SS was observed in wheat crop (Wh-1021). Another significant enzyme

involved in sucrose metabolism is invertase, hydrolyzes sucrose to hexose as a fuel for

cell growth, elongation and other metabolic processes (Roitsch and Gonzalez, 2004).

Invertase plays vital role in sucrose accumulation and have been well documented in

sugarcane plant since five decades (Gayler and Glasziou, 1972; L. Wang et al., 2017).

Invertases isoforms are classified according to subcellular location and optimum pH and

designated cell wall invertase at apoplectic space, vacuolar invertase at in vacuoles and

cytoplasmic invertase in cytoplasm (Ma et al., 2000). These enzymes declined the

expression under heat stress, some increase upon recovery. In this study the enzyme

activity of invertase isoforms (CyIN, CWIN and VIN) of cultivar S2003-US-633 and SPF-238

were carried out at various growth phases under heat stress conditions. Results showed

that when heat stress was applied for different episodes (T24, T48 and T72) both cultivars

declined the activity of cytoplasmic invertase at all stages. Comparatively, highest CyIN

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activity exhibited at maturity stage in SPF-238. Similar results reported that the

cytoplasmic invertase activity or their expression was increased while maturation process

as this play important role is in sugars accumulating at maturity stage (Sachdeva et al.,

2011). In sugarcane, invertase activity is higher in juvenile tissue where fast cleavage

happens to provide fuel for cell growth or biosynthesis and other metabolic activities (A

Chandra et al., 2015). Lower CyIN activity was observed in cultivar S2003-US-633 at this

maturity stage (Fig 16). In contrast, results were suggested that minimum CyIN activity

was showed in mature tissues in early maturity sugarcane cultivar (Rossouw et al., 2010).

Cell wall invertase that is believed to be present in sugarcane plant (Vorster and Botha.,

1998) have variable functions on the bases of specificity and enzymatic characterization

(Wan et al., 2018). CWIN plays imperative role for plant growth and development,

reproduction, germination and ovary activity in plants (Goetz et al., 2017; Wan, Wu, Yang,

Zhou, and Ruan, 2018; Lv et al., 2018). This study reported the significant decrease in the

activity of CWIN from 24-72 h after heat stress treatments in both cultivars (S2003-US-

633 and SPF-238). In tomato crop, CWIN activity increased under heat stress conditions,

which suppresses reactive oxygen species independent in cell death (Liu et al., 2016).

Among cultivars, the activity of CWIN was substantially greater in SPF-238 than cultivar

S2003-US-633 at vegetative and maturity stages with low sucrose accumulation (Fig 17).

It is proposed that the highest level of CWIN associated with low level of sucrose

accumulating cultivar in juvenile tissues while lowest level of activity was associated with

higher level of sucrose accumulating cultivar in mature sugarcane stem or stalk (Zhu et

al., 1997). CWIN activity is consider as the most significant enzyme regulating the sucrose

content in sugarcane stalk as compared to other sucrose metabolizing enzymes (SPS, SS

138

and CyIN). Maximum activity of soluble acid invertase (VIN) and insoluble acid invertase

(CWIN) in immature tissues of S2003-US-633 were observed (Batta et al., 2008). It is

assumed that CWIN play important role in this variety in sugar signaling and transporting,

supplying nutritional for elongation of pollen tubs (Goetz et al., 2017). Comparatively

CWIN activity, acid invertase and cytoplasmic invertase were declined in mature tissues

S2003-US-633 due to least growth and development processes (Verma et al., 2011). This

low invertase activity may govern active and maximum sucrose accumulation in the

vacuoles and in storage sinks (Chandra et al., 2012). For example, knock out of cell wall

invertase genes declined grain mass in corn (Miller and Chourey, 1992), inhibited

expression of cell wall invertase genes led to declined root growth in carrot (Tang et al.,

1999). Regarding vacuolar invertase activity, present study revealed that both cultivars

(S2003-US-633 and SPF-238) declined VIN activity but during control, heat shock and

recovery treatments, maximum reduction was observed in cultivar S2003-US-633 at

maturity stage. As the short-term response of heat stress might differ from long term

responses but 72h might be a significant time point for initiating long term heat stress

response in sugarcane crop. Acid invertase cleavage sucrose during cane maturity and

post-harvest (Chandra et al., 2015), may be due to this reason minimum VIN activity was

exhibited in cultivar S2003-US-633 at maturity stage. It is assumed that storage of sucrose

in vacuoles leads to high sucrose accumulation or high sink strength in sugarcane stalk

(Fig 18). On the other hand, sugar metabolizing gene inhibition, disturbed biochemical

activity and deactivated regulatory network eventually result decline growth rate, sugar

production or yield in sugarcane due to heat shock. The activity of VIN depended on

multitude of signals, sugars hormones and other environmental stresses and exhibited

139

maximum activity when rapid growth observed (Lontom et al., 2008; Koch, 2004). In case

of down regulation of VIN genes in sugarcane at maturity stage may enhance sucrose

content or sugar recovery percentage along with regulation of source to sink strength of

sucrose by physiological and biochemical alteration (Tauzin and Giardina, 2014). It is also

reported other crop that the VIN invertase gene suppression can be minimize or could

control sugar-end defect formation in potato (Zhu, 2014). Cleavage of sucrose in the

intracellular space and vacuole affect the sucrose content in sugarcane stalk (Wang et al.,

2013).

5.5.2. Qualitative Analysis of Invertase Isozymes

In the present study cytoplasmic invertase (CyIN) of multiple molecular weights were

identified (134,150 and 160 kDa) in both sugarcane cultivars (S2003-US-633 and SPF-

238) at different growth phases. Although, increasing temperature declined the

expression of invertase activity but minimum CyIN expression was exhibited in

cultivar S2003-US-633 as compared to SPF-238 at vegetative and maturity stages due

to presence of invertase inhibitors (Fig 19). It is suggested that invertase inhibitor

gene homologs (18.17 kDa ShINH1) and (19.97 kDa ShINH2) were identified in

sugarcane (Shivalingamurthy et al., 2018). Regarding cell wall invertase that multiple

CWIN molecular mass band proteins (140, 150 and 160 kDa) were expressed in

cultivar S2003-US-633 at vegetative and grand growth stages suggesting is

involvement in thermotolerance (Fig 20). Only 160 kDa molecular mass CWIN found

in SPF-238 at all growth stages (Pressman et al., 2006). Among VIN, three types of

molecular weight (134, 155 and 160 kDa) were exhibited in both cultivars at different

140

growth phases. Only 160 kDa molecular mass was observed at vegetative and

maturity stages in both cultivars. While only 134 kDa molecular mass VIN band was

observed at grand growth stage in both cultivars. Previous study also reported that

60, 120, 240 kDa (Vorster and Botha 1999), 150 kDa was found in yeast Candida utilis

(Chavez et al., 1997), 218 kDa in sugarcane (Rahman et al., 2004), 52.94 kDa

molecular weight of VIN was observed in sugarcane (L. Wang et al., 2017). It is also

reported that 55 to 70 kDa molecular weight VIN was observed which is N-

glycosylated at multiple sites (Tymowska et al., 1998). First, genotypic evidence that

maximum VIN activity are likely to play key role in heat resistant in sugarcane.

Moreover, VIN activity was significantly higher in juvenile tissue in sugarcane. This

result suggested that VIN activity declined under heat stress as well maturity stages

(Fig 21 and 22) in high sucrose accumulating cultivar while at maturity stage VIN

activity increased in low sucrose accumulating variety (Gayler and Glasziou., 1972).

However, complete information about invertase enzymes is unclear, so further study

is required to explore thermotolerant mechanism in sugarcane crop under heat stress

conditions.

141

5.6. SDS-PAGE Protein Profiling

Proteins play vital role in energy production, growth and development by prevention

of severe damage to the photosynthetic machinery in plant cells (Ford et al., 2011).

In this study, quantitative analysis of total soluble protein contents was declined in

both cultivars under heat stress, but drastic reduction was exhibited in cultivar SPF-

238 as compared to S2003-US-633. After SDS-PAGE analysis differential protein

expression (high molecular weight proteins) was observed during heat stress (Fig 25-

27) in both cultivars at all stages but higher expression were found at vegetative

stage. It is assumed that these expressed protein may belong to class of stress

proteins such as HSPs, dehydrins and osmotins etc. Heat shock protein (HSPs)

involved in transcription and translation might be important in response to heat

stress (Kültz, 2003; Hasanuzzaman et al., 2003). Multiples bands protein molecular

mass such as 60, 90 100 kDa these proteins were higher expression under stress

conditions in both cultivars S2003-US-633 and SPF-238 but the level of expression

was higher in cultivar S2003-US-633 under heat stress conditions. This result

indicating that these molecular mass protein might also be significant in the response

of sugarcane crop to heat stress conditions. These differential expression protein

might be different types of heat shock proteins which expression was higher in heat

stress conditions. Under heat shock treatments, expression of heat shock or stress

proteins are significant adaptation to cope with abiotic stresses. Most of the heat

shock protein water soluble so contribution to stress resistance apparently through

hydration of cellular structure (Wahid and Close, 2007). Molecular weight of heat

shock proteins ranging from 10 kDa to 250 kDa, have chaperon like role and under

142

heat stress it is involved in signal transduction (Shinozaki, 1999), protect cellular

structure (Sanmiya et al., 2004) and protein folding (Cho and Choi, 2009) and act as

molecular chaperones (Li et al., 2013). Heat shock protein play important role in

conferring heat, drought and light stresses resistant in plants (Montero-Barrientos et

al., 2010). For example, in sugarcane overexpression of HSP70 heat stress protein

confers abiotic stress (Augustine et al., 2015). These HSPs support other large number

of proteins in stress condition to protect other protein and maintain the functions of

biomolecules in plant cell (Cho and Choi, 2009). These epigenetic alteration can

regulate the expression of important genes related to thermotolerance (Grativol et

al., 2012) and can activate the transcription of genes that respond to environmental

stress (Probst and Scheid, 2015); Rausell et al., 2003). Thermotolerant sugarcane

cultivars present a more abundance of protein involved in protein biosynthesis,

proposed that these proteins are significant in the response of sugarcane crop under

heat stress conditions (Pacheco et al., 2013).

5.7. Correlation

Results revealed that total sugar negatively associated with VIN and CWIN activities

at maturity stage this results agreement witw previous study a negative and

significant correlation between CyIN and SAI with sucrose accumulating (Siswoyo et

al., 2016) and maximum activity of acid invertase was correlated with low levels of

sucrose (Lontom et al., 2008; Pan et al., 2009). Present results indicating that

invertase activity was lowest in cultivar S2003-US-633 with high sucrose

accumulation and invertase was highest in cultivar SPF-238 with low sucrose

accumulation (Table 9). Invertase my play important role in sucrose accumulation in

143

sugarcane crop and also stated that invertase activity had negative association with

and significant in the accumulation of sucrose. Cell wall invertase increased at

maturity stage. Our results lined with (Botha et al., 1996). However, it is suggested

hydrolyze and resynthesize model for sucrose unloading and storage in sugarcane

stem (parenchyma) where sucrose is hydrolyzed in to hexose (glucose and fructose).

Incontrast, it is demonstrated that sucrose could be transported in sugarcane but did

not hydrolyzed and resynthesize played no role in sugarcane storage (Lingle, 1989).

CWIN was negatively correlated with sugar recovery and sucrose content in both

cultivars. Regarding sugar recovery rate, pol, °brix were showed strongly positive

association with sugar recovery at both growth stages. Same results also reported (Ali

et al., 2019). So, CWIN may be a good candidates for high sugar recovery rate and

sugar accumulation in sugarcane crop.

144

Key Findings

1. Morphological study showed that maximum nodes, tillers while minimum internodal

distance, stem diameter, fresh to dry weight ratio, shoot and root length were

observed in cultivar SPF-238 with compared to S2003-US-633. This proposed that

SPF-238 was susceptible cultivar.

2. Thermotolerant damage indicators studies revealed that maximum cell membrane

thermostability, hydrogen peroxide, MDA contents were accumulated under heat

stress treatments in SPF-238. However, maximum proline content was accumulated

in cultivar S2003-US-633. This higher accumulation of proline play a protective role

under heat stress suggested that cultivar S2003-US-633 has ability to cope any harsh

environmental stress conditions. But this character or ability was lack in SPF-238.

3. Sugar quantification revealed that higher total sugar and nonreducing sugar contents

were exhibited in cultivar S2003-US-633 while maximum reducing sugar content

found in SPF-238 under heat shock treatments. This higher concentration of reducing

sugar in cultivar SPF-238 may be sucrose cleavages into hexose sugar due to higher

expression of invertase in sugarcane culm at maturity stage resulting sugar recovery

rate loss.

4. Sugar recovery rate is positively associated with attributes with SPS, SS and total

sugar content while negatively associated with invertase isozymes and reducing

sugar.

5. Sugar recovery parameters study revealed that exposure of heat stress severely

effected on sugar recovery rate, pol and brix in both cultivars. Both the cultivars had

different thermotolerant potential. Maximum sugar recovery rate was observed in

145

cultivar S2003-US-633 at all treatments. This indicated that cultivar S2003-US-633 has

higher sink strength (less sucrose hydrolysis in stem at maturity stage) and less fiber

content in stem.

6. Different molecular mass protein bands (70, 90 and 100 kDa) may be heat shock

protein which involved under heat stress conditions in both cultivars. This heat shock

protein (HSPs) help to folding denatured proteins and provide cellular protection

against heat induced damages. This indicated that cultivar S2003-US-633 had better

performance under heat stress and upon recovery treatments, quick recovery rate

was exhibited as compared to SPF-238.

7. Native PAGE and zymography studies revealed that the differential expression of

invertase isozymes at various growth phases in both cultivars. Their expression or

activity to be growth stage specific. The vegetative stage of plant growth phase

appears to be more affected by heat shock as compared to other stages. Both

cultivars indicated three types of cytoplasmic invertase (134, 150 and 160 kDa) at

grand growth stage. Cultivar S2003-US-633 was better performance under heat stress

as compared to SFP-238.

8. Regarding cell wall invertase, multiple bands (140, 150 and 160 kDa) were observed

at vegetative and grand growth stages due to higher demand of sugars, juvenile cells

use this sugars as fuel for growth and development during growth phase. While

vacuolar invertase expression pattern was same in both cultivars at all growth stages.

146

Future Directions

Due to complex genome structure of sugarcane, marker assisted breeding, genetic

transformation and genome editing approaches require the complete information of

all heat stress tolerant mechanisms as well as sucrose metabolism enzymes

characterization particularly invertase isozymes for improved sugar recovery rate

and thermotolerance. Many commercial agriculture biotechnological companies

have been heavily invested in developing high yielding as well thermotolerant

commercial sugarcane cultivars. So there is urgent need to grow thermotolerant crop,

unfortunately there is limited thermotolerant varieties have been developed. For the

development of high yielding thermotolerant varieties, enzymes controls points

involved in sucrose metabolic pathway, degree of phloem loading and unloading and

rate of sucrose assimilation needs to be explore for increasing sucrose accumulation.

Engineering of sugar metabolizing enzymes through genetic transformation may lead

to the increased sucrose accumulation and sugar recovery rate. At this point

fundamental research plays vital role by providing molecular physiology of the plant

heat stress response and can speed up biotechnological modification of heat tolerant

traits.

Biotechnology intervention for vacuolar targeted expression of sucrose metabolizing

enzymes may not only depict enhanced sucrose transport but sink strength.

Post-transcriptional gene silencing can be done to target suppress the expression of

invertase and its isozymes in maturity stage for more accumulation of sucrose.

147

Conclusion

Environmental fluctuations are the major limitations for growth, yield and production

of sugarcane plant. The cultivar S2003-US-633 revealed to show thermotolerant

under heat stress than SPF-238. More osmolytes, sugars and total soluble protein

content were evident in S2003-US-633. While less amount of hydrogen peroxide,

MDA and EC content in cultivar S2003-US-633 suggesting that directly associated with

osmotic homeostasis in sugarcane during the exposure of heat stress. Heat shock

proteins (HSPs) and their differential expression in cultivar S2003-US-633 might be

directly play in maintenance of pant growth and development under heat stress. The

multiple molecular bands proteins and different enzymes (multiple types of invertase

isozymes) identified during heat stress and their linked biochemical pathways provide

new avenue regarding sugarcane improvement programme with respect to high

temperature. Cultivar S2003-US-633 is ranked in high yielding and high sugar

recovery rate variety due to high °brix, pol and total sugar content despite severe

environmental condition.

148

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A-Oral Presentations

1. Faisal Mehdi*, Kazim Ali, Nesheman Huma, Abid Azhar and Saddia Galani. Biochemical

analysis of high and low sucrose accumulation sugarcane varieties at formative stage

under heat stress. Dynamic Trends in Plant Sciences: Fostering Environment and Food

Security, May 9-11, 2017. Organized by Pakistan Botanical Society (PBS) at Sardar

Bahadur Khan Women's University (SBK), Quetta, Pakistan.

2. Faisal Mehdi*, Kazim Ali, Nesheman Huma, Abid Azhar and Saddia Galani.

Quantitative and qualitative analysis of invertase isozymes regulating sucrose mechanism

in heat shock sugarcane. “Molecular biosciences: research and innovations” Fourteenth

Biennial Conference of Pakistan Society for Biochemistry and Molecular Biology (PSBMB),

December 9-12, 2018. Organized by Pakistan Society for Biochemistry and Molecular

Biology (PSBMB) at KIBGE University of Karachi.

B-Publication

1. Faisal Mehdi*, Kazim Ali, Nesheman Huma, Iqbal Hussain, Abid Azhar and Saddia

Galani (2019). Comparative biochemical analysis of high and low sucrose accumulating

sugarcane varieties at formative stage under heat stress. Accepted to be published in the

Journal of Agricultural Sciences for issue 2020/Vol 26/Issue 1.

C-Poster Presentation

1. Faisal Mehdi1*, Shaghufta Sahar, Maleeha Akbar, Abid Azhar and Saddia Galani.

“Recent Innovations in Molecular Sciences”. The Conference organized by the

University of the Punjab (Quaid-e-Azam Campus), Lahore during November 06-08,

2019.

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