109

http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 109-119© TÜBİTAKdoi:10.3906/biy-1501-100

Overexpression of an H+-PPase gene from Arabidopsis in sugarcane improvesdrought tolerance, plant growth, and photosynthetic responses

Ghulam RAZA1, Kazim ALI1, Muhammad Yasin ASHRAF2,*, Shahid MANSOOR1, Muhammad JAVID3, Shaheen ASAD11National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

2Nuclear Institute of Agriculture and Biology, Faisalabad, Pakistan3Melbourne School of Land and Environment, University of Melbourne, Parkville, Victoria, Australia

* Correspondence: niabmyashraf@gmail.com

1. IntroductionAbiotic stresses are major constrains to sustainable agricultural food productivity worldwide, which needs to be doubled by the year 2050 to meet the ever-growing demands of population. Boyer (1982) reported that environmental factors may limit crop production by as much as 70%. According to FAO (http://www.fao.org/docrep/010/a1075e/a1075e00.htm) 96.5% of the global land area is affected by some environmental constraints. Among the environmental constraints, water deficit and salinity are the major limiting factors for crop productivity. These two environmental stresses affect more than 10% of arable land and cause yield losses of more than 50% in most major field crops (Bartels and Sunkar, 2005). Due to these environmental constraints and world population growth, increasing food and cash crop production has become a top priority. Therefore, it is of strategic significance to develop crop varieties that are stress-tolerant, especially against drought and salt.

Sugarcane (Saccharum officinarum L.) has great economic importance due to its application in the food industry. About 60% of the total world sugar requirements

are being fulfilled from this crop, and it is valuable in the production of ethanol, a less polluting renewable biofuel (Banschbach and Letovsky, 2010). In sugarcane, drought conditions reduce Brix content (% sugar/sucrose g/100 mL of juice), resulting in hindered sugar production due to its glycophytic nature and affecting its growth and cane yield, as well as sucrose quality and contents (Koyro et al., 2012). Drought stress can reduce the sugarcane crop yield up to 50% or more depending upon the drought severity and growth stage of the crop (Hussain et al., 2004; Bloch et al., 2006), and the situation even becomes critical in countries where sugarcane is only grown in irrigated areas.

Plants either tolerate or avoid stress by going through an array of phenological, physiological, and biochemical adjustments (Khan and Beena, 2002; Çulha Erdal and Çakırlar, 2014). Plants with both tolerance and avoidance mechanism(s) or with either of these two mechanisms can survive under drought conditions (Alpert, 2000; Otte, 2001). These mechanism(s) of drought tolerance could be engineered in plants through transgenic approaches. For instance, one way to engineer drought tolerance in plants is to increase solute concentration in the vacuoles

Abstract: This study investigated the integration of Arabidopsis vacuolar H+-pyro-phosphatase (H+-PPase) (AVP1) transgene in transgenic sugarcane plants for drought tolerance. The gene integration was confirmed by polymerase chain reaction (PCR) and Southern blotting. Transgene expression was estimated by Western blotting. When sugarcane plants were grown on soil under 50% reduced water supply, the transgenic sugarcane overexpressing AVP1 produced higher shoot biomass based on cane height, number of millable canes, and Brix (%) compared with wild-type. The overexpression of AVP1 in transgenic sugarcane plants increased tolerance to drought stress, as demonstrated by increased relative water content (RWC) and leaf water (Ψw), osmotic (Ψs), and turgor potential (Ψp). Physiological parameters such as photosynthetic rate (Pn), stomatal conductance (C), and transpiration rate (E) were less affected by water-deficit stress in transgenic AVP1 plants compared with wild-type plants. In conclusion, our results indicated that AVP1 conferred tolerance to drought or water-deficit stress, highlighting potential use of this gene for crop improvement through biotechnological applications.

Key words: Transgenic sugarcane, drought tolerance, AVP1, overexpression, biolistic method, photosynthetic response

Received: 02.02.2015 Accepted/Published Online: 22.05.2015 Final Version: 05.01.2016

Research Article

RAZA et al. / Turk J Biol

110

of plant cells to facilitate osmotic regulation. The increased vascular osmotic pressure associated with a decrease in water potential would favor water movement from soil into plant roots. As a result, maintenance of ion gradient during water stress is very important, because normally cells expend 50% of intracellular energy to maintain the ion gradient across the membranes which regulate several important biological functions (Nelson, 1994). Proton electro chemical gradient (PEG) also facilitates the energy required for the transport of ions across the cell membranes (Sze et al., 1999; Gaxiola et al., 2002).

The generation and utilization of PEG in plants are accomplished by membrane transport proteins (primary and secondary transporters), which represent approximately 5% of the Arabidopsis genome (Mäser et al., 2001). The Arabidopsis H+-PPase is a vacuolar membrane protein that acidifies vacuoles in plant cells by pumping H+ from the cytoplasm into vacuoles with inorganic pyrophosphate (PPi) dependent on H+ transport activity; this could be used to increase solute content in the vacuoles. The level of H+-PPase is upregulated under abiotic conditions such as drought, salt, mineral deficiency, anoxia, and/or cold stress (Carystinos et al., 1995; Maeshima, 2000).

In a number of previous studies, overexpression of H+-PPase gene (AVP1) of Arabidopsis has been shown to enhance drought and/or salt tolerance in transgenic Arabidopsis thaliana (Gaxiola et al., 2002), tomato (Park et al., 2005), rice (Zhao et al., 2006), tobacco (Ibrahim et al., 2009), alfalfa (Bao et al., 2009), cotton (Asad et al., 2008; Pasapula et al., 2011; Shen et al., 2015), peanut (Qin et al., 2013), and barley (Schilling et al., 2014). The AVP1 overexpressing in Arabidopsis greatly enhanced root development by facilitating recovery under water-deficit conditions (Li et al., 2005). The established potential of AVP1 prompted us to widen the effort to develop transgenic sugarcane for drought tolerance. In the current study, we have successfully developed a transgenic sugarcane line overexpressing the full-length genomic AVP1 gene derived from Arabidopsis. We further demonstrated that the resulting transgenic line was more tolerant to water-deficit stress than wild type.

2. Materials and methods2.1. Plant material and genetic transformation of sugarcaneSugarcane cultivar CSSG-668 sourced from the Shakarganj Sugarcane Research Institute (SSRI), Jhang, Pakistan, was used for genetic transformation. The cultivar CSSG-668 was selected based on its good tissue culture responses (Raza et al., 2010; Ali et al., 2015), better yield and quality components, and sensitivity to water deficient conditions (personal communication with the breeder). The plasmid

pZSI (Figure 1) harboring AVP1-H+ full-length genomic clone under regulatory control of 35S promoter with double enhancers (2 × 35S) of the cauliflower mosaic virus and polyadenylation region from the nopaline synthase terminator, were used to transform the genotype CSSG-668 through a biolistic mediated transformation method. Following the method of Raza et al. (2010), transformation protocols including particle gun bombardment, tissue culture, selection, and plant regeneration were performed. The apical discs were retrieved and cultured on callus induction medium (CIM; see Appendix S1) and kept in the dark at 26 °C for 4 weeks. Three days before bombardment, the induced embryogenic calli were picked and arranged in the center of petri plates. The plasmid DNA (1 µg µL–1) was precipitated onto gold particles (3 mg) and bombarded using a particle delivery system (PDS-1000/He, Bio-Rad Laboratories, USA) following the manufacturer’s instructions. The bombarded and unbombarded (control) embryogenic calli were then cultured on fresh CIM and placed in the dark at 26 °C for 3 days. Transgenic calli were selected by culturing on CIM supplemented with 60 mg L–1 Geneticin (G-418, Sigma-Aldrich). After 1 month of vigorous selection, the surviving green calli were shifted to regeneration selection medium (RSM; see Appendix S2), and the plates were placed in the dark at 26 °C. Regenerated plantlets (T0) were placed on rooting selection medium (RTSM; see Appendix S3) in sterilized tissue culture jars. Rooted plantlets were transferred into plastic pots containing peat moss and kept in a growth room for hardening at 26 °C under a 16/8 h light/dark period for 6 weeks and then subjected to further analysis.2.2. Genotyping of transgenic plantsTotal genomic DNA was extracted from fresh leaf material of putative transgenic plants by the CTAB method (Richards et al., 2001). Total genomic DNA was used for PCR amplification of the internal fragment (600 bps) of AVP1 gene using forward (5’-GCAATCTTCATCCCAAGGAA-3’) and reverse (5’-AGCACCAGACCTGAATGCAAAAATGAACGC-3’) primers (Koressaar and Remm, 2007). For Southern blotting, 20 µg of genomic DNA of transgenic and wild-type sugarcane plants were digested overnight with Eco R1 enzyme. The digested samples were then size-fractionated overnight by electrophoresis on 0.8% agarose gel under low voltage. The remaining Southern steps were done following the procedure of Bower and Birch (1992) in sugarcane. To use as a probe, the purified internal fragment (600 bps) of AVP1 gene was labeled using a Rad-Primed labeling kit (GIBCO-BRL, USA) following the manufacturer’s instructions. 2.3. Protein isolation and Western analysisFollowing methods of Gaxiola et al. (2001) and Pasapula et al. (2011), the protein was isolated from the homogenates

RAZA et al. / Turk J Biol

111

of plant-stressed leaves of the selected transgenic sugarcane line (T1). The isolated proteins were dissolved in Laemmli buffer (4% SDS, 20% glycerol, 0.125 M Tris-HCl, and pH 6.8) at 95 °C for 5 min and placed in a boiling water bath. An equal amount of protein, i.e. 100 µg, was separated from selected transgenic lines and wild type by 10% SDS-PAGE and immune-blotted with antibodies raised against keyhole limpet hemocyanin (KLH)-conjugated synthetic peptide derived from Arabidopsis V-PPase (AS121849, Agrisera, Sweden). The AVP1 protein was detected by chromogenic Western blot immuno-detection kit (Invitrogen, USA), following the manufacturer’s instructions.2.4. Water deficit experimentThe transgenic plants confirmed through PCR and Southern analyses were shifted into large earthen pots containing soil and placed in a glasshouse for 8–10 months. When plants were mature, the cane sets (T1) from transgenic plants showing better growth and plant height were retrieved. These cane sets were planted for 1 more year in a micro-plot for multiplication so that enough cane sets could be obtained for the water-stress experiment. After 1 year, cane sets of wild type and the selected transgenic line (SP-27) were planted in three replicates at 5 cm depth in micro-plots (12’ × 4’ × 1.5’) containing homogeneous soil (ECe 1.34, SAR 2.72, and pH 7.8). Water-deficit stress (50% reduced irrigation) was initiated after 2 months (60 days) of cane set germination and continued for 6 months (180 days). Data for shoot and root biomass, yield components (stalk height, millable cane number, Brix %), and physiological parameters were recorded before harvesting the transgenic and wild-type sugarcane plants.

Water relation and gas exchange parameters were measured after 180 days of water-deficit stress. For RWC estimation, the youngest leaf was taken from five plants. Leaves were sampled at midday in a 50 mL falcon tube from transgenic and nontransgenic plants, and fresh weights were immediately recorded. Leaves were then soaked overnight in distilled water, dried on blotting paper, and turgid weights were recorded. Leaves were oven dried at 70 °C for 72 h, and dry weights were measured. RWC percentage was calculated using the following equation:

(Fresh weight – dry weight)RWC (%) = ×100. (Turgid weight – dry weight)

For leaf water potential (Ψw) measurement, the third fully expanded leaf from the top of each plant was excised. The measurements were made between 0830 and 1030 with a Scholander type pressure chamber (Skye Instruments, Llandrindod Wells, UK). For osmotic potential (Ψs) measurement, each leaf used for water

potential was frozen at –20 °C in a 1.5 mL tube. Then the frozen leaf was thawed, and the cell sap was extracted with a clean glass rod. The extracted sap was used to measure the osmotic potential with the calibrated Osmometer Wescor-5500 (Logan, UT, USA). The turgor potential (Ψp) was calculated as the difference between osmotic potential (Ψs) and water potential (Ψw):

(Ψp) = Leaf water potential (Ψw) – osmotic potential (Ψs).

Gas exchange parameters such as photosynthetic rate (Pn), stomatal conductance (C), and transpiration rate (E) were measured within 1 h of solar noon with an infrared gas-exchange analyzer portable photosynthetic system (LI-COR 6400, Inc., Lincoln, NE, USA). Gas exchange measurements were determined on the uppermost fully-expanded leaves directly exposed to solar radiation. Gas exchange measurements were performed on days with minimal-to-absent cloud cover and at times of maximal solar radiation (approximately between 1000 and 1200). 2.5. Statistical analysisAll experiments contained three replicates, and observations were recorded from each replicate. Growth and physiological trait data were analyzed by two-way analysis of variance (P ≤ 0.05) using Statistix 8.1 software.

3. Results3.1. Generation and molecular identification of transgenic sugarcane plantsSugarcane ‘CSSG-668’ was successfully transformed with pZS1 construct (Figure 1) using a biolistic mediated transformation method. From 12 independent transformation batches, five lines with enhanced drought tolerance were identified, and three of these lines (SP27, SP34, and SP58) are described here. The steps involved in generation of transgenic sugarcane plants are detailed in Figures 2a–2i.

The PCR results (Figures 3a–3c) indicated that the target AVP1 fragment (600 bps) was found in representative putative transgenic sugarcane lines and in the positive control (plasmid), while there was no amplification in the wild-type plant (Figure 3a). The PCR-positive transgenic lines showed better growth behavior at T0 stage (regenerated plants from bombarded calli, matured in glasshouse) compared with wild type. Transgenic plants with phenotypic similarities to wild type and better growth during T0 stage were selected for a further micro-plot experiment. Stable integration of transgene AVP1 was confirmed through Southern blot hybridization, which showed multiple transgene copy numbers in the genome of the examined transgenic sugarcane lines at T1 stage (Figure 3b). Western blot analysis further confirmed the presence of AVP1 protein (67 kDa) in the examined

RAZA et al. / Turk J Biol

112

Figure 1. Schematic diagram of plasmid pZSI used for genetic transformation and double enhancer of 35S promoter cauliflower mosaic virus promoter (2 × 35S); Arabidopsis vacuolar pyrophosphatase genomic clone (AVP1); cauliflower mosaic virus terminator sequence (CaMVT); nopaline synthase promoter sequence (Pnos); neomycin phosphotransferase gene II (nptII); nopaline synthase terminator sequence (Tnos).

a cb

d e

g

f

h i

Figure 2. Tissue culture steps involved in generation of transgenic sugarcane; a) embryogenic calli ready for bombardment; b) Geneticin selection of transformed calli; c) nontransformed calli (control) on selection medium (60 mg L–1 Geneticin); d) regeneration from surviving calli under selection (60 mg L–1 Geneticin); e) regenerated shoots placed on rooting selection medium; f) putative transgenic plants transplanted into pots of sandy soil medium for hardening.

RAZA et al. / Turk J Biol

113

transgenic lines under water-stress conditions. The AVP1 expression varied in examined lines (Figure 3c).3.2. Transgenic plants with enhanced AVP1 expression are drought tolerantThree AVP1 transgenic lines of sugarcane (SP27, SP34, and SP58) were analyzed for growth and productivity (Figures 3a–3d). The transgenic sugarcane lines overexpressing AVP1 showed more drought tolerance than the wild-type plants. As described earlier (in Materials and methods), water-deficit stress under irrigation reduced by 50% was initiated after 2 months (60 days) of cane set germination and continued for 6 months (180 days). Shoot and root biomass of the transgenic lines SP27, SP34, SP58, increased significantly (P ≤ 0.05) by 83%, 68%, and 79% and 93%, 73%, and 87%, respectively, relative to the control wild-type plants (Figures 4a and 4b).

The AVP1 overexpressed transgenic sugarcane showed significantly enhanced agronomic traits such as cane height, number of millable canes, and Brix (%) under water-deficit stress. Micro-field trial results showed that cane height in AVP1 overexpressing transgenic lines SP27, SP34, and SP58 increased significantly (P ≤ 0.05) by 51%, 36%, and 43%, respectively, relative to wild type under water-deficit stress (Figure 4c). Similarly, number of millable canes increased by 30% in transgenic line SP27 and 23% in both SP34 and SP58 relative to wild type (Figure 4d).

Brix is an important parameter which has a direct link with sucrose content in cane sugar. Therefore, the effect of

drought on this parameter provides necessary information about sugar recovery, the ultimate goal of sugarcane cultivation. In all transgenic lines, Brix (%) in water-stress conditions was significantly greater: 27% in SP34 followed by 15.5% and 12.5% in SP27 and SP58, respectively (Figure 5).

AVP1 overexpressed transgenic sugarcane possessed greater water holding capacity than wild type in water-stress conditions.

Transgenic plants showed significantly (P ≤ 0.05) better water relations postwater stress than wild type (Figures 6a–6d). The transgenic line SP27 retained the most RWC to 81% followed by 77% in SP34 and SP58, respectively; RWC in wild type was significantly lower (71%; Figure 6a). Furthermore, values of Ψw for wild type were less negative (–1.13 MPa) than the –1.24 and –1.29 MPa registered in transgenic lines SP27 and SP58, respectively (Figure 6d). AVP1 overexpressed transgenic sugarcane showed better osmotic and turgid potential than wild type in water-stress conditions.

Leaf osmotic potential (Ψs) refers to the amount of solutes dissolved in water, and this was assayed in transgenic and wild type. Osmotic potential in wild type is less negative (–1.87 MPa) than in transgenic lines SP34 (–2.12 MPa), SP58 (–2.11 MPa), and SP27 (–2.09 MPa; Figure 6c). Maintenance of turgor is one of the important adaptations to drought stress and was evaluated by measuring the turgor potential (Ψp). The transgenic lines SP58, SP34, and SP27 showed 1.02, 1.01, and 0.85 MPa increased turgor

Figure 3. Molecular analysis of transgenic and wild-type sugarcane plants; a) genotyping of partial AVP1 gene fragment (600 bps) in transgenic and wild-type (WT) sugarcane plants; b) Southern blot analysis of transgenic and wild-type (WT) sugarcane plants; c) Western blotting of transgenic and wild-type (WT) sugarcane plants in water-stress conditions, where L is 1 kb DNA ladder, and P is positive plasmid.

RAZA et al. / Turk J Biol

114

potential (Ψp), respectively, compared with 0.74 MPa in wild type (Figure 6b). AVP1 overexpressed transgenic sugarcane lines showed less damage to photosynthetic machinery than wild type under limited water conditions.

In order to evaluate this, photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (C) were measured before harvesting the sugarcane lines after water stress treatment (Figures 7a–7d). Transgenic line SP27 overexpressing AVP1 showed significantly (P ≤ 0.05) higher Pn (18 µmol m

–2 s–1), followed by 12 and 11 µmol m–2

s–1 in SP34 and SP58, respectively, relative to Pn of wild type (7 µmol m–2 s–1) (Figure 7a). The stomatal conductance (C) in transgenic line SP34 increased to a maximum of 11.85 mmol m–2 s–1, closely followed by 9.84 and 8.57 mmol m–2 s–1 in SP34 and SP27, respectively; in wild type it was 6.34 mmol m–2 s–1 (Figure 7b). The transpiration rate (E) in SP27 increased to 1.05 mmol m–2 s–1 followed by 0.97 mmol m–2 s–1 in SP34 and SP58, whereas wild type showed a relatively low (0.54 mmol m–2 s–1) transpiration rate (Figure 7c).

a

b b b

0

50

100

150

200

250

Shoo

t bio

mas

s (g

plan

t–1 )

a

bb

b

0

5

10

15

20

25

30

Root

bio

mas

s (g

plan

t–1)

a

b b b

0

25

50

75

100

125

150

Stal

k he

ight

(cm

pla

nt–1

)

b

a

ba

0

2.5

5

7.5

Control SP27 SP34 SP58

Mill

able

cane

num

ber (

plan

t–1)

a

b

c

d

Figure 4. Growth responses of wild-type and the AVP1 transgenic sugarcane plants at harvesting stage after applying limited water-stress conditions; a) shoot biomass (g); b) root biomass; c) cane height (cm); d) number of millable canes. Values are means ± SE (n = 6), and bars with a different letter are significantly different at P ≤ 0.05.

RAZA et al. / Turk J Biol

115

ab

cb

0

5

10

15

20

Control SP27 SP34 SP58

Brix

(%)

Figure 5. Comparison of total soluble salts in transgenic and wild-type sugarcane lines. Values are means ± SE (n = 6), and bars with a different letter are significantly different at P ≤ 0.05.

a

c

b b

70

75

80

85

RWC

(%)

ab

c c

0

0.5

1

Turg

or p

oten

tial (

MPa

)

ab b b

0

0.5

1

1.5

2

2.5

Osm

otic

pot

entia

l (-M

Pa)

ab

ab

0

0.5

1

1.5

Control SP27 SP34 SP58

Leaf

wat

er p

oten

tial (

-MPa

)

d

c

b

a

Figure 6. Responses of wild-type and the AVP1 transgenic sugarcane plants after 180 days under limited water-stress conditions; a) relative water content (%); b) leaf water potential (-MPa); c) osmotic potential (-MPa); d) turgor potential (-MPa). Values are means ± SE (n = 6), and bars with a different letter are significantly different at P ≤ 0.05.

RAZA et al. / Turk J Biol

116

4. Discussion Plant biotechnology has shown a profound impact on agricultural productivity through crop improvement. In recent years, this technology has been extensively used to improve agronomically important crops against abiotic stresses. Among these stresses, drought is one of the major causes of reduced crop productivity and is prevalent in crops with high water requirements such as sugarcane. In the present study, we successfully developed drought tolerant transgenic sugarcane plants by overexpressing the H+-PPase of A. thaliana (AVP1 gene). The AVP1 gene incorporation and overexpression was confirmed through PCR, Southern, and Western analyses (Figures 3a–3c). The AVP1 overexpressed transgenic sugarcane lines efficiently avoided water-deficit stress compared with wild type, which did not show AVP1 expression (Figure 3c).

The enhanced drought tolerance in the transgenic line compared with wild type was reflected in the increased shoot and root biomass and better agronomic sugarcane traits such as cane height, number of millable canes, and Brix (%), which are directly linked to cane sugar and yield (Landell and Silva, 2004) (Figures 4a–4c and Figure 5). Previously, studies have shown that transgenics overexpressing AVP1 in Arabidopsis (Gaxiola et al., 2001) and cotton (Pasapula et al., 2011) contributed to better plant growth compared with nontransgenic plants. In the current study, enhanced biomass (e.g., cane height and millable cane number) in transgenic plants compared with wild type under water-deficit stress, was aided by increased retention of water and solute accumulation and efficient photosynthetic activity (Figures 6a–6d and 7a–7d). The higher water retention in transgenic plants

a

c

bb

0

5

10

15

20

25

Phot

osyn

thet

ic ra

te

(µm

ol m

–2s–

1 )

a

bb

c

0

5

10

15

Stom

atal

cond

ucta

nce

(m m

ol m

–2 s–

1 ) (m

mol

m–2

s–1 )

a

cb b

0

0.5

1

Control SP27 SP34 SP58

Tran

spira

tion

rate

a

b

c

Figure 7. Responses of wild-type and the AVP1 transgenic sugarcane plants after 180 days under limited water-stress conditions; a) photosynthetic rate (µmol m–2s–1); b) transpiration rate (mmol/m2s–1); c) stomatal conductance (mmol m–2 s–1). Values are means ± SE (n = 6), and bars with a different letter are significantly different at P ≤ 0.05.

RAZA et al. / Turk J Biol

117

was presumably due to better root growth and solute accumulation (Gaxiola et al., 2001). Similar responses have previously been observed in overexpressing transgenic Arabidopsis (Gaxiola et al., 2001) and alfalfa (Bao et al., 2009) plants under drought conditions.

Root growth is an important parameter to assess the capability of plants to avoid stress. Results of this study indicated that AVP1 overexpressing transgenic sugarcane lines SP27, SP34, and SP58 showed significantly better root growth compared with wild type under water-deficit stress. It might be due to enhanced stimulation of auxin polar transport in root system of AVP1 transgenic sugarcane plants (Li et al., 2005), which increased the plant’s ability to capture water and adapt to drought conditions. Therefore, similar mechanism(s) may have been operating in transgenic sugarcane as a tolerance response to water-deficit stress.

Under water-deficit stress conditions, significantly lower values in Brix (%) were noted in wild type compared with AVP1 overexpressed transgenic plants (Figure 5). This could be due to impaired photosynthetic processes found in wild-type plants (Figure 7a). Previously, the work by Lv et al. (2009) showed increased total sugar and amino acid content in transgenic lines overexpressing H+-PPase (TsVP) under drought stress. Thus, an increase in Brix (%) in transgenic lines could reflect better sugar recovery under drought/water-deficit stress, which is pivotal for sugarcane cultivation.

Stomatal conductance is directly linked to a reduction in RWC in plants facing water-deficit stress, as foliar transpiration is controlled by stomatal opening and closure (Taiz and Zeiger, 2006). In our study, RWC was reduced by 10% in wild-type sugarcane plants compared to the AVP1 overexpressing transgenic sugarcane, which clearly indicated a reduction in stomatal conductance (Figure 7b) due to reduced water absorption (Lopez-Climent et al., 2008).

Lowering of RWC, Ψw, and Ψs is common in almost all plant species (Stoyanov, 2005; Amini et al., 2014). However, plants having the genetic potential to cope with water stress are successful in maintaining higher RWC by osmotic adjustment. During osmotic adjustment heavy molecules of organic compounds are broken into smaller and soluble compounds (proline, betaine, free amino acids, polyols, and organic acids), resulting in the lowering of Ψw and Ψs (Hussain et al., 2010). This creates osmotic pull in roots for water absorption (Shahid et al., 2013) from deeper soil, and the plants were successful in growing under stress. In the present case, AVP1 overexpressed transgenic plants acquired this genetic potential and adjusted themselves osmotically.

Our results demonstrated that AVP1 overexpressing transgenic sugarcane plants showed greater decreased Ψw

and Ψs, and increased turgor pressure than wild type under water-deficit stress (Figures 6a–6d). Osmotic adjustments are contributed to by accumulation of organic or inorganic solutes within the cell which helps maintain turgor under drought stress (Ashraf et al., 1994). Furthermore, increased solute accumulation and osmo-regulatory capacity of the transgenic sugarcane plant may have been aided by better osmotic adjustment and turgor pressure (Bao et al., 2009; Koyro et al., 2012). In contrast, a decrease in Ψw, as found in wild-type sugarcane plants, was related to a reduction in the plant photosynthesis rate (Figure 7a), indicating that during water-deficit stress less carbon assimilation may have occurred in wild-type compared with transgenic sugarcane plants. Hence, osmotic adjustment could be one of the important processes in the adaptation of transgenic plants to drought/water-deficit stress. A positive relationship found among cane height, millable cane, and turgor potential (Figures 5a–5d) further indicated that the AVP1 overexpressed transgenic sugarcane showed better tolerance to a water-deficit regime compared to wild type.

The AVP1 overexpressing transgenic sugarcane plants maintained a higher photosynthetic rate and stomatal conductance and better transpiration rate than wild type in water-deficit stress (Figures 7a–7d). A change in stomatal resistance may have a greater effect on transpiration than on photosynthesis, because it constitutes a larger proportion of the total resistance to water vapor diffusion (Singh et al., 2014). However, the higher photosynthetic rate is one of the major factors contributing to better productivity, because it facilitates the raw material and the energy required for growth and development, as measured in AVP1 overexpressing transgenic plants compared with wild-type sugarcane under water-deficit stress.

In conclusion, our data demonstrated that the overexpression of Arabidopsis AVP1 gene in sugarcane could be one of the successful strategies to enhance drought tolerance in this economically important crop. Significantly improved growth and physiological parameters, such as photosynthesis rate, stomatal conductance, RWC, osmotic, and turgor potentials in transgenic plants, compared to wild type under water-deficit stress, clearly indicated the putative functional role of AVP1 gene for drought tolerance. However, further research is needed to elucidate the performance of transgenic sugarcane plants under field conditions.

AcknowledgmentsWe are thankful to Higher Education Pakistan (HEC) and the Punjab Agriculture Research Board (PARB) of Pakistan for providing funds to carry out the current research. We are highly obliged to the Sugarcane Research Institute (SRI), Faisalabad and the Shakarganj Sugarcane

RAZA et al. / Turk J Biol

118

Research Institute (SSRI), Jhang for providing the explants to conduct the experiment. Furthermore, we are thankful to Dr Zahid Mukhtar, NIBGE, Faisalabad, for providing

AVP1 genomic clone construct and Mr Muhammad Arshad, NIBGE, Faisalabad, for his technical expertise on initial experimental set-up.

References

Ali K, Raza G, Mukhtar Z, Mansoor S, Asad S (2015). Ideal in-vitro culture and selection conditions for sugarcane genetic transformation. J Agri Sci 52: 43–49.

Alpert P (2000). The discovery, scope, and puzzle of desiccation tolerance in plants. Plant Ecol 151: 5–17.

Amini H, Arzani A, Karami M (2014). Effect of water deficiency on seed quality and physiological traits of different safflower genotypes. Turk J Biol 38: 271–282.

Asad S, Mukhtar Z, Nazir F, Hashmi JA, Mansoor S, Zafar Y, Arshad M (2008). Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants. Molecular Biotechnology 40: 161–169.

Ashraf MY, Azmi A, Khan A, Naqvi S (1994). Water relations in different wheat (Triticum aestivum L.) genotypes in water deficits. Acta Physiol Plant 16: 231–240.

Banschbach VS, Letovsky R (2010). The use of corn versus sugarcane to produce ethanol fuel: a fermentation experiment for environmental studies. The American Biology Teacher 72: 31–36.

Bao A, Wang S, Wu G, Xi J, Zhang J, Wang C (2009). Over-expression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Sci 76: 232–240.

Bartels D, Sunkar R (2005). Drought and salt tolerance in plants. Critical Rev Plant Sci 24: 23–58.

Bloch D, Hoffmann CM, Märländer B (2006). Impact of water supply on photosynthesis, water use and carbon isotope discrimination of sugar beet genotypes. European J Agron 24: 218–225.

Bower R, Birch R (1992). Transgenic sugarcane plants via micro-projectile bombardment. The Plant J 2: 409–416.

Boyer JS (1982). Plant productivity and environment. Science 218: 443–448.

Carystinos GD, MacDonald HR, Monroy AF, Dhindsa RS, Poole RJ (1995). Vacuolar H+-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiol 108: 641–649.

Çulha Erdal Ş, Çakırlar H (2014). Impact of salt stress on photosystem II efficiency and antioxidant enzyme activities of safflower (Carthamus tinctorius L.) cultivars. Turk J Biol 38: 549–560.

Gaxiola R, Li J, Undurraga S, Dang L, Allen G, Alper S, Fink G (2001). Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. P Natl Acad Sci USA 98: 11444–11449.

Gaxiola RA, Fink GR, Hirschi KD (2002). Genetic manipulation of vacuolar proton pumps and transporters. Plant Physiol 129: 967–973.

Hussain A, Khan ZI, Ghafoor MY, Ashraf M, Parveen R, Rashid MH (2004). Sugarcane, sugar metabolism and some abiotic stresses. Intl J Agric Biol 6: 732–742.

Hussain S, Saleem MF, Ashraf MY, Cheema MA, HAQ MA (2010). Abscisic acid, a stress hormone helps in improving water relations and yield of sunflower (Helianthus annuus L.) hybrids under drought. Pak J Bot 42: 2177–1289.

Ibrahim M, Khan SA, Zafar Y, Mansoor S, Arsalan Y, Mukhtar Z (2009). Expression of a full length Arabidopsis vacuolar H+-pyrophosphatase (AVP1) gene in tobacco (Nicotiana tabbacum) to increase tolerance to drought and salt stresses. Intl J Phytol 1: 433–440.

Khan MA, Beena N (2002). Seasonal variation in water relations of desert shrubs from Karachi, Pakistan. Pak J Bot 34: 329–340.

Koyro H-W, Ahmad P, Geissler N (2012). Abiotic stress responses in plants: an overview. In: Ahmad P, Prasad MNV, editors. Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change. New York, NY, USA: Springer, pp. 1–28.

Landell M, Silva M (2004). As estratégias de seleção da cana em desenvolvimento no Brasil. Visão Agrícola 1.

Li J, Yang H, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL (2005). Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 310: 121–125.

Lopez-Climent MF, Arbona V, Pérez-Clemente RM, Gómez-Cadenas A (2008). Relationship between salt tolerance and photosynthetic machinery performance in citrus. Environ Expt Bot 62: 176–184.

Lv S-L, Lian L-J, Tao P-L, Li Z-X, Zhang K-W, Zhang J-R (2009). Overexpression of Thellungiella halophila H+-PPase (TsVP) in cotton enhances drought stress resistance of plants. Planta 229: 899–910.

Maeshima M (2000). Vacuolar H+-pyrophosphatase. Biochimica et Biophysica Acta (BBA)-Biomembranes 1465: 37–51.

Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis FJ, Sanders D (2001). Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol 126: 1646–1667.

Nelson N (1994). Energizing porters by proton-motive force. J Exptl Biol 196: 7–13.

Otte ML (2001). What is stress to a wetland plant? Environ Exptl Bot 46: 195–202.

http://dx.doi.org/10.1023/A:1026513800380http://dx.doi.org/10.1023/A:1026513800380http://dx.doi.org/10.3906/biy-1308-22http://dx.doi.org/10.3906/biy-1308-22http://dx.doi.org/10.3906/biy-1308-22http://dx.doi.org/10.1007/s12033-008-9072-5http://dx.doi.org/10.1007/s12033-008-9072-5http://dx.doi.org/10.1007/s12033-008-9072-5http://dx.doi.org/10.1007/s12033-008-9072-5http://dx.doi.org/10.1007/s12033-008-9072-5http://dx.doi.org/10.1525/abt.2010.72.1.8http://dx.doi.org/10.1525/abt.2010.72.1.8http://dx.doi.org/10.1525/abt.2010.72.1.8http://dx.doi.org/10.1525/abt.2010.72.1.8http://dx.doi.org/10.1080/07352680590910410http://dx.doi.org/10.1080/07352680590910410http://dx.doi.org/10.1016/j.eja.2005.08.004http://dx.doi.org/10.1016/j.eja.2005.08.004http://dx.doi.org/10.1016/j.eja.2005.08.004http://dx.doi.org/10.1016/j.eja.2005.08.004http://dx.doi.org/10.1111/j.1365-313X.1992.00409.xhttp://dx.doi.org/10.1111/j.1365-313X.1992.00409.xhttp://dx.doi.org/10.1126/science.218.4571.443http://dx.doi.org/10.1126/science.218.4571.443http://dx.doi.org/10.1104/pp.108.2.641http://dx.doi.org/10.1104/pp.108.2.641http://dx.doi.org/10.1104/pp.108.2.641http://dx.doi.org/10.1104/pp.108.2.641http://dx.doi.org/10.3906/biy-1401-33http://dx.doi.org/10.3906/biy-1401-33http://dx.doi.org/10.3906/biy-1401-33http://dx.doi.org/10.1073/pnas.191389398http://dx.doi.org/10.1073/pnas.191389398http://dx.doi.org/10.1073/pnas.191389398http://dx.doi.org/10.1104/pp.020009http://dx.doi.org/10.1104/pp.020009http://dx.doi.org/10.1104/pp.020009http://dx.doi.org/10.1126/science.1115711http://dx.doi.org/10.1126/science.1115711http://dx.doi.org/10.1126/science.1115711http://dx.doi.org/10.1126/science.1115711http://dx.doi.org/10.1016/j.envexpbot.2007.08.002http://dx.doi.org/10.1016/j.envexpbot.2007.08.002http://dx.doi.org/10.1016/j.envexpbot.2007.08.002http://dx.doi.org/10.1016/j.envexpbot.2007.08.002http://dx.doi.org/10.1007/s00425-008-0880-4http://dx.doi.org/10.1007/s00425-008-0880-4http://dx.doi.org/10.1007/s00425-008-0880-4http://dx.doi.org/10.1007/s00425-008-0880-4http://dx.doi.org/10.1016/S0005-2736(00)00130-9http://dx.doi.org/10.1016/S0005-2736(00)00130-9http://dx.doi.org/10.1104/pp.126.4.1646http://dx.doi.org/10.1104/pp.126.4.1646http://dx.doi.org/10.1104/pp.126.4.1646http://dx.doi.org/10.1104/pp.126.4.1646http://dx.doi.org/10.1016/S0098-8472(01)00105-8http://dx.doi.org/10.1016/S0098-8472(01)00105-8

RAZA et al. / Turk J Biol

119

Park S, Li J, Pittman JK, Berkowitz GA, Yang H, Undurraga S, Morris J, Hirschi KD, Gaxiola RA (2005). Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants. P Natl Acad Sci USA 102: 18830–18835.

Pasapula V, Shen G, Kuppu S, Paez-Valencia J, Mendoza M, Hou P, Chen J, Qiu X, Zhu L, Zhang X et al. (2011). Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions. Plant Biotechnology Journal 9: 88–99.

Qin H, Gu Q, Kuppu K, Sun L, Zhu X, Mishra N, Hu R, Shen G, Zhang J, Zhang Y, Zhu L, Zhang X, Burow M, Payton P, Zhan H (2013). Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance. Plant Biotechnol Rep 7: 345–355.

Raza G, Ali K, Mukhtar Z, Mansoor S, Arshad M, Asad S (2010). The response of sugarcane (Saccharum officinarum L.) genotypes to callus induction, regeneration and different concentrations of the selective agent (Geneticin-418). Afr J Biotech 9: 8739–8747.

Richards E, Reichardt M, Rogers S (2001). Preparation of genomic DNA from plant tissue. In: Current Protocols in Molecular Biology. John Wiley & Sons, Inc.

Schilling RK, Marschner P, Shavrukov Y, Berger B, Tester M, Roy SJ, Plett DC (2014). Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field. Plant Biotech J 12: 378–386.

Shahid MA, Ashraf MY, Pervez MA, Ahmad R, Balal RM, Garcia-Sanchez F (2013). Impact of salt stress on concentrations of Na+, Cl- and organic solutes concentration in pea cultivars. Pak J Bot 45: 755–761.

Shen G, Wei J, Qiu X, Hu R, Kuppu S, Auld D, Blumwald E, Gaxiola R, Payton P, Zhang H (2015). Co-overexpression of AVP1 and AtNHX1 in cotton further improves drought and salt tolerance in transgenic cotton plants. Plant Molecular Biology Reporter 33: 167–177.

Singh M, Ranjan S, Verma KK, Pathre UV, Shirke PA (2014). Photosynthetic characteristics of red and green leaves in growing seedlings of Jatropha curcas. Turk J Biol 38: 457–468.

Stoyanov ZZ (2005). Effects of water stress on leaf water relations of young bean (Phaseolus vulgaris L.). J Central Eur Agri 6: 5–14.

Sze H, Li X, Palmgren MG (1999). Energization of plant cell membranes by H+-pumping ATPases: regulation and biosynthesis. The Plant Cell Online 11: 677–689.

Taiz L, Zeiger E (2006). Stress physiology. In: Taiz L, Zeiger E, editors. Plant Physiology. Sunderland, MA, USA: Sinauer Associates, Inc., pp. 671–681.

Zhao F-Y, Zhang X-J, Li P-H, Zhao Y-X, Zhang H (2006). Co-expression of the Suaeda salsaSsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1. Molecular Breed 17: 341–353.

http://dx.doi.org/10.1073/pnas.0509512102http://dx.doi.org/10.1073/pnas.0509512102http://dx.doi.org/10.1073/pnas.0509512102http://dx.doi.org/10.1073/pnas.0509512102http://dx.doi.org/10.1111/j.1467-7652.2010.00535.xhttp://dx.doi.org/10.1111/j.1467-7652.2010.00535.xhttp://dx.doi.org/10.1111/j.1467-7652.2010.00535.xhttp://dx.doi.org/10.1111/j.1467-7652.2010.00535.xhttp://dx.doi.org/10.1111/j.1467-7652.2010.00535.xhttp://dx.doi.org/10.1111/j.1467-7652.2010.00535.xhttp://dx.doi.org/10.1007/s11816-012-0269-5http://dx.doi.org/10.1007/s11816-012-0269-5http://dx.doi.org/10.1007/s11816-012-0269-5http://dx.doi.org/10.1007/s11816-012-0269-5http://dx.doi.org/10.1007/s11816-012-0269-5http://dx.doi.org/10.1002/0471142727.mb0203s27http://dx.doi.org/10.1002/0471142727.mb0203s27http://dx.doi.org/10.1002/0471142727.mb0203s27http://dx.doi.org/10.1111/pbi.12145http://dx.doi.org/10.1111/pbi.12145http://dx.doi.org/10.1111/pbi.12145http://dx.doi.org/10.1111/pbi.12145http://dx.doi.org/10.1111/pbi.12145http://dx.doi.org/10.1007/s11105-014-0739-8http://dx.doi.org/10.1007/s11105-014-0739-8http://dx.doi.org/10.1007/s11105-014-0739-8http://dx.doi.org/10.1007/s11105-014-0739-8http://dx.doi.org/10.1007/s11105-014-0739-8http://journals.tubitak.gov.tr/biology/issues/biy-14-38-4/biy-38-4-4-1312-98.pdfhttp://journals.tubitak.gov.tr/biology/issues/biy-14-38-4/biy-38-4-4-1312-98.pdfhttp://journals.tubitak.gov.tr/biology/issues/biy-14-38-4/biy-38-4-4-1312-98.pdfhttp://dx.doi.org/10.2307/3870892http://dx.doi.org/10.2307/3870892http://dx.doi.org/10.2307/3870892http://dx.doi.org/10.1007/s11032-006-9005-6http://dx.doi.org/10.1007/s11032-006-9005-6http://dx.doi.org/10.1007/s11032-006-9005-6http://dx.doi.org/10.1007/s11032-006-9005-6

RAZA et al. / Turk J Biol

1

Appendix S1. Callus induction medium (CIM).CIM composed of: 4.43 g L–1 MS salts with vitamins (M519; Phytotechnology Laboratories),

4 mg L–1 2,4-D (D8407; Sigma-Aldrich), 4 mg L–1 thiamine- HCl (T4625; Sigma-Aldrich ), 0.5 g L–1 casein hydrolysate (22090; Sigma-Aldrich), 100 mg L–1 myo-inositol (I3011; Sigma-Aldrich), 3% sucrose (S7903; Sigma-Aldrich), 4 mg L–1 L-arginine (A8094; Sigma-Aldrich), 6 g L–1 agar (A1296; Sigma-Aldrich), pH 5.7.

Appendix S2. Regeneration selection medium (RSM).RSM composed of: 4.43 g L–1 MS salts with vitamins, 0.5 mg L–1 2,4- D, 1 g L–1 myo-inositol,

3% sucrose, 2 mg L–1 BAP (B3408; Sigma-Aldrich)], 1 mg L–1 NAA (N0640; Sigma-Aldrich), 0.5 mg L–1 Kinetin (K0753; Sigma-Aldrich), 60 mg L–1 Geneticin, 6 g L–1 agar, pH 5.7.

Appendix S3. Rooting selection medium (RTSM). RTSM composed of: 4.43 g L–1 MS salts with vitamins, 0.5 mg L–1 2,4- D, 1 g L–1 myo-

inositol, 4% sucrose, 0.5 mg L–1 BAP, 0.5 mg L–1 NAA, 3 mg L–1 IBA (I5386; Sigma-Aldrich), 40 mg L–1 Geneticin, 6 g L–1 agar, pH 5.7.