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Seaweed extracts as plant biostimulants:Benefits for biotic and abiotic stress
Balakrishnan PrithivirajDepartment of Plant and Animal Sciences
Nova Scotia Agricultural CollegeCanada
There are about 10,500 species of seaweeds6,500 are red algae (Rhodophyta)
Seaweeds are marine macroalgae
Seaweeds
Green
Red
Brown
Atlantic CanadaAscophyllum nodosum (Rockweed, Kelp)Chondrus crispus (Irish Moss)Furcellaria lumbricalisFucus vesiculosus (Bladderwrack)Laminaria digitata (Kelp)
• Widely used as plant biostimulants
• Minor nutritional component
• Alleviates Abiotic stress (salinity,drought, temperature extremes)
• Imparts resistance to pest anddiseases
Ascophyllum nodosum from cold waters -Benefits
Ascophyllum nodosumTolerant to Extreme conditionsTemperature -30 to +30Varying lightSalinity
But How?
Nature Chemical Biology 1, 64-66 (2005)
Bioactive components in A. nodosum extracts from cold waters
•• Small genome (114.5 /125 Mb)Small genome (114.5 /125 Mb)
•• Extensive genetic and physicalExtensive genetic and physicalmaps of all 5 chromosomesmaps of all 5 chromosomes
•• A rapid life cycle (about 6A rapid life cycle (about 6weeks)weeks)
•• Prolific seed production andProlific seed production andeasy cultivation in restrictedeasy cultivation in restrictedspacespace
•• Efficient transformationEfficient transformation --AgrobacteriumAgrobacterium tumefacienstumefaciens
•• A large number of mutant linesA large number of mutant linesand genomic resourcesand genomic resources
Arabidopsis thaliana model tobioprospect seaweed components
Ascophyllum extracts from cold water AlleviateSalinity stress in Arabidopsis thaliana
150mMNaCl
150mMNaCl +SWEfraction 1
150mMNaCl +SWEFraction 4
Control SWE NaCl NaCl + Frac 1
Asco Water
0
2
4
6
8
10
12
14
16
control 100mM NaCl 150mM NaCl
Water 5221 1186
0
5
10
15
20
25
30
Control 100mM NaCl 150mm NaCl
Water 5221 1186
0
3
6
9
12
15
18
21
Control 100mM NaCl 150mM NaCl
Water 5221 1186
Lea
fA
rea/
Pla
nt
(Cm
2 )P
lan
tH
eig
ht
(Cm
)
Lea
fN
um
be
r/P
lan
tP
lan
tF
W(g
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Control 100mM NaCl 150 mM NaCl
Water 5221 1186
Ascophyllum extracts improves plant performanceunder NaCl stress in Arabidopsis thaliana
0.854641.6095128.71200.0000205.350380.08581.10582.50991.25410.60483.5080#4 +NA +
Frac1
0.528340.8031137.88452.4877219.686889.81250.82312.27240.75100.60264.0020#3 +NA+Frac 4
1.142637.4608120.65234.0673116.178759.02691.07452.43201.54120.59183.1100#2 +NA
CONTROL
0.176136.391899.094811.195994.148382.65681.30272.51111.71160.69383.3010#1 -NA
CONTROL
SODIUM %BORON
ppmZINC ppmCOPPER ppm
MANGANESE ppmIRON ppm
MAGNESIUM %
CALCIUM%POTASSIUM %
PHOSPHORUS%
NITROGEN %SAMPLE
Effect on Cellular Ion Content
o Method of Analysis- AOAC 968.08
0 . 0
1 . 0
2 . 0
% D r y W t
( - N a c t r l ) ( + N a c t r l ) E t A c M e t h a n o l
T r e a t m e n t s
S o d iu m
P o ta s s iu m
Asco
Con
trol
l
NHX2
NHX5
Actin
DAY 1 DAY 5
Control Plants
treated with 150 mM NaCl
TotalRNA
TotalRNA
Plants treated with150 mM NaCl andextracts added
cRNA synthesis cRNA synthesis
Hybridization
Scanning
Affy Microarray Chip
Categorization offunctional categoriesand RT-PCR
Global Transcriptome Changes
inducible (>1.5 fold) with Org Frac1 extract treatment.
(repression) with Org Frac 1treatment.
Few genes altered
Global Transcriptome Changes elicited byModerately active fraction
Repression Genes < 1.5 foldwith Org Fra 2 treatment.
inducible Genes (>1.5 fold)with Org Fra 2 treatment
More genes altered
Global Transcriptome Changes elicited byactive fraction (active)
Active Frac/NaCl controls –Day 5
Less Active/NaCl controls-Day 5
Microarray analysis reveals the changes in theexpression of specific subset of genes
Abiotic factors
Cellular Organization andbiogenesis
Unknown
Metabolism
Others
Signal transduction
Transcription factors
Cellular defense and rescue
Transporters
Energy
Abiotic factors
Cellular Organization andbiogenesis
Unknown
Metabolism
Others
Signal transduction
Transcription factors
Cellular defense and rescue
Transporters
Energy
Day 1 Day 5
Induced Genes
Mechanisms?
DREB 1/ CBF 1, 2, 3)
SALT STRESS
SignallingABA dependent Gene expression
MYC/ MYB bZIPCBF4
MYBRS MYCRS
Myb recognition site MYC recognitionsite
(rd22)
ABA independent Geneexpression
DREB 2
DRE (rd29A)
Physiological and biologicalchanges
LEADING TO SALT STRESSTOLERANCE
DAY 5ABRE
(rd 29B)
DAY 1
LEA can be induced by both ABAand ABA independent pathway
KO of some of the genes down regulated by A. nodosumextract treatment showed NaCl tolerance phenotype
0m
MN
aCl
125
mM
NaC
lCol-0 Salk_072421 Col-0 Salk_007858C
wtpmei1-1
wtpmei1-1
wtpmei1-1
0 mM NaCl 75 mM NaCl 100 mM NaCl
wt pmei 1-1wtpmei 1-1
A
B C
pmei1 is resistant to NaCl stress
pmei 1-1 is tolerent to moderate NaCl stress
WTpmei 1-1
0 mM 50 mM 75 mM 100 mM 125 mM 150 mM
After 3 days
**
*
0 mM 50 mM 75 mM 100 mM 125 mM 150 mM
After 7 days
pmei 1-1WT
*
*
*
pmei1-1 is specific to NaCl
Mannitol
200, 250 mM
KCl
100, 125 mM
LiCl
10, 12.5
Characterization of pmei1-1
Col-0
75 mMNaCl
100 mMNaCl
pmei 1-1A
B
Cor 15A
ADH
RAB 18
RD 29 A
18 S
At1g62760
0 3 24 0 3h 24 (h)
Col-0 pmei 1-1
WTLMUL
WTS
MUS
PME supplementation Reverts NaCl Toxicity
L-Phenylalanine
Trans-cinnamate
4-coumarate
Ammonia
phenylalanineammonia-lyase At-PAL1AT2G37040.1
At-PAL 2 AT3G53260.1
Cinnamate 4-hydroxylase (
4-coumarate-coA ligase
4CL1, 2, 3, 44CL 3AT1G650604CL 1AT5G38120
4-Coumaroyl CoA
naringeninchalcone
naringeninchalconesynthetase
AT5G13930
naringenin
Chalconeisomerase
AT3G55120 ;
AT5G05270.1
Flavanoid pathway induction in Day 1 ofthe treatment
naringenin 3-dioxygenase AT3G51240.1
dihydrokaempferol
Flavanoid 3-hydroxylaseAT5G07990
Dihydroquercetin
quercetin
Flavanol synthetase FLS1 AT5G08640
leucopelargonidin
Dihydroflavanol 4-reductaseAT5G42800
leucocyanidin
cyanidin
leucoanthocyanidin dioxygenase,putative / anthocyanidin synthaseAT4G22870
ANTHOCYANIN
Disease resistance
Nodulation
Ascopyllum extracts Impart Freezing Tolerancein Arabidopsis thaliana Col-0
Control Fraction 2Fraction 1 Fraction 3
Ascopyllum extracts Impart Freezing Tolerancein Arabidopsis thaliana Col-0
Control ANE EtoAc
RD29A
CBF1
Cor15a
ESK1
FAD8
18s
0°C -2°C 4°C 22°C
1 2 3 1 2 3 1 2 3 1 2 3
Control SWE-1
Ascophyllum elicits disease resistance
PR3
PR5ICS1
AOS
6h 24h 48h
COI1
PDF1.2
18S
Ctrl INA ANE C E Ctrl ANE 5221 C E Ctrl INA ANE C E
PR1
SA
JA24h
96 h
Control ANE1 Chl
PR
1::G
US
AO
S::
GU
S
24h
72h
INA ANE
Ascophyllum elicits Jasmonic acid dependentdisease resistance in Arabidopsis
Nature Chemical Biology 1, 64-66 (2005)
Small molecules in Ascophyllum extracts affect growthby affecting fundamental biological processes
Bioactive components in A. nodosum extracts
Summary Points
• Ascophyllum nodosum extracts have been used for many years as a plantstimulant
• Our research has established cause and effect relationships usingAscophyllum extract fractions against salinity stress
• Specifically, components in Ascophyllum extracts affects the regulation ofspecific subset of genes resulting in stress tolerance
• Abiotic stress resistance is a polygenic trait – additional challengestrangenic approach – Ascophyllum seaweed extract an alternate?
• May lead to unraveling novel functions of genes in stress tolerance andgrowth
Abiotic and Biotic stress: A Global problem!
http://www.ciseau.org
Development of cropvarieties tolerant toSalinity, drought,Freezing, pest anddiseases
EnvironmentalStress factorsHuman activity –climate change
•Conventional Breeding
•Molecular Approaches
10 Years
PitfallsTime to developmentPolygenicConsumer concerns - GMOs
Oligogenic traits
Strategies for the development of plants tolerantto Abiotic stresses
Our method:Use of existing high yielding cvsLess complicatedFeasibleCan be used as needed thus reducing theenergy requirements –no reduction ineconomic yield
Acknowledgements
Dr. Pragya Kant (Post Doc)Dr. Wajahat Khan (Post Doc)Dr. Jithesh Narayan (Post Doc)Sowmya Subramanian (Graduate Student)Prasanth Rayarath (Graduate Student)Amir Farid (Research Assistant)Ruijie Zhai (Undergraduate)
CollaboratorsDr. Ravi Palanivelu, University of Arizona, TucsonDr Mark Hodges, AAFC, Kentville