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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