Seaweed extracts as plant biostimulants:
Benefits for biotic and abiotic stress
Balakrishnan Prithiviraj
Department of Plant and Animal Sciences
Nova Scotia Agricultural College
Canada
Seaweeds
Seaweeds are marine macroalgae
Green
Red
Brown
There are about 10,500 species of seaweeds
6,500 are red algae (Rhodophyta)
Atlantic Canada
Ascophyllum nodosum (Rockweed, Kelp)
Chondrus crispus (Irish Moss)
Furcellaria lumbricalis
Fucus vesiculosus
(Bladderwrack)
Laminaria digitata (Kelp)
Ascophyllum nodosum from cold waters Benefits
• Widely used as plant biostimulants
• Minor nutritional component
• Alleviates Abiotic stress (salinity,
drought, temperature extremes)
• Imparts resistance to pest and
diseases
Ascophyllum nodosum
Tolerant to Extreme conditions
Temperature -30 to +30
Varying light
Salinity
But How?
Nature Chemical Biology 1, 64-66 (2005)
Bioactive components in A. nodosum extracts from cold waters
Arabidopsis thaliana model to
bioprospect seaweed components
• Small genome (114.5 /125 Mb)
• Extensive genetic and physical
maps of all 5 chromosomes
• A rapid life cycle (about 6
weeks)
• Prolific seed production and
easy cultivation in restricted
space
• Efficient transformation Agrobacterium tumefaciens
• A large number of mutant lines
and genomic resources
Ascophyllum extracts from cold water Alleviate
Salinity stress in Arabidopsis thaliana
150mM
NaCl
Control
150mM
NaCl +
SWE
fraction 1
SWE
Asco
150mM
NaCl +
SWE
Fraction 4
NaCl
Water
NaCl + Frac 1
30
0.7
Water
25
5221
1186
20
15
10
5
5221
1186
0.5
0.4
0.3
0.2
0.1
0
0
Control
100mM NaCl
150mm NaCl
Control
21
16
Water
18
5221
1186
15
12
9
6
3
0
Leaf Number/ Plant
Plant Height (Cm)
Water
0.6
Plant FW (g)
Leaf Area/ Plant (Cm2)
Ascophyllum extracts improves plant performance
under NaCl stress in Arabidopsis thaliana
100mM NaCl
Water
14
150 mM NaCl
5221
1186
12
10
8
6
4
2
0
Control
100mM NaCl
150mM NaCl
control
100mM NaCl
150mM NaCl
Asco
l
2 .0
Control
Effect on Cellular Ion Content
NHX2
% D r y W t 1 .0
S o d iu m
P o ta s s iu m
NHX5
0 .0
(- N a c trl) (+ N a c trl)
E tA c
M e th a n o l
Actin
T r e a tm e n ts
DAY 1
DAY 5
SAMPLE
NITROG
EN %
PHOSPH
ORUS%
POTASSIUM %
CALCIUM
%
MAGNESIU
M%
IRON ppm
MANGANE
SE ppm
COPPE
R ppm
ZINC ppm
BORON
ppm
SODIUM %
#1 -NA
CONTROL
3.3010
0.6938
1.7116
2.5111
1.3027
82.6568
94.1483
11.1959
99.0948
36.3918
0.1761
#2 +NA
CONTROL
3.1100
0.5918
1.5412
2.4320
1.0745
59.0269
116.1787
4.0673
120.6523
37.4608
1.1426
#3 +NA
+Frac 4
4.0020
0.6026
0.7510
2.2724
0.8231
89.8125
219.6868
2.4877
137.8845
40.8031
0.5283
#4 +NA +
Frac1
3.5080
0.6048
1.2541
2.5099
1.1058
80.0858
205.3503
0.0000
128.7120
41.6095
0.8546
o Method of Analysis- AOAC 968.08
Global Transcriptome Changes
Control Plants
treated with 150 mM NaCl
Plants treated with
150 mM NaCl and
extracts added
Total
RNA
Total
RNA
Affy Microarray Chip
cRNA synthesis
cRNA synthesis
Hybridization
Scanning
Categorization of
functional categories
and RT-PCR
Global Transcriptome Changes elicited by
Moderately active fraction
inducible (>1.5 fold) with Org Frac
1 extract treatment.
Few genes altered
(repression)
treatment.
with
Org
Frac
1
Global Transcriptome Changes elicited by
active fraction (active)
inducible Genes (>1.5 fold)
with Org Fra 2 treatment
More genes altered
Repression Genes < 1.5 fold
with Org Fra 2 treatment.
Microarray analysis reveals the changes in the
expression of specific subset of genes
Active Frac/NaCl controls –
Day 5
Less Active/NaCl controlsDay 5
Induced Genes
Abiotic factors
Abiotic factors
Day 1
Cellular Organization and
biogenesis
Day 5
Cellular Organization and
biogenesis
Unknown
Unknown
Metabolism
Metabolism
Others
Others
Signal transduction
Signal transduction
Transcription factors
Cellular defense and rescue
Transporters
Energy
Transcription factors
Cellular defense and rescue
Transporters
Energy
Mechanisms?
SALT STRESS
Signalling
ABA dependent Gene expression
ABA independent Gene
expression
CBF4
MYC/ MYB
ABRE
(rd 29B)
MYBRS
DREB 2
bZIP
DAY 5
DREB 1/ CBF 1, 2, 3
)
DRE (rd29A)
MYCRS
Myb recognition site MYC recognition
site
(rd22)
LEA can be induced by both ABA
and ABA independent pathway
Physiological and biological
changes
LEADING TO SALT STRESS
TOLERANCE
DAY 1
KO of some of the genes down regulated by A. nodosum
extract treatment showed NaCl tolerance phenotype
125 mM NaCl
0 mM NaCl
Col-0
Salk_072421
Col-0
Salk_007858C
pmei1 is resistant to NaCl stress
A
wt
wt
pmei1-1
wt
pmei1-1
0 mM NaCl
pmei1-1
75 mM NaCl
100 mM NaCl
C
B
wt
pmei 1-1
wt
pmei 1-1
pmei 1-1 is tolerent to moderate NaCl stress
WT
pmei 1-1
WT
pmei 1-1
*
*
*
*
*
*
0 mM
50 mM
75 mM
100 mM
After 3 days
125 mM
150 mM
0 mM
50 mM
75 mM
100 mM 125 mM
After 7 days
150 mM
pmei1-1 is specific to NaCl
Mannitol
KCl
LiCl
200, 250 mM
100, 125 mM
10, 12.5
Characterization of pmei1-1
Col-0
A
0
75 mM
NaCl
100 mM
NaCl
B
Cor 15A
ADH
RAB 18
RD 29 A
18 S
MU
L
WTL
MUS
WTS
pmei 1-1
Col-0
pmei 1-1
At1g
62760
3
24
0 3h
24
(h)
PME supplementation Reverts NaCl Toxicity
Flavanoid pathway induction in Day 1 of
the treatment
L-Phenylalanine
phenylalanine
ammonia-lyase At-PAL1
AT2G37040.1
Ammonia
Trans-cinnamate At-PAL 2 AT3G53260.1
Cinnamate 4hydroxylase (
4-coumarate
4-coumarate-coA ligase
4CL1, 2, 3, 4
4CL 3AT1G65060
4CL 1AT5G38120
4-Coumaroyl CoA
Disease resistance
Nodulation
naringenin
chalcone
synthetase
AT5G13930
naringenin
chalcone
Chalcone AT3G55120 ;
isomerase
naringenin
AT5G05270.1
naringenin 3-dioxygenase AT3G51240.1
dihydrokaempferol
Flavanoid 3-hydroxylase
AT5G07990
Dihydroquercetin
Flavanol synthetase FLS1 AT5G08640
quercetin
Dihydroflavanol 4-reductase
AT5G42800
leucopelargonidin
ANTHOCYANIN
leucocyanidin
leucoanthocyanidin dioxygenase,
putative / anthocyanidin synthase
AT4G22870
cyanidin
Ascopyllum extracts Impart Freezing Tolerance
in Arabidopsis thaliana Col-0
Control
Fraction 1
Fraction 2
Fraction 3
Ascopyllum extracts Impart Freezing Tolerance
in Arabidopsis thaliana Col-0
Control
ANE
EtoA
c
0°C
-2°C
4°C
22°C
Cor15a
RD29A
CBF1
ESK1
FAD8
18s
1
2
3
1
2
3
1
2
3
1
2
3
Ascophyllum elicits disease resistance
Control
SWE-1
Ascophyllum elicits Jasmonic acid dependent
disease resistance in Arabidopsis
6h
24h
48h
PR1::GUS
24h
PR1
PR3
72h
PR5
ANE
AOS::GUS
INA
SA
ICS1
AOS
24h
COI1
PDF1.2
18S
96 h
Ctrl INA ANE C E
Control
ANE1
Chl
Ctrl ANE 5221 C E
Ctrl INA ANE C E
JA
Small molecules in Ascophyllum extracts affect growth
by affecting fundamental biological processes
Nature Chemical Biology 1, 64-66 (2005)
Bioactive components in A. nodosum extracts
Summary Points
•
Ascophyllum nodosum extracts have been used for many years as a plant
stimulant
•
Our research has established cause and effect relationships using
Ascophyllum extract fractions against salinity stress
•
Specifically, components in Ascophyllum extracts affects the regulation of
specific subset of genes resulting in stress tolerance
•
Abiotic stress resistance is a polygenic trait – additional challenges
trangenic approach – Ascophyllum seaweed extract an alternate?
•
May lead to unraveling novel functions of genes in stress tolerance and
growth
Abiotic and Biotic stress: A Global problem!
Environmental
Stress factors
Human activity –
climate change
http://www.ciseau.org
Development of crop
varieties tolerant to
Salinity, drought,
Freezing, pest and
diseases
Strategies for the development of plants tolerant
to Abiotic stresses
Pitfalls
•Conventional Breeding
Time to development
Polygenic
Consumer concerns - GMOs
•Molecular Approaches
Oligogenic traits
Our method:
Use of existing high yielding cvs
Less complicated
Feasible
Can be used as needed thus reducing the
energy requirements –no reduction in
economic yield
10 Years
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)
Collaborators
Dr. Ravi Palanivelu, University of Arizona, Tucson
Dr Mark Hodges, AAFC, Kentville