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Identification of drought-induced genes in giant leucaena (Leucaena leucocephala).
Michael Honda
Department of Molecular Biosciences and Bioengineering
University of Hawaii at Manoa
Outline
• Introduction
• Methods
• Results
• Microarray
• qRT-PCR
• Conclusion
• Questions
2
Leucaena leucocephala (leucaena)• Fast growing
• Tree-legume
• Native to Southern Mexico and Central America
• Tropics and sub-tropics
• Hawaii• ‘Common leucaena’ or ‘Koa haole’
• Subsp. leucocephala (a & b)
• Invasive
• ‘Giant leucaena’
• Subsp. glabrata (c & d)
• K636
• Not invasive 3
Giant leucaena
4
Uses• Agroforestry
• Agriculture
• Fuel
• Human food
• Animal fodder
5
https://www.flickr.com/photos/iita-media-library/6916352253
http://www.tropicalforages.info/key/Forages/Media/Html/Leucaena_leucocephala.htm
https://www.daf.qld.gov.au/plants/field-crops-and-pastures/research/pastures-and-grazing/growing-leucaena
http://tropical.theferns.info/image.php?id=Leucaena+leucocephala
http://thekitchenbuzzz.com/2016/09/green-salsa-with-guajes.html/
http://polyploid.net/leucaena/pages/Leucaena_leu.html
Leucaena in different environmental conditions
• Salt
• UV light
• Flooded soils
• Acidic soils
• Alkaline soils
• Eroded soils
• Drought
Drought• Global warming
• Crop loss
• Billions of $ world wide
• Reduction of global food supply
• Drought-tolerant crops
• Leucaena can tolerate prolonged drought
https://archive.epa.gov/climatechange/kids/impacts/signs/droughts.html
How does leucaena tolerate prolonged drought conditions?
8
• Cope with drought stress (primary stress)
• Cope with oxidative and osmotic stress (secondary stress)
• Posses some unique traits or characteristics
• Unique genes
• Unique gene expression patterns
Objective
1. Identify genes that are upregulated during leucaena drought stress
a) Microarray
b) Validation of microarray by qRT-PCR
Methods: Microarray
https://www.slideshare.net/AustralianBioinformatics/ken-mcgrath-next-gen-sequencing-game-of-thrones-edition
https://commons.wikimedia.org/wiki/File:DNA_microarray.svg
Transcriptome sequencing and assembly, and microarray analysis on drought-treated leucaena root and shoot
Root and shoot of drought-treated leucaena
RNA
Methods: qRT-PCR
Scarification
Germination
Mature leucaenaseeds K636
Methods: qRT-PCR
15% polyethylene glycol MW = 6,000
Drought-treatment
Untreated control Water
One-month old seedlings
Methods: qRT-PCR
Leaf
Embryonic leaf
Stem
RootHarvest leucaena after 48 h
Total RNA extraction
Separate organs and tissues
Methods: qRT-PCR
2-ΔΔCT
Dnase treatment cDNA synthesis
qRT-PCRData analysis
Results: Microarray analysis revealed 110 gene sequences showing > 5-fold upregulation in drought-treated leucaeana compared to untreated leucaena (control).
• 2 in both root and shoot
• 71 in root only
• 37 in shoot only
Results: Microarray analysis
15
5
6
7
10
10
9
13
14
0 2 4 6 8 10 12 14
Membrane-bound/signal-transduction
Protein regulation/interaction
Sorting/trafficking/transport
Metabolism
Hyp no homology
Other
Hyp
Nucleic acid regulation/interaction
1
2
4
4
4
7
8
9
0 2 4 6 8 10
Hyp
Protein regulation/interaction
Sorting/trafficking/transport
Nucleic acid regulation/interaction
Membrane-bound/signal-transduction
Hyp with no homology
Defense/stress-related
Metabolism Root
Shoot
Results: Functional annotation
Results: qRT-PCR of root
• 71 genes • 24 genes selected• 12 of 24 validated
• > 2-fold upregulation
17
4.1
7.2 8.4
9.312.3
18.0
25.3 32.442.4
52.453.6
83.9
1
10
100
Seri
ne
ca
rbo
xyp
ep
tid
ase
Tra
nsm
em
bra
ne
pro
tein
PR
A1
Pero
xin
β-a
myrin
syn
tha
se
9-c
is-e
poxyca
rote
no
idd
ioxyg
ena
se
Pen
tatr
icop
eptid
e r
ep
ea
t-co
nta
inin
g p
rote
in M
RL1
Fe
-S c
luste
r asse
mb
lyfa
cto
r N
AR
1
Zin
c f
ing
er
pro
tein
CC
Hd
om
ain
con
tain
ing
pro
tein
Tra
nscrip
tion
facto
rT
CP
14
Me
mb
rane
-an
cho
red
pro
tein
CO
BR
A
Ser/
Thr
pro
tein
kin
ase
AB
C tra
nspo
rte
r C
Fo
ld c
ha
nge
Root
n = 3p < 0.05
2.2 2.32.3 2.4
4.0
6.7
19.025.0
25.4 30.836.7 46.2
71.7
1
10
100
Rib
onucle
ase H
TM
V r
esis
tance
pro
tein
N
Dis
ease r
esis
tance
pro
tein
TIR
-NB
-LR
Rre
cepto
r pro
tein
Rpp4
Fla
vonoid
3-O
-gala
cto
syl
transfe
rase
Me
mbra
ne p
rote
inU
PF
049
6
Zin
c fin
ger
CC
CH
dom
ain
-conta
inin
gpro
tein
Kin
esin
mo
tor
dom
ain
-conta
inin
gpro
tein
Inactive p
rote
inkin
ase
GD
SL
este
rase/lip
ase
Pa
tatin
-11 p
rote
in
Rham
nose
reducta
se
UD
P-
gly
cosyltra
nsfe
rase
Fold
change
Leaf
2.12.7 3.3 3.3 3.3
3.64.8 4.9 5.5
337.3
1
10
100
Pa
tatin
-11 p
rote
in
Dis
ease r
esis
tance
pro
tein
Inactive p
rote
in k
inase
UD
P-
gly
cosyltra
nsfe
rase
TM
V r
esis
tance
pro
tein
N
An
kyrin
repeat
con
tain
ing p
rote
in
GD
SL
este
rase/lip
ase
Me
mbra
ne p
rote
inU
PF
049
6
Gag-p
ol poly
pro
tein
Caffeoyl-C
oA
O-
me
thyltra
nsfe
rase
Fold
change
Embryonic leaf
5.4
7.710.1
10.6
11.111.3
13.3 16.6
32.950.6 54.7
1
10
100
Rham
nose
reducta
se
TIR
-NB
-LR
Rre
cepto
r pro
tein
Rpp4
Rib
onucle
ase H
Zin
c fin
ger
CC
CH
dom
ain
-con
tain
ing
pro
tein
Inactive p
rote
inkin
ase
Me
mbra
ne
pro
tein
UP
F049
6
Pa
tatin
-11
pro
tein
Fla
vonoid
3-O
-gala
cto
syl
transfe
rase
GD
SL
este
rase/lip
ase
TM
V r
esis
tance
pro
tein
N
Kin
esin
mo
tor
dom
ain
-con
tain
ing
pro
tein
Fold
change
Stem
18
• 37 genes
• 23 genes selected
• Validated (> 2-fold upregulation)
• 13 of 23 in leaf
• 10 of 23 in embryonic leaf
• 11 of 23 in stem
• 16 total validated in shootn = 3p < 0.05
Results: qRT-PCR analysis of shoot
Results: qRT-PCR of root and shoot
1. Zinc finger CCCH domain-containing protein
a) Validated in root (32.4-fold)
b) Validated in shoot
a) Leaf and stem (19.0 and 10.6-fold, respectively)
2. UDP-glycosyltransferase
a) Not validated in root
b) Validated in shoot
a) Leaf and embryonic leaf (71.7 and 3.3-fold, respectively)
Leucaena drought response model
20
Four different categories
1. Sensor and signaling proteins
2. Transcription factors
3. Degradation pathway enzymes
4. Biosynthesis and structural proteins
Conclusion:
• Validated microarray results
• Root and shoot
• Different parts of shoot have different expression patterns
• Important in leucaena drought response
• Important to plant breeders
• Develop drought tolerant crops
• Interesting to transform these genes in other plants
• Do gene silencing
21
Thank you
• Dr. Dulal Borthakur
• Dr. James Brewbaker
• Lab mates
22
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Mahalo!
Questions?