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?