Mer

ged

Mar

ker

B

rig

ht-

fiel

d G

FP

-ch

ann

el

C D

35S:sGFP 35S:HvEXPB7-sGFP

0

0.4

0.8

1.2

Root Stem Leaf Seed

Rel

ativ

e ex

pre

ssio

n l

evel

Actin

HvEXPB7

Root Leaf Stem SeedA B

35S:sGFP 35S:HvEXPB7-sGFP 35S:HvEXPB7-sGFP

(Plasmolysis)

Mer

ged

B

rig

ht-

fiel

d

G

FP

-ch

ann

el

Actin

HvEXPB7

A

B

0

0.5

1

1.5

2

2.5

3

Rel

ativ

e ex

pres

sion

leve

l

(1)

BSMV:γ

Control

(2)

BSMV:γ

Drought

(3)

BSMV:HvEXPB7

Control

(4)

BSMV:HvEXPB7

Drought

ab

c c

0

20

40

60

80

K+

conc

entr

atio

n (m

g g-1

DW

) C

Actin

HvPDS

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0.2

0.4

0.6

0.8

1

1.2

Control BSMV:HvPDS

Rel

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A B

Drought is one of the most severe abiotic factors affecting crop productivity worldwide. The identification of drought tolerant crop germplasm and understanding the

underlying tolerance mechanisms is necessary, therefore, to improve the plant adaptation to drought-prone environments. As a single cell type, root hair is an

important organ in uptake water and nutrients, and plays an important role in drought tolerance. However, there is a very little information about the genetic and

molecular basis of root hair development in monocots in response to drought. Tibetan wild barley is rich in genetic diversity and provides elite genes for crop

improvement in such abiotic stress tolerance as drought. In this study, the drought-tolerant Tibetan wild barley XZ5 was used. Based on the genotypic differences of

root hair development and root hair transcriptome in response to drought stress, the root hair development-regulated gene, HvEXPB7 being associated with drought

tolerance in XZ5, was isolated and cloned, and the molecular drought-tolerant mechanism in wild barley was studied.

Results

Acknowledgments

Xiaoyan He, Feibo Wu

College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China. Corresponding author E-mail: wufeibo@zju.edu.cn

Identification, Cloning and Characterization of HvEXPB7 in Root Hairs

of Tibetan Wild Barley under Drought Stress

XZ5 XZ54 Tadmor XZ5 XZ54 Tadmor

Control Drought

1 d

3 d

5 dOxidative stress ATP and nitrogen

XLOC_014532

XLOC_085743

XLOC_019100

XLOC_004739

XLOC_016879

XLOC_012447

XLOC_013896

XLOC_006853

XLOC_013018

Antioxidant

Drought tolerance in XZ5

Log2 （fold change）

-2 0 +2

Drought

Stress responseCell wall Root hair

XLOC_012543

XLOC_031639

XLOC_024622

XLOC_088601

XLOC_078554

XLOC_061217

XLOC_088358

XLOC_086498

DefenseRoot hair

development

Cell wall

modificationEnergy

Left, XZ5

Middle, XZ54

Right, Tadmor

Fig. 7 Silencing of the phytoene desaturase (PDS) gene in wild barley XZ5 using BSMV-VIGS.

1014 bp

396 93 525

UTR ATG Exon Intron TGA

5' 3'

GT AG2000 bp

1000 bp

2000 bp

1000 bp

M cDNA M DNAA B

C

1 aatctccccaacggcattaaccaccagagtagggtcatcgatcagtaaccaacgagaccagcaagcgagccatggcagcggccttttcctM A A A F S S

91 cctactccgtcgcggccgctttcctctgcctgctcgccgccaacggctgctgcggctgcccctggtttctccctgctacattctgccctgY S V A A A F L C L L A A N G C C G C P W F L P A T F C P E

181 aaccgacacccgaccccatacctacacctgaccccgctccagctacacctccgcctgctctggctacccccagcccaggctccggctctgP T P D P I P T P D P A P A T P P P A L A T P S P G S G S G

271 gcagcgccaacggttccatgggcggctggctggacgccagggccacatggtacggcgctccggatggcgccgggccgttggacaacggtgS A N G S M G G W L D A R A T W Y G A P D G A G P L D N G G

361 gcgcgtgcgggttcaagaacgtgaacctgccgcccttcaacgccatgacgtcgtgcggcaacgagccgatcttcaaggacggcaagggatA C G F K N V N L P P F N A M T S C G N E P I F K D G K G C

451 gcggctcctgctaccagataaggtgcgtgggcaaggttcacccagcgtgctccggcgaccccgagacggtgatcatcaccgacatgaactG S C Y Q I R C V G K V H P A C S G D P E T V I I T D M N Y

541 actacccggtcgcccgctaccacttcgacctcagcggcactgccttcggcgccatggccaaggacggccgcaacgacgagctccgccacgY P V A R Y H F D L S G T A F G A M A K D G R N D E L R H A

631 ccggcatcatcgacatgcagttcaagagggtgccgtgccagtacacagggctgtccgtgacgtttcacgtggagaaggggtcgaacccgaG I I D M Q F K R V P C Q Y T G L S V T F H V E K G S N P N

721 attacctggcgatcctggtggagtacgggaacggcgacggcgacgtggcgcaggtggacctcatggacggcggcgagccaacggtgtcctY L A I L V E Y G N G D G D V A Q V D L M D G G E P T V S W

811 ggaggccgatgagggagtcctggggctccatctggcgcctggacacgcggcgcccgcttcgggggcccttctcgctgcgggtcaccaacgR P M R E S W G S I W R L D T R R P L R G P F S L R V T N G

901 ggtccggcaggtcgctcgtcgcggaccaggtcatccccgccgactggcagcccgacaccgtgtacagctccgacgtccagttcgatgaatS G R S L V A D Q V I P A D W Q P D T V Y S S D V Q F D E *

991 gatcccgtccgatgatccatggaaggcatgcacgtacgtccatgcctgcatggattttttgtgccaagctatatagctgagacgtcgcct1081 gtgacttccggtgcaagtcctcgtcgtgtgctacctatgctacatatgtacacgtagtacataacatacgtggcagctttaattagtttg1171 tgggatctggagtaggcagcgagtagtatatattgctcgcagctataaatttacgtttgtaatttctcctgtattacacaagtttattat1261 gtcactggtgtgtataaa

Fig. 6 Phylogenetic tree, tissue expression pattern and subcellular

localization of HvEXPB7.

+1 +72

ATGE2rE1

-2320 -1669 -1458 -1454 -1125 -639 -497 -468 -434 -167

E3 E4 E5 E6r E7r E8r E9 E10

E2rE1 E3 E4 E5r E6r E7 E8

E2rE1 E3 E4 E5r E6r E7r E8 E9

+1 +71

ATG

+1 +72

ATG

-2318 -1667 -1452 -1123 -637 -495 -432 -165

-2316 -1666 -1451 -1122 -636 -494 -465 -431 -165

A

B

C

Fig.5 Relative positions of RHEs in the HvEXPB7 promoter regions of XZ5,

XZ54 and Tadmor.

Conclusion

Fig. 3 A predicted root hair DEGs based drought tolerance associated model in Tibetan wild

barley XZ5 in response and adaptation to drought stress.

Fig.1 Root hair morphology of XZ5, XZ54 and cv. Tadmor under control

(column a, b, c) and drought condition (column d, e, f).

Fig. 4 Gene cloning of HvEXPB7 in XZ5.

The drought-tolerant Tibetan wild barley

XZ5 has significantly well-developed root

hairs compared to XZ54 and cv. Tadmor

under drought stress; Thirty-six drought-

tolerance-related DEGs are identified, and

the full length sequence of a novel β-

expansin gene named HvEXPB7 is cloned;

The HvEXPB7 sequence from XZ5 has

abundant genetic diversity compared to cv.

Tadmor; BSMV-VIGS is successfully carried

out on wild barley; HvEXPB7 expression

plays an important role in root hair

development under drought stress. These

results, to a degree, illustrate the drought

tolerance mechanism of XZ5 and its

differences from cv. Tadmor, and HvEXPB7

could be applied in biotechnology to improve

drought tolerance via root hair growth in

barley.

Fig. 8 Functional assessment of HvEXPB7 in wild barley XZ5 via BSMV-VIGS.

The project was supported by National Natural Science Foundation of China (31171488), and the National 863 Program (2012AA101105).

BSMV vector is kindly provided by Prof. Peidu Chen, College of Agriculture, Nanjing Agricultural University. The binary vector pCAMBIA

1300 containing a CaMV 35S promoter::GFP cassette is kindly provided by Prof. De-An Jiang, College of Life Science, Zhejiang University.

Fig. 2 Distribution of root hair DEGs in XZ5, XZ54 and Tadmor in

response to drought stress and their function prediction.

XZ5 XZ54 Tadmor0

20

40

60

180

200

220

240

Up-regulated

Down-regulated

Nu

mb

er

of

gen

es

30

186

3

16

29

48

+13

- 167

+16

- 11

+1

- 3

+12

- 29

+1

- 5

+0

- 5 +1

- 3

XZ5

Tadmor

XZ54

A B

structu

ral m

olecule

activ

ity

bindin

g

catal

ytic ac

tivity

electr

on carri

er ac

tivity

antio

xidan

t acti

vity

transp

orter a

ctivity

05

1015202530

45

50 XZ5

XZ54

Tadmor

Num

ber

of g

enes

C