a soybean expansin gene gmexpb2 intrinsically involved in ... soybean b-expansin... · keywords:...

A soybean b-expansin gene GmEXPB2 intrinsically involvedin root system architecture responses to abiotic stresses

Wenbing Guo†,‡, Jing Zhao†, Xinxin Li, Lu Qin, Xiaolong Yan and Hong Liao*

Root Biology Centre, South China Agricultural University, Guangzhou 510642, China

Received 9 August 2010; revised 5 January 2011; accepted 21 January 2011; published online 7 March 2011.*For correspondence (fax +86 20 85281829; e-mail [email protected]).†These authors contributed equally to this work.‡Present address: School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA.

SUMMARY

Root system architecture responds plastically to some abiotic stresses, including phosphorus (P), iron (Fe) and

water deficiency, but its response mechanism is still unclear. We cloned and characterized a vegetative

b-expansin gene, GmEXPB2, from a Pi starvation-induced soybean cDNA library. Transient expression of

35S::GmEXPB2-GFP in onion epidermal cells verified that GmEXPB2 is a secretory protein located on the cell

wall. GmEXPB2 was found to be primarily expressed in roots, and was highly induced by Pi starvation, and

the induction pattern was confirmed by GUS staining in transgenic soybean hairy roots. Results from intact

soybean composite plants either over-expressing GmEXPB2 or containing knockdown constructs, showed

that GmEXPB2 is involved in hairy root elongation, and subsequently affects plant growth and P uptake,

especially at low P levels. The results from a heterogeneous transformation system indicated that over-

expressing GmEXPB2 in Arabidopsis increased root cell division and elongation, and enhanced plant growth

and P uptake at both low and high P levels. Furthermore, we found that, in addition to Pi starvation, GmEXPB2

was also induced by Fe and mild water deficiencies. Taken together, our results suggest that GmEXPB2 is a

critical root b-expansin gene that is intrinsically involved in root system architecture responses to some abiotic

stresses, including P, Fe and water deficiency. In the case of Pi starvation responses, GmEXPB2 may enhance

both P efficiency and P responsiveness by regulating adaptive changes of the root system architecture. This

finding has great agricultural potential for improving crop P uptake on both low-P and P-fertilized soils.

Keywords: b-expansin, soybean, root system architecture, cell-wall protein, abiotic stress.

INTRODUCTION

Roots are the major organ subjected to below-ground abi-

otic stresses in nature, and are responsible for acquisition

of water and nutrients from the soil. The root system archi-

tecture is regulated by environmental factors (Karban, 2008),

including phosphorus (P), nitrogen (N), iron (Fe) and water

starvation (Zhang and Forde, 1998; Lopez-Bucio et al., 2003;

Ward et al., 2008; Fang et al., 2009; Wang et al., 2010). The

mechanisms determining root system architecture have

been classified into intrinsic and response pathways

(Malamy, 2005). Under environmental stress, intrinsic and

response pathways interact with each other and then regu-

late the root system architecture responses. Response

pathways affect root system architecture in response to

environmental factors via intrinsic pathways, and intrinsic

pathways determine the extent of the response. However,

the molecular mechanisms of alterations in root system

architecture in response to abiotic stress, particularly in

crops, remain unclear.

Soybean (Glycine max (L.) Merr.) is one of the most widely

grown leguminous crops in the world. However, soybean

production is limited by various edaphic conditions, espe-

cially low P availability in soils (Bureau et al., 1953). As

phosphorus is one of the least-available nutrients, with very

low mobility in soils (Vance et al., 2003), plants can only

absorb Pi from the soil regions directly explored by roots.

Thus the root system architecture critically functions in

both the P efficiency (biomass and/or P uptake under low-P

conditions) and P responsiveness (biomass and P uptake

under high-P conditions) of plants (Lynch, 1995, 1998, 2007;

Wang et al., 2010). It has been reported that P-efficient

soybean genotypes respond to Pi starvation via alterations

in the root system architecture and morphology associated

ª 2011 South China Agricultural University 541The Plant Journal ª 2011 Blackwell Publishing Ltd

The Plant Journal (2011) 66, 541–552 doi: 10.1111/j.1365-313X.2011.04511.x

with enhanced exploration for soil P (Zhao et al., 2004;

Yan et al., 2006). Recently, we identified 215 Pi starvation-

induced genes from a P-efficient soybean genotype using a

suppression subtractive hybridization (SSH) technique. One

of these candidate P-responsive genes, RL42, was similar to

the soybean b-expansin gene, Cim1, and may be a member

of the expansin family (Guo et al., 2008).

To date, two expansin sub-families have been identified,

named a-expansins (EXPA or EXP) and b-expansins (EXPB).

Although a- and b-expansins share only 20–25% amino acid

identity, they contain a number of conserved residues and

characteristic motifs (Cosgrove et al., 1997). Members of the

b-expansin sub-family include the group I allergens that are

abundantly and specifically expressed in grass pollen, and

related genes expressed in vegetative tissues called ‘vege-

tative EXPBs’ (Cosgrove, 2000). Most EXPBs are found in

monocots (see http://www.bio.psu.edu/expansins), but only

12 have been identified in dicots, including Cim1 from

soybean (Crowell et al., 1990), PPAL from tobacco (Pezzotti

et al., 2002) and ten from Arabidopsis (Li et al., 2002).

Previous studies have focused on regulation of processing

of the Cim1 protein by plant hormones (Crowell et al., 1990;

Downes and Crowell, 1998; Downes et al., 2001), and

information is still limited regarding its biological functions.

PPAL was the first identified generative EXPB in dicots, and

is pistil-specific (Pezzotti et al., 2002). However, no func-

tional analysis of PPAL has been performed. Li et al. (2002)

identified 38 expansin sequences in Arabidopsis, in which

there are 10 EXPBs. AtEXP-b1 is sensitive to salt stress;

however, the mechanisms underlying its enhanced salt

sensitivity is still unknown (Kwon et al., 2008).

Expansins may play important roles in root growth and

development. The first root-specific soybean expansin gene,

GmEXP1, was identified by Lee et al. (2003). Ectopic expres-

sion of GmEXP1 in transgenic tobacco lines accelerated root

growth and cell enlargement at the seedling stage, implying

that GmEXP1 may function in root development. Expression

of two Arabidopsis expansin genes, AtEXP7 and AtEXP18,

was found to be closely linked to root hair initiation (Cho and

Cosgrove, 2002). Similarly, expression of the b-expansin

(EXPB) gene HvEXPB1 was root-specific and associated with

root hair formation in barley (Kwasniewski and Szarejko,

2006). These findings suggest that expansins, with their

proposed functions in cell division and elongation, may play

important roles in the cell-wall synthesis that takes place

during cell division to create new transverse cell wall

separating daughter cells (Muller et al., 2007). More recently,

it has been found that the changes in root development in

response to P deficiency involve changes in cell division and

elongation in Arabidopsis (Sanchez-Calderon et al., 2005;

Lai et al., 2007). Therefore, we hypothesize that expansin

genes may function in adaptive root system architecture

changes in response to Pi starvation. To test this hypothesis,

we cloned and characterized the soybean b-expansin gene,

GmEXPB2, which was a candidate P-responsive gene

isolated from our Pi starvation-induced cDNA library con-

structed from a P-efficient soybean genotype. Its expression

patterns in response to P deficiency in soybean hairy roots

were investigated using promoter–GUS fusion analysis.

GmEXPB2 over-expression and knockdown in a homoge-

neous transformation system, leading to soybean compos-

ite plants with transgenic hairy roots on wild-type shoots,

as well as heterogeneous expression in transgenic Arabi-

dopsis, were used to evaluate the possible functions of

GmEXPB2 in root growth and P uptake in response to Pi

starvation. Moreover, GmEXPB2 expression under several

other abiotic stresses, including N, potassium (K), Fe and

water deficiency, was also measured to determine whether

GmEXPB2 is involved in the intrinsic control of root growth.

RESULTS

Cloning and identification of GmEXPB2 in soybean

Based on the sequence of the cDNA fragment, RL42, from

a suppression subtractive library (Guo et al., 2008), we

obtained the full-length cDNA of a new gene by RACE-PCR

and named it as GmEXPB2, i.e. the second Glycine max

expansin B. GmEXPB2 is a 1048 bp cDNA (accession num-

ber EU362626), and its deduced protein consists of 227

amino acid residues with a predicted molecular weight of

29.5 kDa. A search of a protein database (http://www.ncbi.

nlm.nih.gov/) showed that it shares 79% amino acid

sequence identity to the soybean b-expansin protein Cim1

(accession number AAA50175), and 58% identity to AtEXPB2

(accession number NP_564860) in Arabidopsis. Comparative

analysis of the deduced amino acid sequence for GmEXPB2

with the known EXPBs in plants using the Clustal W method

revealed that conserved motifs were shared among all

EXPBs (Figure S1), including C (cysteine) residues in the

N-terminal region, an HFD (His-Phe-Asp) motif in the centre,

and W (tryptophan) residues in the putative cellulose-binding

domain in the C-terminal region (Thompson et al., 1994).

Using GmEXPB2 and other b-expansin protein sequences,

we constructed a phylogenetic tree using neighbor-joining

analysis in the MEGA 4.1 program (Figure S2) (Tamura et al.,

2007). The phylogenetic results indicated that the monocot

and dicot EXPBs are divided into two distinct groups, with

strong bootstrap support, suggesting that the most recent

common ancestor of EXPBs was probably a single-copy

EXPB in the plant kingdom, which was duplicated in both

monocots and dicots. Furthermore, EXPBs in monocots

could also be separated into two sub-groups, representing

EXPBs expressed in vegetative or generative tissues (Fig-

ure S2). Due to the lack of information on EXPBs from dicots,

it was hard to further separate the EXPBs in dicots into sub-

groups. However, the generative pistil-specific b-expansin in

tobacco, PPAL (AAG52887), was separated from the other

sub-branches in the phylogenetic tree, far from GmEXPB2

542 Wenbing Guo et al.

ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552

and Cim1, implying that GmEXPB2 may belong to the

vegetative EXPB group.

Using the programs PSORT (Nakai and Kanehisa, 1992)

and TargetP (http://www.cbs.dtu.dk/services/TargetP/), it

was predicted that GmEXPB2 had a signal peptide for entry

into the secretory pathway and secretion to the cell wall.

To verify the subcellular localization of GmEXPB2, the

GFP reporter gene translationally fused to the GmEXPB2

coding region was used in a transient assay using onion

epidermis cells. After cell plasmolysis by addition of 30%

sucrose solution, laser confocal scanning microscope was

used to check whether GmEXPB2 was located on the cell

wall or the plasma membrane. The results clearly indicate

that GmEXPB2 is located on the cell wall and Hechtian

strands and in the cytoplasm (Figure 1).

We obtained the promoter sequence of GmEXPB2 using

TAIL-PCR (Liu and Whittier, 1995). A 1225 bp region

upstream from the transcription start site was isolated

and deposited in the GenBank database (accession number

FJ461673). In silico analysis of the promoter sequence

was performed using the software programs TPSS-TCM

(Shahmuradov et al., 2005), NSITE-PL (http://www.softberry.

com) and PLACE (Higo et al., 1999). The TATA box was

predicted to be located at positions -21 to -29 upstream of

the transcription start site. Some known hormone-related

motifs were predicted to be present in the promoter region,

including three GA-responsive elements (GARE-AT, GATA

motif, pyrimidine box), an ABA-responsive element (ABRE),

an auxin-responsive element (AUX-D4), a cytokinin-

enhanced protein binding motif (CPB) and an ethylene-

responsive element (ERE). Some putative cis-elements

regulated by environmental factors such as dehydration

(MYC), elicitors (ElRE), light (GT-1), pathogens and salt

(GT-1), and low temperature (LTRE) were also present

(Table S1).

GmEXPB2 expression occurred primarily in the roots

and was up-regulated by Pi and Fe starvation

Using a quantitative real-time PCR technique to evaluate

the temporal and spatial expression patterns of GmEXPB2

in response to Pi starvation, we found that the transcripts

of GmEXPB2 were primarily localized in the root and were

up-regulated by Pi starvation (Figure 2a). GmEXPB2 was

most abundantly expressed in roots, followed by expression

in hypocotyls, but not in leaves. The GmEXPB2 expression

levels in roots increased with increasing treatment time,

especially under Pi starvation. This implied that GmEXPB2

may be involved in adaptive changes of roots in response to

P deficiency.

To determine the response specificity of GmEXPB2 to

nutrient stresses, 10-day-old soybean seedlings subjected

to various nutrient deficiencies were used for quantitative

real-time PCR analysis. GmEXPB2 expression in roots was

significantly up-regulated by Pi and Fe starvation but not

by lack of the other macronutrients, including N and K

(Figure 2b).

Histochemical detection of GmEXPB2 promoter activity

in soybean transgenic hairy roots

Expression of a 500 bp region of the GmEXPB2 pro-

moter::GUS reporter construct by soybean transgenic hairy

roots was confirmed by PCR analysis. These hairy roots

were treated with and without P for a week, and then har-

vested for GUS activity staining. Promoter::GUS activity was

clearly induced in the transgenic hairy roots under Pi star-

vation, but was almost undetectable under high-P condi-

tions (Figure 3). Staining of longitudinal sections showed

that GmEXPB2 was mainly expressed in the root cap and

stele (Figure 3a), while staining of cross-sections indicated

that expression of GmEXPB2 in the stele occurred mostly

in the phloem and pericycle cells (Figure 3b). Strong GUS

staining in response to P deficiency was also seen in

GFP PI Merged

GFP

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

GFP-GmEXPB2

GFPplasmolysis

GFP-GmEXPB2plasmolysis

Figure 1. Subcellular localization of GmEXPB2 fused to GFP in epidermal

onion cells.

For the plasmolyzed cells in (d–f) and (j–l), plasmolysis was induced by adding

30% sucrose solution prior to confocal scanning. Cells were observed by

green GFP fluorescence of the GmEXPB2 protein and red propidium iodide

(PI) fluorescence of the cell wall. Scale bar = 150 lm.

GmEXPB2 involved in root system architecture responses 543


emerged lateral root primordia originating from pericycle

cells (Figure 3b), suggesting that GmEXPB2 may be directly

involved in lateral root development under Pi starvation.

Altered expression of GmEXPB2 in soybean led to

contrasting responses of transgenic composite plants

to Pi starvation

As GmEXPB2 is mainly expressed in roots, it is practicable

to use a ‘composite plant system’ that we have recently

developed, comprising transgenic hairy roots attached to

wild-type shoots for ‘whole-plant’ functional analysis. A

highly efficient protocol has been described for Agrobacte-

rium rhizogenes-mediated transformation of soybean to

develop transgenic composite plants, in which 25–80% of

the hairy roots were co-transformed (Kereszt et al., 2007). In

the present study, we further improved the transformation

efficiency by wrapping the infection sites with rock-

wool containing antibiotics. Non-transformed roots were

efficiently inhibited using this modification. GUS staining of

hairy roots carrying the control construct (pCAMBIA1305.2)

indicated that transformation of more than 90% of the hairy

roots could be achieved in the transgenic soybean com-

posite plants (Figure S3). Considerable increases and de-

creases in GmEXPB2 transcripts were detected in hairy roots

carrying GmEXPB2 over-expression and RNAi constructs,

respectively, at low P, but not at high P (Figure S4).

The expression levels of GmEXPB2 in transgenic hairy

roots were consistent with the composite plant growth.

Transgenic soybean composite plants with GmEXPB2 over-

expressing hairy roots grew much better than those

expressing the empty vector control and GmEXPB2 knock-

down hairy roots at low P (Figure 4). Over-expression of

GmEXPB2 resulted in 28 and 24% increases in fresh weight

and plant P content, respectively, at low P, while knockdown

of GmEXPB2 resulted in 19 and 22% reductions, respectively

(Figure 4a,b). This indicated that GmEXPB2 expression in

roots enhances P efficiency in soybean. As expected, the

roots of GmEXPB2 over-expressing transgenic soybean

composite plants were 36% longer, and those of knockdown

lines were 20% shorter compared with control plants at low

P (Figure 4c). This implies that the expression of GmEXPB2

in soybean roots can dramatically regulate root develop-

ment, and thus influence plant P efficiency.

Over-expression of GmEXPB2 in Arabidopsis promoted

root growth and P efficiency

In order to better understand the functions of GmEXPB2

in root growth as well as P efficiency in plants, the same

construct used for GmEXPB2 over-expression in composite

soybean hairy roots was introduced into Arabidopsis

ecotype Col-0 by Agrobacterium tumefaciens-mediated

transformation. After identifying the transgenic plants on

selection medium, three transgenic lines with varying levels

of expression of GmEXPB2 were used for further studies

(Figure S5a). We also detected expression of AtCYCB1:1

(At4g37490, a B-type cyclin), a marker gene for cell division

(Nacry et al., 2005; Sanchez-Calderon et al., 2005). In con-

trast to wild-type, all three transgenic lines had much higher

AtCYCB1:1 expression (Figure S5b). Ectopic expression of

GmEXPB2 obviously caused accelerated growth in Arabid-

opsis on the P-rich growth medium (Figure S6). To further

analyze the responses of the root system and plant P effi-

ciency as effected by GmEXPB2 in Arabidopsis, short- and

long-term Pi starvation experiments were performed in agar

and hydroponic culture, respectively. Short-term low P

supply significantly inhibited plant growth, especially root

growth, as indicated by much shorter primary and lateral

root length in low P-grown plants (Figure 5a). However, the

roots of the GmEXPB2 over-expressing lines grew much

better than those of wild-type plants, as indicated by longer

primary and lateral roots, especially under low-P conditions

(Figure 5). After 16 days of low-P treatment, the primary and

(b) 2.0

1.5

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15 25 35 15 25 35 15 25 35

Leaves Hypocotyls Roots

–P

+P

Tissue

DAT

(a)

Figure 2. Expression pattern analysis for GmEXPB2 under low and high P

availability.

(a) Temporal and spatial expression patterns. Plants were grown on )P (no P

added) and +P (1 mM P added as KH2PO4) for 15, 25 and 35 days. Samples

from leaves, hypocotyls and roots were analyzed.

(b) Response of GmEXPB2 to various nutrient stresses. Ten-day-old soybean

seedlings were subjected to P ()P), N ()N), K ()K) or Fe ()Fe) deficiency (see

Experimental Procedures). Seedlings grown in half-strength Hoagland’s

solution were used as controls (CK, 0.25 mM P added as KH2PO4). The

expression levels in shoots and roots were analyzed by quantitative real-time

PCR. Values are the means of three biological replications � standard error.



lateral roots of the three GmEXPB2 over-expressing lines

were 1.7 and 4.1 times longer, respectively, than those of

wild-type plants at low P, and 1.2 and 2.4 times longer at

high P (Figure 5b,c). These results show a significant

enhancement effect of GmEXPB2 on root growth under both

low- and high-P conditions.

(a)

(b)

Figure 3. Histochemical detection of GUS activ-

ity under the control of the GmEXPB2 promoter

in transgenic soybean hairy roots at two P levels.

(a) GUS staining of transgenic hairy roots,

including whole root staining (left) and longitu-

dinal sections from the root tip region (right).

(b) Cross-sections from the elongation zone of

hairy roots.

)P: no P added; +P: 1 mM P added as KH2PO4.

8.0

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(m/p

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(b)

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OX RNAi CK OX RNAi CK

+P

Figure 4. Effects of over-expression and knock-

down of GmEXPB2 on root growth and P

efficiency of transgenic soybean composite

plants.

(a) Increase in fresh weight; (b) plant P content;

(c) total hairy root length. OX, GmEXPB2 over-

expressing plant; RNAi, GmEXPB2 knockdown

plant; CK, empty vector plant. )P: no P added, +P:

1 mM P added as KH2PO4. The increase in fresh

weight was calculated as the fresh weight after

harvest minus the fresh weight before treatment.

Each transgenic soybean composite plant had

more than 90% transgenic roots (Figure S3), and

each transgenic root represented one indepen-

dent transgenic line. One independent trans-

genic plant was considered as one biological

replication. Values are the means of 10 biological

replications � standard error.



To demonstrate whether the changes in root growth were

caused by modifications in root cell division and elongation

in GmEXPB2 over-expressing Arabidopsis, we measured

the size of the meristematic and elongation zones, and the

number and mean lengths of cortex cells in the meristematic

and elongation zones. We found that over-expressing

GmEXPB2 in Arabidopsis dramatically increased the size of

both the meristematic and elongation zones at both P levels

(Figure 6a,b). Compared to wild-type plants, the three

GmEXPB2 over-expressing lines showed 66, 69 and 80%

increases in meristem size at low P, and 53, 41 and 34%

increases at high P. The increases in the size of the

elongation zone were 37, 44 and 47% increase at low P,

and 34, 31 and 31% at high P (Figure 6c,d). In the meristem,

over-expressing GmEXPB2 in Arabidopsis mainly promoted

cell division, as indicated by more cortex cells in the three

GmEXPB2 over-expressing lines compared to wild-type

under low P (47, 55 and 55% increases) and high P (56, 51

and 46% increases) (Figure 6e). The mean length of the

cortex cells in the meristem increased by 13, 9 and 16%

at low P in GmEXPB2 over-expressing lines, but no such

increase was seen at high P (Figure 6g). In the elongation

zone, over-expressing GmEXPB2 facilitated both cell divi-

sion and elongation as indicated by increases in the number

of cortex cells of 23, 18 and 21% at low P, and 16, 14 and 8%

at high P. The lengths of the cortex cells in the elongation

zone increased by 11, 21 and 21% at low P and by 16, 14 and

21% at high P in the three GmEXPB2 over-expressing lines

compared to wild-type (Figure 6f,h).

Consistent with the results of the short-term experiments,

the GmEXPB2 over-expressing plants also grew much better

than the wild-type plants in long-term experiments. This

growth improvement in the transgenic lines was seen not

only at low P but also at high P. Dramatic increases in shoot

fresh weight (29%), root fresh weight (139%), primary root

length (83%) and plant P content (20%) were found under

low-P conditions, and 52, 6, 12 and 35% increases for the

same parameters were found under high-P conditions

(Figure 7). These findings further confirm that GmEXPB2

affects plant P efficiency by regulating root growth.

Soybean root growth and GmEXPB2 expression was

enhanced by mild water deficiency and external

auxin supply

In order to evaluate whether GmEXPB2 expression specifi-

cally responds to Pi and Fe starvation, 3-day-old soybean

seedlings subjected to treatment with 0.2% PEG and 0.5 lM

IAA were used for determination of relative root length and

quantitative real-time PCR analysis. The results showed that

root elongation was enhanced by 11 and 20% by 0.2% PEG

and 0.5 lM IAA, respectively (Figure 8a). Compared to the

control treatment, GmEXPB2 expression was significantly

induced by PEG and IAA supplied simultaneously (Fig-

ure 8b), implying an intrinsic function of GmEXPB2 in

alterations of the root system architecture in response to

some abiotic stresses.

DISCUSSION

The responses of the root system architecture play a fun-

damental role in plant growth and adaptation to a variety of

abiotic stresses, such as nutrient deficiency and drought

(Malamy, 2005; Hodge et al., 2009). In the present study, we

cloned and characterized a b-expansin gene from soybean,

named GmEXPB2, for which a cDNA fragment was isolated

from our Pi starvation-induced cDNA library constructed

from a P-efficient soybean genotype (Guo et al., 2008). The

function of GmEXPB2 in regulation of the response of the

root system architecture to abiotic stress, especially Pi star-

vation, has been analyzed.

(a)

WT OX1 OX2 OX3 WT OX1 OX2 OX3

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5.0

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Figure 5. Short-term effects of Pi starvation on root growth and development

of Arabidopsis GmEXPB2 over-expressing lines.

(a) Phenotypic responses of wild-type (WT) and three 35S:GmEXPB2 over-

expression lines (OX).

(b) Primary root length.

(c) Lateral root length.

Plants were grown on agar plates at two P levels for 16 days. )P: no P added;

+P: 1 mM P added as KH2PO4. Values are the means from three independent

experiments � standard errors. Each experiment had 10 biological replica-

tions (n = 10).



In contrast to other expansin-encoding genes, GmEXPB2

is a b-expansin gene that has been cloned from and

functionally analyzed in dicots. Three major features of this

expansin suggest that it is indeed a member of the

b-expansin family: (i) it possesses the conserved motifs of

EXPBs based on comparison of its deduced amino acid

sequence with those of other b-expansins (Figure S1); (ii) it

exhibits high homology to classical EXPBs such as Cim1

(79%) from soybean and AtEXPB2 (58%) from Arabidopsis

(Wieczorek et al., 2006) (Figure S2); and (iii) it is localized, at

least in part, on the cell wall (Figure 1), as has been predicted

for other EXPBs By in silico prediction and shown directly for

OsEXPB3 by immunogold labeling (Lee and Choi, 2005).

Phylogenetic tree analysis showed that GmEXPB2 belongs

to the dicot group of b-expansins and is closely related to the

other vegetative EXPBs, including Cim1 and AtEXPB2, but is

quite distant in sequence from PPAL, which is a generative

dicot EXPB (Figure S2). Furthermore, the high transcript

abundance of GmEXPB2 in vegetative tissues such as roots

and hypocotyls shows that GmEXPB2 is indeed a vegetative

b-expansin (Figures 2 and 3).

It has been suggested that expansin may be involved in

cell division and elongation, as cell-wall synthesis takes

place during cell division to create new transverse separat-

ing daughter cells (Muller et al., 2007). Here, we present the

direct evidence that GmEXPB2 could promote cell division

and elongation in root apex as indicated by the increased

cortex cell number and length in the roots of Arabidopsis

GmEXPB2-overexpression lines. The numbers of cortex

cells in meristem and elongation zone were increased under

both low P and high P conditions. Interestingly, the cortex

cells in elongation zone were longer under both conditions,

while the ones in meristem zone were longer only under low

P condition (Figure 6). This was confirmed by the higher

expression levels of the cell division marker gene AtCYCB1:1

in Arabidopsis GmEXPB2 over-expressing lines (Fig-

ure S5b). Furthermore, both quantitative real-time PCR

nalysis in soybean roots and histochemical promoter–GUS

activity staining using transgenic soybean hairy roots

showed that GmEXPB2 expression occurs primarily in the

root and is up-regulated by Pi starvation, especially in the

primary and lateral root elongation zones (Figures 2 and 3).

15

10

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60

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)C

orte

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mbe

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/roo

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µm)

Meristem zone Elongation zone1500

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WT OX1 OX2 OX3 WT OX1 OX2 OX3 WT OX1 OX2 OX3 WT OX1 OX2 OX3

–P +P –P +P

(d)(c)

(f)(e)

(h)(g)

(a) (b)

WT OX –P WT OX +P

Figure 6. Effects of over-expressing GmEXPB2

on root cell division and elongation of transgenic

Arabidopsis lines as regulated by P availability.

(a, b) Root apex. The red and green lines repre-

sent the meristematic and elongation zones,

respectively. Scale bar = 50 lm.

(c, d) Sizes of the meristematic and elongation

zones.

(e, f) Number of cortex cells in the meristematic

and elongation zones.

(g, h) Mean length of cortex cells in the meriste-

matic and elongation zones.

Wild-type (WT) and three 35S:EXPB2 over-

expression lines (OX) were grown on agar plates

at two P levels for 7 days ()P: no P added; +P:

1 mM P added as KH2PO4). Values are the means

from three independent experiments � standard

errors. Each experiment had 10 biological repli-

cations (n = 30).



GUS activity driven by the GmEXPB2 promoter in transgenic

hairy roots was particularly high in the pericycle cells of the

stele and in the root cap (Figure 3). As pericycle cells are

specialized cells that form lateral roots, and the root cap can

mediate root architectural changes (Tsugeki and Fedoroff,

1999; Lopez-Bucio et al., 2005), this result implied that

GmEXPB2 may be involved in lateral root development

and root architectural responses to P deficiency. Moreover,

strong expression of GmEXPB2 was also observed in the

vascular tissues (Figure 3), indicating that GmEXPB2 may

have unknown functions in addition to involvement in

regulating root meristematic activity and lateral root forma-

tion in response to Pi starvation in soybean.

Interestingly, expression of GmEXPB2 is not only induced

by P deficiency, but also strongly induced by Fe and mild

water deficiency as well as external IAA supply in soybean

roots (Figures 2 and 8). It is well accepted that P and Fe

deficiency have similar effects on the differentiation of

epidermal cells, and subsequently affect root growth, such

as lateral root and root hair formation (Schmidt and Schik-

ora, 2001; Lopez-Bucio et al., 2003; Nacry et al., 2005; Lai

et al., 2007). In our study, we also found that over-express-

ing GmEXPB2 in Arabidopsis significantly facilitated root

hair growth (as indicated by root hair density) and initiation

of lateral root primordia (as indicated by primordia number)

under both low- and high-P conditions (Figure S7). As auxin

has been shown to play a major role in regulation of the root

system architecture (Hodge et al., 2009), the induction of

GmEXPB2 expression by external auxin supply implied an

intrinsic function of GmEXPB2 in root system architecture

changes. Furthermore, over-expressing GmEXPB2 in both

soybean composite plants and Arabidopsis induces root

elongation even under nutrient-sufficient conditions (Fig-

ures 4,5 and 7). Expression of GmEXPB2 was induced by

long- but not short-term Pi starvation (Figure 2), and also

induced in a P-inefficient genotype (Figure S8). All the

evidence strongly supports the conclusion that GmEXPB2

is an intrinsic regulator of root system architecture changes.

WT OX WT OX

–P +P

(a)

(b) 0.7

0.6

0.5

0.4

0.1

0

Shoo

t fre

sh w

eigh

t(g

/pla

nt)

(c) 70

50

30

10

Roo

t fre

sh w

eigh

t(m

g/pl

ant)

14.0

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leng

th(c

m/p

lant

)

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–P +P

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l P c

onte

nt(μ

g/pl

ant)

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–P +PWT OX WT OX

16.0

12.0

8.0

4.0

0

Figure 7. Long-term effects of Pi starvation on growth of Arabidopsis

GmEXPB2 over-expressing lines.

Ten-day-old plants were grown in hydroponic culture with P (+P: 1 mM P

added as KH2PO4) or without P ()P: no P added) for 50 days.

(a) Phenotypic responses of wild-type (WT) and 35S:GmEXPB2 over-expres-

sion lines (OX).

(b) Shoot fresh weight.

(c) Root fresh weight.

(d) Primary root length.

(e) P content in plants.

Values are the means of four biological replications � standard error.

130

120

110

100

90

80

Rel

ativ

e ro

ot le

ngth

(%

)

CK 0.2% PEG 0.5 µM IAA

(a)

CK 0.2% PEG 0.5 µM IAA

(b) 1.5

1.0

0.5

0R

elat

ive

expr

essi

on v

alue

Shoots

Roots

Figure 8. Effect of mild water deficiency and IAA supply on soybean root

growth and GmEXPB2 expression.

(a) Relative root length.

(b) Relative GmEXPB2 expression.

Three-day-old soybean seedlings were treated with 0.2% PEG or 0.5 lM IAA

for 5 days (see Experimental Procedures). The expression levels in shoots and

roots were analyzed by quantitative real-time PCR. Values are the means of

three biological replications � standard error.



In addition to the intrinsic involvement of GmEXPB2 in

root system architecture changes in response to abiotic

stress, it is reasonable to speculate that GmEXPB2 may have

great potential in improving plant P efficiency. GmEXPB2

was cloned from HN89, a P-efficient genotype, which was

previously shown to acquire more P through improved root

system architecture (Zhao et al., 2004; Yan et al., 2006; Guo

et al., 2008), and expression of GmEXPB2 was much higher

in HN89 (Figure S8a). In addition, the growth reduction

of primary roots was much more severe in HN112 (a

P-inefficient genotype) than in HN89 (Figure S8b). More-

over, over-expression of GmEXPB2 in both hairy roots of

transgenic soybean composite plants and Arabidopsis sig-

nificantly attenuates the root growth inhibition caused by Pi

starvation, and thus increases P efficiency, as indicated by

greater plant biomass and P uptake under low-P conditions

(Figures 4 and 7). We therefore conclude that GmEXPB2

could affect plant P efficiency by regulating root adaptive

changes in response to Pi starvation.

In summary, we report here the cloning and functional

characterization of a vegetative b-expansin gene, GmEXPB2,

in soybean, and show that this gene may function in the

intrinsic control of root system architecture changes in

response to some abiotic stresses, enhancing P efficiency

and P responsiveness. These findings could have significant

agricultural potential for improving crop P uptake on soils

with both low and high P availability.

EXPERIMENTAL PROCEDURES

Soybean materials and growth conditions

Soybean cv. HN89 plants were used in this study. Surface-sterilizedseeds were germinated and grown in silicon sand containingmodified half-strength Hoagland solution (Hoagland and Arnon,1938) for 10 days, with 0.25 mM KH2PO4 as the P supply. Thenutrient solution contained 2.5 mM Ca, 3.25 mM K, 1.0 mM Mg,7.5 mM NO�3 , 0.25 mM SO2�

4 (macronutrients) and 82 lM Fe-EDTA,4.57 lM Mn, 0.38 lM Zn, 1.57 lM Cu, 0.54 lM NHþ4 , 0.63 lM MoO�4 ,23.13 lM B, 9.14 lM Cl) (micronutrients). For temporal and spatialanalysis on the expression patterns of GmEXPB2 in response to Pistarvation, uniform seedlings were grown in half-strength of Hoa-gland solution for 10 days, and then treated with P supply (+P: 1 mM

P added as KH2PO4) and without P supply ()P: no P added) for 15, 25and 35 days. Leaves, hypocotyls and roots were harvested sepa-rately for further analysis.

To assess the responses of GmEXPB2 to nutrient stresses,ten-day old seedlings grown under normal conditions (0.25 mM

KH2PO4) were treated under N-, P-, K- or Fe-deficient conditionsfor 30 days. For N deficiency, KNO3 was replaced by 2.5 mM

K2SO4, Ca(NO3)2 was replaced by 2.5 mM CaSO4 and(NH4)6Mo7O24 was replaced by 0.54 lM Na2MoO4. For P deficiency,KH2PO4 was replaced by 0.125 mM K2SO4. For K deficiency, KNO3

was replaced by 1.25 mM Ca(NO3)2, K2SO4 was replaced by0.25 mM CaSO4 and KH2PO4 was replaced by 0.25 mM NaH2PO4.For Fe deficiency, Fe-EDTA was omitted. Plants grown undernormal conditions were used as a contrast check (CK). Eachtreatment had three biological replicates. Leaves and roots werecollected separately for total RNA extraction and quantitative real-time PCR analysis.

To determine whether GmEXPB2 is the intrinsic control for rootgrowth responses to abiotic stress, seeds of soybean were germi-nated on germination paper (Anchor paper Co., http://www.anchorpaper.com) for 3 days, then seedlings were transplantedinto nutrient solution containing 0.2% PEG or 0.5 lM IAA. Five daysafter treatment, roots and shoots were harvested separately for totalRNA extraction and quantitative real-time PCR analysis. Relativeroot length was calculated as the percentage of root length underPEG or IAA treatment relative to control.

Isolation of the full-length cDNA of GmEXPB2

The cDNA fragment of GmEXPB2 isolated from the subtractivelibrary was 447 bp long (Guo et al., 2008). Based on the sequencesof the cDNA fragment, the gene-specific primers EXPB2GSPF andEXPB2GSPR, together with two nested primers EXPB2GSPnestFand EXPB2GSPnestR, were designed (Table S2) to obtain the full-length cDNA by RACE-PCR (Frohman, 1990). RACE was performedusing a GeneRacerTM kit and SuperscriptTM reverse transcriptase(Invitrogen, http://www.invitrogen.com/) according to the manu-facturer’s instructions. The resultant PCR product was cloned intothe pGEM-T Easy vector (Promega, http://www.promega.com/) andthen sequenced. After alignment, the complete GmEXPB2cDNA sequence was submitted to the National Center for Biotech-nology Information GenBank database under accession numberEU362626.

Amplification of promoter and TAIL-PCR procedure

The full-length cDNA sequence of GmEXPB2 was used to isolate theunknown 5¢ flanking regions via TAIL-PCR (Liu and Whittier, 1995).Total genomic DNA for use as the template was extracted fromsoybean shoots using the standard cetyltrimethylammonium bro-mide (CTAB) method (Murray and Thompson, 1980). TAIL-PCR wasperformed as previously described, and the arbitrary degenerate(AD) primers were the same as those used by Liu and Whittier(1995). The promoter of GmEXPB2 was obtained through two timesof TAIL-PCR. The gene-specific primers EXPB-R1, EXPB-R2 andEXPB-R3 were designed according to the sequence of the 5¢ UTR ofGmEXPB2. The sequence of the isolated fragment was further usedto isolate its upstream sequence, and further specific primers EXPB-R4, EXPB-R5 and EXPB-R6 were designed. All the primer sequencesare given in Table S2.

Subcellular localization

The coding region for GmEXPB2 was amplified using the oligonu-cleotide primers 5¢-GCATGTCGACATGGCTCCTACACTTCAACG-TGCA-3¢ and 5¢-GCATCCATGGTTAGCTTGATGGAGAATGGTGC-3¢.After digestion with SalI and NcoI (underlined in primer sequences),the fragments were fused to and cloned in-frame with the GFPcoding sequence in a pUC18-based vector and placed under thecontrol of the CaMV 35S promoter (Cormack et al., 1996). Afterconfirmation by sequencing, the constructs were transiently trans-formed into onion epidermal cells (Scott et al., 1999) on agar platesusing a helium-driven accelerator (PDS/1000, Bio-Rad, http://www.bio-rad.com/). Bombardment parameters were as follows:1100 psi bombardment pressure, 1.0 mm gold particles, a distanceof 9 cm from macrocarrier to the samples, and a decompressionvacuum of 1000 psi. A day later, the bombarded epidermal cellswere plasmolyzed by adding 30% sucrose solution for 20 minbefore confocol scanning. GFP expression was viewed using aconfocal scanning microscope system (ZEISS 510 META, http://www.zeiss.com/) with 488 nm laser light for fluorescence excitationof GFP, and detection using a 515–545 nm filter (green; GFP fluo-rescence) and a 610 nm filter (red; propidium iodide fluorescence).



Quantitative real-time RT-PCR analysis

For quantitative real-time PCR analysis, the soybean housekeepinggene TefS1 encodingelongation factor EF-1a (Accession number:X56856) was used as a reference gene. The optimal primersequences for GmEXPB2 and TefS1 are given in Table S2. RNA wastreated with DNase I to remove contaminating genomic DNA beforesynthesizing first-strand cDNA using MMLV reverse transcriptase(Promega) according to the manufacturer’s instructions. The cDNAproducts were used as templates. A series of dilutions of mixedcDNAs were used to produce the standard curve. PCR reactionswere performed in a 20 ll volume containing 2 ll 1:100 diluted re-verse transcription product, 0.2 lM primers and 10 ll QuantiTectTM

SYBR� Green PCR master mix (Qiagen, http://www.qiagen.com/).All the reactions were performed on a Rotor-Gene 3000 (CorbettResearch, http://www.corbettlifescience.com/).

Transgene constructs

For the GmEXPB2 promoter–GUS fusion, a 519 bp fragmentcorresponding to the GmEXPB2 promoter and including thefirst 19 bp of the GmEXPB2 coding sequence was amplifiedusing primers 5¢-TCTAGGATCCTGGTTTGAGCTTGACCTTTT-3¢ and5¢-GCATCCATGGGTTGAAG TGTAGGAGCCAT-3¢ with soybeangenomic DNA as template. The 5¢ and 3¢ primers contain BamHIand NcoI sites, respectively (underlined in primer sequences). ThePCR fragment was digested and ligated into the BamHI and NcoIsites of the pCAMBIA3301 vector (CAMBIA, http://www.cambia.org), resulting in a translational fusion of the first six amino acidsof the GmEXPB2 protein to the uidA open reading frame(Figure S9a). The pCAMBIA3301 vector containing the GmEXPB2promoter region was transformed into Agrobacterium rhizogenesstrain K599 by electroporation. For the over-expression construct,the ORF region of GmEXPB2 was amplified and inserted into apCAMBIA1305.2-based vector with a CaMV 35S promoter (Fig-ure S9b). The resulting construct was used for transformation intoboth soybean hairy roots and Arabidopsis. For the RNAi construct,400 bp of the GmEXPB2 coding region was cloned and insertedinto the vector in the sense and antisense orientations as shown inFigure S9c. The over-expression and RNAi constructs were basedon the same vector, modified from pCAMBIA1305.2.

Induction of transgenic soybean hairy roots

For sterile hairy root induction, plant inoculation was performed asdescribed by Cho et al. (2000) with some modifications. Briefly,soybean seeds were surface-sterilized by incubating overnight in achlorine gas atmosphere produced by a mixture of 100 ml hypo-chlorite and 4.2 ml HCl, and then germinated on half-strength solidMS medium in the dark for 4 days to collect cotyledons. The coty-ledonary nodes were wounded with a scalpel previously dipped intoovernight cultures of A. rhizogenes strain K599 containing theconstructed plasmid. The wounded cotyledons were then incubatedabaxial side up on filter paper pre-moistened in sterile water for4 days. After incubation, cotyledons were transferred to solidMS medium containing 500 lg ml)1 carbenicillin disodium and5 lg ml)1 ammonium glufosinate (Sigma, http://www.sigmaaldrich.com/) to produce transgenic hairy roots.

For production of transgenic soybean composite plants, consist-ing of a wild-type shoot with transgenic hairy roots, the hypocotylinjection method described by Kereszt et al. (2007) was used withsome modifications. Sterilized seeds (1 min in 3% H2O2, three rinseswith sterile water) were germinated in a pot filled with sands andgrowth medium at a ratio of 1:1 v/v. Five-day-old seedlings withunfolded cotyledons were infected on the hypocotyl with A. rhiz-ogenes strain K599 carrying the gene construct, and the plants were

kept under high humidity conditions. To increase transformationefficiency, the infection sites were wrapped with rockwool contain-ing 20 mg L)1 hygromycin. After approximately 30 days, when theemerged hairy roots were approximately 10 cm long, the mainroots were removed and the hairy roots were collected for RNAextraction and quantitative real-time PCR. Selected transgenicsoybean composite plants were used for further P treatments.

Histochemical GUS staining and tissue sections

For histochemical analysis of GUS expression, hairy roots wereincubated in GUS staining solution (0.1 M Na2HPO4/NaH2PO4

buffer, pH 7.0, 1 mM X-Gluc) at 37�C for 16 h, followed by wash-ing in 70% ethanol, as described by Jefferson et al. (1987). AfterGUS staining, root segments (approximately 5 mm long) weresampled and immediately fixed in FAA fixative (formaldehyde5 mL, glacial acetic acid 5 mL, 70% ethanol 90 mL) for 24 h, thendehydrated gradually in a graded ethanol series (50, 65, 75, 85 and95%). After dehydration, root samples were separately infiltratedin half- and full-strength 7022 Leica Histeresin for 24 h. Rootsamples were then embedded in an embedding solution(7022:hardener = 15:1 v:v), and then sectioned transversely to athickness of 4 lm using a microtome for observation under a lightmicroscope.

Phosphorus treatment of transgenic soybean composite

plants and determination of plant growth performance

and P efficiency

After removal of tap roots, all composite plants (GmEXPB2 over-expression, RNAi constructs and empty vector control) were cul-tured in half-strength Hoagland solution with 1 mM KH2PO4 for1 week for root growth, then treated with P (+P: 1 mM P added asKH2PO4) or without P ()P: no P added) for 20 days. In orderto reduce the growth differences caused by transformation pro-cesses the increase in fresh weight was used as a measure ofplant growth performance, calculated as the fresh weight afterharvest minus the fresh weight before treatment. Sampled rootswere scanned as digital images using a specialized color scanner(Epson Expression 800; Seiko Epson, http://global.epson.com/).Root length was quantified using a computer image program(WinRhizo Pro; Regent Instruments, http://www.regent.qc.ca/). Pcontent was analyzed as described above. More than 90% of theroots of each transgenic soybean composite plant were transgenic(Figure S3), and each transgenic root represented one indepen-dent transgenic line. One independent transgenic plant carryingthe same construct was considered as one biological replication.For each construct in each treatment, ten transgenic plants weretested.

Transgenic Arabidopsis over-expressing GmEXPB2

The over-expression construct was transformed into Agrobacte-rium tumefaciens strain Gv3101 by electroporation and then intro-duced into Arabidopsis plants as previously described (Clough andBent, 1998) to obtain GmEXPB2 over-expressing seeds. After iden-tification on selection medium and by quantitative real-time PCRanalysis, homozygous T3 transgenic seeds were used for furtherstudies. The GmEXPB2 over-expressing lines OX1, OX2 and OX3with various levels of expression of GmEXPB2 (Figure S5a) wereused for agar culture in the short-term experiment. OX2 was sub-sequently used for the long-term hydroponics experiment.

Arabidopsis growth and analysis of root traits

For the short-term experiment on agar, ecotype Columbia (Col-0,wild-type) and GmEXPB2 over-expressing seeds were surface-



sterilized, and then germinated on 13 · 13 cm Petri dishes con-taining sterile modified Murashige and Skoog medium with low P()P: no P added) or high P (+P: 1 mM P added as KH2PO4). Seedswere firstly grown in Petri dishes under 16 h/8 h (dark/light) at 24�Cfor 5 days, then the seedlings with uniform growth were selectedand transplanted to new dishes containing fresh medium for16 days. For microscopic observation, 7-day-old seedlings werevisualized using DIC optics (BX51; Olympus, http://www.olympus-global.com/). The number and length of cortex cells in the rootmeristematic and elongation zones were determined as describedpreviously (Sanchez-Calderon et al., 2005), i.e. by counting thecortical cells in files extending from the quiescent centre of thechloral hydrate-cleared seedlings. Root parameters were measuredusing IMAGE J software (http://rsb.info.nih.gov/ij/).

The long-term hydroponics experiment was performed asdescribed by Gibeaut et al. (1997). Col-0 and GmEXPB2 over-expressing seeds were germinated and grown in hydroponicculture with high P supply (1 mM P added as KH2PO4) in a growthchamber under 16 h/8 h (dark/light), 21�C/17�C (day/night), 80%relative humidity and 150 mmol m)2 sec)1 irradiation for 10 days,and then uniform seedlings were transplanted into treatmentsolutions (+P: 1 mM P added as KH2PO4 or )P: 0 mM P added) foran additional 50 days. The pH value was adjusted to 5.7. Shoots androots were harvested separately, and P content and root parameterswere measured as described above.

Statistical analyses

All the data were analyzed statistically using Microsoft Excel 2000(http://www.microsoft.com/) for calculating mean and standarderror, and the SAS system for windows v6.12 (SAS Institute Inc.,http://www.sas.com/) for two-way ANOVA.

ACKNOWLEDGEMENTS

This work was jointly supported by the grants from the NationalNatural Science Foundation of China (grant numbers 30890131 and31000931) and the National Key Basic Research Special Funds ofChina (grant number2011CB100301). We are grateful to Dr Yao-guang Liu (College of Life Sciences, South China Agricultural Uni-versity) for providing the vectors, Dr Peter Gresshoff (University ofQueensland) for providing A. rhizogenes strain K599, Dr XiurongWang, Drs Chuxiong Zhuang and Lizhen Tao (College of Life Sci-ences, South China Agricultural University) and Ms Xinlan Xu(South China Botanical Garden, Chinese Academy of Sciences) fortechnical assistance, and Drs Leon Kochian and Jiping Liu (CornellUniversity), Dr. Yongchao Liang (Institute of Agricultural Resourcesand Regional Planning), Drs Lixing Yuan and Jianbo Shen (Collegeof Resources and Environmental Sciences, China Agricultural Uni-versity), Dr Yiping Tong (Institute of Genetics and DevelopmentBiology, Chinese Academy of Sciences), Dr Xuewen Hou (Collegeof Life Sciences, South China Agricultural University) for criticalreview of the manuscript.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Alignment of GmEXPB2 amino acid sequence to those ofother EXPBs in plants.Figure S2. Phylogenetic analysis of GmEXPB2 in plants.Figure S3. GUS staining on the hairy roots of transgenic soybeancomposite plants containing the empty vector (pCAMBIA1305.2).Figure S4. Relative expression value of GmEXPB2 in GmEXPB2over-expressing, RNAi and empty vector (control) lines of trans-genic soybean composite plants at two P levels.

Figure S5. Relative expression values of GmEXPB2 and AtCYCB1:1in various transgenic Arabidopsis lines at two P levels.Figure S6. GmEXPB2 over-expressing and wild-type Arabidopsislines grown on P-rich growth medium.Figure S7. Effects of over-expressing GmEXPB2 on root hair andlateral root formation in Arabidopsis.Figure S8. Expression levels of GmEXPB2 and primary root lengthof the two contrasting soybean genotypes as regulated by Pavailability.Figure S9. Binary constucts for over-expression and promoteranalysis.Table S1. Putative responsive cis-elements in the GmEXPB2promoter.Table S2. Oligonucleotides used in this study.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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The accession numbers for the GmEXPB2 cDNA sequence and GmEXPB2 promoter sequence are EU362626 and FJ461673,

respectively.



a soybean expansin gene gmexpb2 intrinsically involved in ... soybean b-expansin... · keywords:...

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