A soybean expansin gene GmEXPB2 intrinsically involved in ... soybean b-expansin... · Keywords:...
Transcript of 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
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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
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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
1.0
0.5
0
Rel
ativ
e ex
pres
sion
val
ue
CK –P –N –K –FeTreatment
ShootsRoots
1.6
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pres
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val
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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.
544 Wenbing Guo et al.
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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
6.0
4.0
2.0
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esh
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g/pl
ant)
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mg/
plan
t)
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t len
gth
(m/p
lant
)
(a)
(b)
(c)
60
40
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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.
GmEXPB2 involved in root system architecture responses 545
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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
–P +P
OW
Prim
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root
leng
th (
cm/p
lant
) L
ater
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oot
leng
th (
cm/p
lant
)
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030
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20
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8 10 12 14 16 8 10 12 14 16 (d)
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4.0
3.0
2.0
1.0
0
6.0
5.0
4.0
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0
–P +P
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).
546 Wenbing Guo et al.
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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
5
0
90
60
30
0
600
400
200
045
30
15
0
Size
(µm
)C
orte
x ce
ll nu
mbe
r(#
/roo
t)
Ave
rage
leng
th o
fco
rtex
cel
ls (
µm)
Meristem zone Elongation zone1500
1000
500
018
12
6
0
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).
GmEXPB2 involved in root system architecture responses 547
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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
12.0
7.0
5.0
Prim
ary
root
leng
th(c
m/p
lant
)
(d)
WT OX WT OX
–P +P
Tota
l P c
onte
nt(μ
g/pl
ant)
(e)
–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.
548 Wenbing Guo et al.
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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).
GmEXPB2 involved in root system architecture responses 549
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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-
550 Wenbing Guo et al.
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552
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.
552 Wenbing Guo et al.
ª 2011 South China Agricultural UniversityThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 541–552