Plant Physiology and Biochemistry 78 (2014) 53e62

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Research article

The involvement of expansins in responses to phosphorus availability in wheat, and its potentials in improving phosphorus efficiency of plants Yang-yang Han a, b,1, Shan Zhou a,1, Yan-hui Chen a, Xiangzhu Kong a, Ying Xu a, Wei Wang a, * a State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, PR China b Plastic Surgery Institute of Weifang Medical University, Weifang, Shandong 261041, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 September 2013 Accepted 22 February 2014 Available online 3 March 2014

Phosphorus (P) is a critical macronutrient required for numerous functions in plants and is one of the limiting factors for plant growth. Phosphate availability has a strong effect on root system architecture. Expansins are encoded by a superfamily of genes that are organized into four families, and growing evidence has demonstrated that expansins are involved in almost all aspects of plant development, especially root development. In the current study, we demonstrate that expansins may be involved in increasing phosphorus availability by regulating the growth and development of plant roots. Multiple expansins (five a- and nine b-expansin genes) were up- or down-regulated in response to phosphorus and showed different expression patterns in wheat. Meanwhile, the expression level of TaEXPB23 was upregulated at excess-P condition, suggesting the involvement of TaEXPB23 in phosphorus adaptability. Overexpression of the TaEXPB23 resulted in improved phenotypes, particularly improved root system architecture, as indicated by the increased number of lateral roots in transgenic tobacco plants under excess-P and low-P conditions. Thus, these transgenic plants maintained better photosynthetic gas exchange ability than the control under both P-sufficient and P-deficient conditions. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Expansin Phosphorus availability Root system architecture Transgenic tobacco Wheat

1. Introduction Phosphorus (P) is one of the six essential macronutrients that plants acquire from soil and utilize for their growth and development (Ribot et al., 2008). Although the total amount of phosphorus in soil may be high, inorganic phosphate (Pi), the main form of phosphorus assimilated by plants, is relatively inaccessible to plant roots due to its low solubility and high-sorption capacity in soil, and thus Pi availability often limits plant growth. To maintain Pi concentrations within the critical limit required for optimal development, plants have evolved a wide variety of responses at the

Abbreviations: DW, dry weight; DTT, dithiothreitol; E, transpiration rate; EDTA, ethylenediaminetetra acetate; FW, fresh weight; Gs, stomatal conductance; IAA, indole-3-acetic; K, potassium; LR, lateral root; P, phosphate; Pi, inorganic phosphate; Pn, net photosynthesis; RSA, root system architecture; WT, wild type. * Corresponding author. Tel.: þ86 538 8246166; fax: þ86 551 8242288. E-mail addresses: [email protected], [email protected] (W. Wang). 1 The two authors contributed equally to this paper. http://dx.doi.org/10.1016/j.plaphy.2014.02.016 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.

morphological, physiological, biochemical, and gene expression levels aimed at regulating and optimizing Pi acquisition from the soil solution and its distribution to various organs and subcellular compartments (Secco et al., 2010). Over the past several years, some studies aimed at analyzing gene expression patterns using microarrays have revealed a complex network of genes that are up- or down-regulated at various points following Pi starvation either in roots or shoots (Morcuende et al., 2007; Müller et al., 2007). Using Arabidopsis ATH1 arrays, Morcuende et al. found that phosphorus deprivation leads to induction or repression of >1000 genes involved in many processes. Among these genes, an expansin gene (GenBank ID: At2g18660) was found to be strongly induced (>20-fold) under phosphorus starvation (Morcuende et al., 2007). Expansins are encoded by a superfamily of genes that are organized into four families (Lee et al., 2001). These proteins loosen the cell wall in a pH-dependent manner and are hypothesized to break hydrogen bonds between hemicelluloses and cellulose microfibrils, thereby allowing turgor-driven cell enlargement

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(McQueen-Mason and Cosgrove, 1994). Previous reports have provided evidences that expansins are associated with environmental stress tolerance in plants. Overexpression of an expansin gene, RhEXPA4, in Arabidopsis confers strong drought tolerance to transgenic plants (Dai et al., 2012). In the resurrection plant Craterostigma plantagineum, an increase in expansin activity is accompanied by enhanced cell wall extensibility during the dehydration and rehydration processes, suggesting a role of expansin proteins in increasing wall flexibility and promoting leaf growth under drought stress (Jones and McQueen-Mason, 2004). In addition to responding to various environmental stresses, increasing evidences have demonstrated that expansins are involved in almost all aspects of plant development, especially root development. Wu et al. reported that at least two expansin genes are upregulated in apical regions of the root elongation zone in maize at low water potential, suggesting that expansins play an important role in maintaining root growth under water stress (Wu et al., 2001). The soybean b-expansin gene GmEXPB2 was demonstrated to be intrinsically involved in root system architecture (RSA), as overexpressing GmEXPB2 in Arabidopsis increases root cell division and elongation and increases plant growth and Pi uptake under both low- and high-P conditions (Guo et al., 2011). Using RNA interference (RNAi) to examine the biological function of AtEXPA7, Lin et al. demonstrated that the root hairs of RNAi transformant lines were 25e48% shorter and exhibited a broader range of lengths than those of the control, thus providing evidence that AtEXPA7 is required for root hair tip growth (Lin et al., 2011). A similar result was obtained in rice: the suppression of OsEXPA17 by RNAi confirmed the requirement for this gene in root hair elongation (Yu et al., 2011). Roots are the major organ subjected to belowground abiotic stress in nature and are responsible for the acquisition of nutrients from the soil (Guo et al., 2011), especially for phosphorus (Ward et al., 2008; Fang et al., 2009; Wang et al., 2010a). In a previous study, we demonstrated that the wheat b-expansin gene TaEXPB23 was expressed in the roots of ProTaEXPB23::GUS transgenic tobacco plants (Han et al., 2012). Transgenic tobacco plants overexpressing TaEXPB23 had more roots than the control plants (Xing et al., 2009). Therefore, we hypothesized that TaEXPB23 may play a role in adaptive RSA changes in response to different P treatments. In this study, we first analyzed the expression patterns of multiple expansin genes affected by P application in wheat. Then, we examined the function of TaEXPB23 in plant adaptation to P by examining the transgenic tobacco plants overexpressing TaEXPB23. Our results demonstrate that the expression level of TaEXPB23 is altered in response to P application. In addition, transgenic tobacco plants overexpressing TaEXPB23 have improved RSA, thus maintaining enhanced photosynthetic ability under P-sufficient and Pdeficient conditions.

2. Materials and methods 2.1. Plant materials, growth conditions, and treatments The experimental design was based on that of Guo et al. with some modifications (Guo et al., 2011). Wheat (Triticum aestivum L.) seedlings grown under normal conditions were cultured in fullstrength Hoagland solution, which was composed of 5 mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 1 mM KH2PO4, 0.1 mM FeEDTA (Na), 46 mM H3BO3, 9.15 mM MnCl2$4H2O, 0.765 mM ZnSO4$7H2O, 0.32 mM CuSO4$5H2O, and 0.89 mM H2MoO4$H2O (Hoagland and Arnon, 1950). The nutrient solution was adjusted to pH 5.7e5.8. Two levels of P treatment were applied: 0.1 mM KH2PO4 (LP) and 10 mM KH2PO4 (EP). For the K experiment, KNO3

Table 1 Primer sequences. Name

Sequence (50 e30 )

Length (bp)

TaEXPB23-F TaEXPB23-R Tubulin-F Tubulin-R TaEXPA1-F TaEXPA1-R TaEXPA4-F TaEXPA4-R TaEXPA5-F TaEXPA5-R TaEXPA8-F TaEXPA8-R TaEXPA9-F TaEXPA9-R TaEXPB1-F TaEXPB1-R TaEXPB2-F TaEXPB2-R TaEXPB3-F TaEXPB3-R TaEXPB4-F TaEXPB4-R TaEXPB5-F TaEXPB5-R TaEXPB7-F TaEXPB7-R TaEXPB8-F TaEXPB8-R TaEXPB9-F TaEXPB9-R

CATGCGCATCACCAACGAGT TGGACGATGGAGCGGTAGAAG ATCTGTGCCTTGACCGTATCAGG GACATCAACATTCAGAGCACCATC CTTCTCCACAGGCATAAGCATT TTAGCAGCCTCAGCGTAGCC ATCGTGCGGCATGTGCTT GGCGGGCAGAAGTTGGTT CACCATCAACGGGCACAG AGTTCCGGCTCATCTCCATC CCGTACCACCACGTCCAA TCCTCCGCTGACACTCTACAC CGAGATCCGGTGCGTGAA GCATGGCGAGGTCGAAGTG GCACTGTTCTCCCTCCTCGTC GCACCACCATCGTCGTCAG CGGCAAGTGGACGAGGAT GGATGCGGATGGAGAAGG GCACTGTTCTCCCTCCTCGTC CACCACCATCGTCGTCAGG TTCTTCCTCGTCGGTGCTG AGGCGATGCCGTGATGTT GTGGATACCGCTGACCTTGT TGGCGATGAGCTTCTTGC ACCACCAGTGGCAACCCT AGACATACGCGGCAGCAA CAACCCTTCCTGCTCTGG GGAGCTTGTCGTTGAGGC ACATCGTCATCACCGACCTG ATGGCACCCTCTTGTACTGC

20 21 23 24 22 20 18 18 18 20 18 21 18 19 21 19 18 18 21 19 19 18 20 18 18 18 18 18 20 20

was used to supply K as follows: 0.5 mM KNO3 (LK) and 50 mM KNO3 (EK). For temporal and spatial analysis of the expression patterns of expansins in response to P, wheat seedlings were cultivated in an incubator in the dark for 36 h (h) at 26  C after the seeds germinated, and the seedlings were then subjected to various treatments using different P or K concentrations for 12 or 24 h. Seedlings incubated in half-strength Hoagland solution at 26  C were used as the controls. Coleoptiles were harvested at different time points for analyses. The transgenic tobacco plants were produced as described previously by Xing et al. (2009), and the T3 homogeneous transgenic tobacco plants were identified as described by Li et al. (2011). For phenotypic comparisons between WT and transgenic tobacco seedlings, one-month-old seedlings grown under normal conditions were cultured in a greenhouse (16 h light/8 h dark at 75% humidity and 25  C) and watered with different concentrations of P for 10 days until phenotypic differences were evident between the transgenic plants and the controls. Fully grown tobacco plants were obtained as described by Li et al. (2011). Detection of photosynthetic gas exchange parameters was performed on three-month-old tobacco plants. Hoagland solution containing different concentrations of P was used to irrigate the roots for one month. Seedlings irrigated with normal Hoagland solution were used as a control.

2.2. Extraction of cell wall proteins and detection of expansin activity Extraction of cell wall proteins and detection of expansin activity were carried out according to McQueen-Mason et al. (1992) and Gao et al. (2007). Plant materials were homogenized in a blender in cold buffer [20 mM sodium acetate, pH 4.5, 2 mM disodium ethylenediaminetetra acetate (EDTA)]. Cell wall material

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was collected on a nylon screen (50 mm mesh). Proteins were extracted from cell wall material for 2 h on ice in extraction buffer [1 M NaCl, 25 mM HEPES, pH 7.0, 3 mM sodium metabisulfite, 2 mM EDTA, 5 mM dithiothreitol (DTT)] and were then squeezed through nylon mesh. The cell wall fragments were further extracted by suspending in the same extraction buffer for an additional 1 h. The proteins in the combined supernatants were slowly precipitated with 0.39 g of ammonium sulfate per mL for 12 h. Precipitated proteins were pelleted by centrifugation (20,800  g, 15 min, 4  C) and subsequently resuspended in sodium acetate (50 mM, pH 4.5). For detection of expansin activity, frozen wheat coleoptiles were thawed, abraded, and boiled in water (20 s) to eliminate endogenous expansin activity, and then pressed and clamped on the extensometer. After pretreatment with sodium acetate buffer (50 mmol/L, pH 4.5) for 30 min, the solution was replaced by the same buffer containing expansin proteins of wheat coleoptiles (2 mg/mL) and the extension was recorded for a further 60 min. Expansin activity was assayed by measuring the wall extension rate after adding the protein extracts.

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2.7. Measurements of Pi concentration and biomass of shoot Seven-week-old tobacco plants grown under normal conditions were irrigated with Hoagland nutrition solution containing different concentrations of P (CK: 1 mM KH2PO4, EP: 10 mM KH2PO4, and LP: 0.1 mM KH2PO4). Then the leaves of both WT and the transgenic plants were harvested for determination of Pi contents after 10 days. The Pi contents of leaves were detected using the photocolorirmetric method (Murphy and Riley, 1962). For the biomass, the full shoot of plants were harvested and weighed. 2.8. Measurements of photosynthetic gas exchange parameters The photosynthetic gas exchange parameters including net photosynthesis (Pn), transpiration rate (E) and stomatal conductance (Gs) were measured with a portable photosynthetic system (CIRAS-2, PP Systems, Herts, UK) according to Wang et al. (Wang et al., 2010b). 2.9. Statistical analysis

2.3. RNA extraction and cDNA synthesis Total RNA was extracted from the wheat coleoptiles with the Trizol reagent (TaKaRa, Japan) according to the manufacturers’ protocol, and then was treated with DNaseⅠ (RNase-free, Promega). The total RNA was subjected to first-strand cDNA synthesis with RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA) according to the manufacturers’ protocol. 2.4. Primers Primers used in this study were listed in Table 1. 2.5. Gene expression analysis by qRT-PCR TaEXPB23 expression was followed by a 96-bp fragment amplified with the primer RT23-F and RT23-R (Table 1). The tubulin cDNA was used as a control reference. Tubulin has been accepted widely as a house keeping gene in growing plants (Coker and Davies, 2003). PCR was carried out in a 25 mL reaction containing 1  SYBR Green PreMix (TaKaRa, Japan), 200 nM primers (for each forward and reverse primer), and two-twenty-fifth of the RT reaction. Quantitative analysis was performed using the Bio Rad CFX Manager system. The absence of primer-dimer formation was examined in single and no-primer controls. Each sample was examined in triplicate using relative quantification analysis. This method normalizes the expression of the specific gene versus the OOCT control reference with the formula 2 , where OCT ¼ CT specific gene  CT reference gene, and OOCT ¼ OCT  arbitrary constant. The threshold cycle value (CT) is defined as the PCR cycle number that crosses an arbitrarily placed threshold line. 2.6. Wheat growth and analysis of coleoptiles cell The wheat seeds were first geminated under 16 h/8 h (light/ dark) at 25  C for 24 h. After that, the seedlings with uniform phenotypes were selected and transplanted to new dishes containing full-strength Hoagland solution for 36 h in dark, and then treated with modified Hoagland solution with low-P (LP: 0.1 mM KH2PO4) or excess-P (EP: 10 mM KH2PO4). After treatment with 12 h, coleoptiles at the same place (2e3 mm from the top of coleoptiles) were visualized using DIC optics for microscopic observation. The length and width of cortex cells of coleoptiles were detected using IMAGE J software (http://rsb.info.nih.gov/ij).

Statistical analysis was conducted using the procedures of DPS (data processing system, Zhejiang University, China). Statistical significance was tested using Duncan’s test at 0.05 or 0.01 probability levels. 3. Results 3.1. Cell elongation in wheat coleoptiles is regulated by different concentrations of P We first used wheat coleoptiles to detect the effects of P on cell growth. As shown in Fig. 1A, the coleoptiles were significantly longer under excess phosphorus (EP) conditions than under control (CK) conditions, while they were shorter under low phosphorus (LP) conditions. To examine whether the changes observed in coleoptile growth were caused by cell elongation, we measured the lengths and widths of cells in the elongation zone. We found that EP treatment significantly increased the length and width of cells compared to CK conditions (Fig. 1BeD), while there were no obvious differences in cell length between the coleoptile cells of wheat under LP and CK conditions (Fig. 1B and C), although the cell width increased under LP conditions. 3.2. Expansin activity is upregulated under both EP and LP conditions We measured the expansin activity in cell wall protein extracts from eight-day-old wheat coleoptiles from seedlings grown under different P conditions (Fig. 2). Extracts from wheat seedlings grown under normal conditions showed a basal level of expansin activity. However, the expansin activity was higher under both EP and LP conditions compared with normal conditions, and the expansin activity under EP conditions was higher than that under LP conditions. These results indicate that both P-sufficient and P-deficient conditions can increase expansin activity in wheat. 3.3. Multiple expansin genes are regulated by P in wheat To determine whether P influences the expression of expansin genes in wheat, we analyzed the expression of five a-expansin genes, EXPA1, EXPA4, EXPA5, EXPA8, and EXPA9, and eight bexpansin genes, EXPB1, EXPB2, EXPB3, EXPB4, EXPB5, EXPB7, EXPB8, and EXPB9 (Lin et al., 2005). As shown in Fig. 3, EP inhibited the

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Fig. 1. Different concentrations of phosphorus affect cell length and width in wheat coleoptiles. (A) Coleoptile length. (B) Longitudinal view of cortex cells in coleoptiles using DIC optics. (C) Length of cells. (D) Width of cells. Wheat seedlings treated with modified Hoagland solution containing low P (LP: 0.1 mM KH2PO4) or excess P (EP: 10 mM KH2PO4) levels 36 h after germination. Samples taken 2e3 mm from the tops of coleoptiles were visualized using DIC optics for microscopic observation after 12 h of treatment (Bar ¼ 20 mm). Each column represents the mean  SE of three biological replicates. * and ** indicate significant differences in comparison to the WT at P < 0.05 and P < 0.01, respectively.

expression of EXPA1, while LP induced the expression of this gene. The expression of EXPA4 was also down-regulated under EP conditions but slightly up-regulated under LP conditions at 12 h. The

expression of EXPA5 was obviously up-regulated under both EP and LP conditions; the maximum expression levels were 7.5-fold higher than those in the CK at 24 h under EP treatment. EXPA8 was strongly up-regulated under EP treatment at 24 h. However, EXPA9 was suppressed under both P levels compared with the control (Fig. 3). As shown in Fig. 4, EXPB2 and EXPB8 were induced under both P levels; the maximum expression level was 2.4- and 2.8-fold higher than that of the CK, respectively. EXPB1, EXPB3, EXPB4 and EXPB7 displayed similar expression patterns, and these genes were induced under EP treatment but suppressed under LP conditions at 24 h. EXPB9 expression was first up-regulated at 12 h and then down-regulated at 24 h under both P levels. The results shown in Figs. 3 and 4 suggest that multiple expansin genes are regulated by various P concentrations in wheat.

3.4. TaEXPB23 expression is regulated by Pi in wheat

Fig. 2. Expansin activities of wheat seedlings grown under different phosphorus concentrations. Cell wall protein extracts were prepared from eight-day-old wheat seedlings grown under various P conditions. Expansin activity was assayed by measuring the increase in extension rate of wheat coleoptiles after the addition of the extract (2 mg/ml). All experiments were repeated at least six times.

A previous report indicated that TaEXPB23 is expressed in wheat coleoptiles (Han et al., 2012), and thus we used wheat coleoptiles to detect the expression patterns of TaEXPB23 under different P concentrations (EP and LP). As shown in Fig. 5, the relative expression level of TaEXPB23 was up-regulated under EP treatment, with an expression level almost 2-fold than that of the CK at 12 h. Under LP conditions, the mRNA level of TaEXPB23 was similar to that of the

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Fig. 3. Expression of a-expansin genes under different phosphorus conditions in wheat, as examined by qPCR. The transcript levels of the genes are indicated relative to the level of a-tubulin expression at 0 h (set at 1) in the same samples. Each column represents the mean  SE of three replicates.

CK at 12 h but was slightly induced compared to the CK at 24 h (Fig. 5). Due to our experimental design, in which different concentrations of P were supplied by KH2PO4: 1 mM (CK), 0.1 mM (LP), and 10 mM KH2PO4 (EP), the levels of potassium (K) also varied under the different P treatments. To verify that the altered expression levels of TaEXPB23 were caused by P treatment, not K treatment, we examined the expression level of TaEXPB23 under different K concentrations (Fig. S1). As shown in Fig. S1, there was almost no difference in TaEXPB23 expression under excess potassium (EK) and low potassium (LK) treatments, although TaEXPB23 expression was slightly down-regulation at 12 h, but it recovered to the control level at 24 h.

lines with overexpression of TaEXPB23. The Pi contents of transgenic plants were significantly higher than WT under EP conditions, and slightly higher than WT under LP conditions (Fig. 6A). However, the biomass of transgenic plants was obviously higher than WT under both EP and LP conditions (Fig. 6B). Under normal conditions, no obvious phenotypic difference was observed between WT and transgenic seedlings (Fig. 7A). In contrast, under EP conditions, the transgenic plants showed obviously increased growth performance compared to WT, especially in terms of root architecture (Fig. 7A), as the transgenic plants had more and longer lateral roots than WT (Fig. 7A and B). Moreover, the number of lateral roots was also significantly higher in transgenic plants than in WT under LP conditions (Fig. 7).

3.5. Overexpressing TaEXPB23 improves growth performance and root system architecture in tobacco plants under both EP and LP conditions

3.6. Transgenic plants show better photosynthetic gas exchange parameters than WT under EP and LP conditions

To better understand the role of TaEXPB23 in Pi efficiency in plants, we examined the Pi contents of three individual transgenic

The effects of Pi on the net photosynthesis rate (Pn), the transpiration rate (E), and stomatal conductance (Gs) were evaluated. As shown in Fig. 8, the Pn of WT and transgenic plants were not

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Fig. 4. Expression of b-expansin genes under different phosphorus conditions in wheat, as examined by qPCR. The transcript levels of the genes are indicated relative to the level of a-tubulin expression at 0 h (set at 1) in the same samples. Each column represents the mean  SE of three replicates.

obviously different under the CK conditions, but this value declined markedly under both EP and LP conditions compared to the CK. However, the Pn of transgenic plants was maintained at a relatively high level compared to WT under both EP and LP conditions (Fig. 8A). Similar results were observed in E (Fig. 8B) and Gs (Fig. 8C). These results suggest that the transgenic plants can maintain greater photosynthetic gas exchange capacity than WT, which is beneficial for growth under P-sufficient and P-deficient conditions. 4. Discussion 4.1. Response to phosphorus levels is a common characteristic of expansins in wheat Recently, increasing evidences have demonstrated that expansins can respond to multiple abiotic and biotic stresses. However, studies examining the expression patterns of expansins under various nutritional conditions are limited. Phosphorus is an essential component of intermediates that function in central and energy metabolism, signaling molecules, and structural macromolecules such as nucleic acids and phospholipids (Morcuende et al., 2007). Plants obtain phosphorus as inorganic phosphate

(Pi), and low availability of Pi often limits plant growth. Pi levels in the soil are often low, since much Pi is covalently or non-covalently bound to the soil, and its mobility is poor (Marschner, 1996). Microarray analysis has indicated that the expansin gene is responsive to Pi levels (Morcuende et al., 2007). However, further studies of this finding have not been reported. Growing results suggest that expansins, in light of their proposed functions in cell division and elongation, may play important roles in cell-wall division to create new transverse cell walls separating daughter cells (Markakis et al., 2012). However, some studies have indicated that some expansins mainly affect cell enlargement but not cell differentiation, e.g., RhEXPA4 (Lü et al., 2013). In the current study, we used wheat coleoptiles to examine the effects of Pi on cell elongation, since cell division rarely occurs during wheat coleoptile growth; wheat coleoptile elongation is primarily attributed to cell expansion (Philippar et al., 1999). As shown in Fig. 1, the wheat coleoptiles showed significant changes in length under different P levels (Fig. 1A). Further study suggested that both P levels may induce the expansion of cortex cell walls in wheat (Fig. 1B and C). In addition, our results suggest that expansin activity is induced under both EP and LP conditions compared to the CK (Fig. 2). Expansins comprise a large superfamily that includes four

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Fig. 5. Expression pattern analysis of TaEXPB23 under excess and low phosphorus conditions. Wheat seedlings were cultivated in an incubator in the dark for 36 h (h) at 26  C after seed germination and subjected to EP (10 mM KH2PO4) or LP (0.1 mM KH2PO4) treatment for 12 or 24 h. Seedlings incubated in full-strength Hoagland solution at 26  C were used for the controls. The expression data correspond to the means of triplicates, normalized to a-tubulin. Expression levels are indicated in arbitrary units  SE.

subfamilies, and EST mining has estimated that there exist at least 30 and 65 a- and b-expansins, respectively in wheat. Here, we choose some expansins which have been isolated by researchers for the further research (Lin et al., 2005). And among them, five a- and nine b-expansin genes were up- or down-regulated and showed dramatically changes of expression levels in wheat (Figs. 3e5). However, the expression patterns of other expansins were not detected, e.g., TaEXPA2, TaEXPA3, TaEXPA6, TaEXPA7and TaEXPB10 (data not shown). These may be caused by the materials of experiment. As a special material, the whole growth period of wheat coleoptiles was mainly the prime 2e3 days accompany with the wheat seedlings, and thus it may lead to the dramatical changes of some gene which were related with the growth of coleoptiles under CK conditions. 4.2. TaEXPB23-overexpressing tobacco plants responding to EP and LP conditions exhibit increased lateral root growth TaEXPB23 is an expansin gene belonging to the b-expansin subfamily in wheat. In a previous study, we found that the expression of TaEXPB23 was up-regulated by water stress (Li et al.,

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2011) and salt stress (Han et al., 2012). Here, our results indicate that the expression level of TaEXPB23 was up-regulated under EP treatment compare to the CK (Fig. 5), and the induction was not caused by K (Fig. S1), suggesting the involvement of TaEXPB23 in Pi adaptability. Reduction in plant growth as a consequence of P limitations has been attributed to a reduced production of assimilates required for growth (Rodríguez et al., 1998). In the current study, TaEXPB23overexpressing tobacco plants showed obviously improved plant growth under both EP and LP levels compared to CK conditions, including the increased biomass of leaves (Fig. 6B) and improved phenotypes (Fig. 7A). This may be one of the reasons for the better photosynthetic traits of transgenic tobacco lines than WT under EP and LP conditions (Fig. 8). P not only affects plant growth, but also affects root growth. In many plant species, low P availability in the external medium strongly alters RSA (López-Bucio et al., 2005). And this is often considered to be an adaptive response, leading to enhanced P uptake capacity in the plant (Nacry et al., 2005). The result in Fig. 6A indicated that the P contents in transgenic plants were slightly higher than WT but not significant under LP conditions. Changes trigged by P limitation are complex, and many studies have led to contrasting and sometimes conflicting conclusions. For example, Al-Ghazi et al. (2003) showed that total root length was affected by P starvation and lateral roots were fewer but elongated faster (AlGhazi et al., 2003), whereas root length was significantly reduced in the experiments of López-Bucio et al. (2005). Similarly, lateral root density appears to be either reduced (Al-Ghazi et al., 2003) or increased (López-Bucio et al., 2005) in response to P starvation. In Arabidopsis, P starvation has been shown to affect the growth of the primary root and the elongation of lateral roots (López-Bucio et al., 2005; Al-Ghazi et al., 2003) and to stimulate the formation of root hairs (Bates and Lynch, 1996). Expansins also play an important role in root development, especially those are mainly expressed in roots (Lin et al., 2011; Yu et al., 2011; Markakis et al., 2012). The result shown in Fig. 7B indicate that TaEXPB23 plays an important role in plant root growth and can help maintain relatively good RSA, as reflected by the relatively high lateral root number observed under both P-rich and P-starvation conditions. This result is in accord with the observed mRNA transcript levels (Fig. 5). Taken together, RSA is one of the primary aspects of plant structure as it influences plant anchorage in the soil and the way plants absorb water and nutrients and in sport of shoot (Lynch, 2005). Therefore, better RSA will lead to the improved phenotype of shoot, and better photosynthetic traits.

Fig. 6. Phosphorus content and biomass of leaves in WT and transgenic plants under different P conditions. 7-week-old tobacco plants were irrigated with Hoagland nutrition solution containing different concentrations of P (CK: 1 mM KH2PO4, EP: 10 mM KH2PO4, and LP: 0.1 mM KH2PO4). Then, (A) Phosphorus content and (B) biomass of leaves were detected 10 days after treatment. T3-1, T3-8 and T3-10 are three individual transgenic lines. Each column represents the mean  SE of three biological replicates. * and ** indicate significant differences in comparison to the WT at P < 0.05 and P < 0.01, respectively.

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Fig. 7. Effects of excess and low phosphorus treatment on the growth of TaEXPB23-overexpressing tobacco lines. Seeds were germinated and grown under normal conditions for one month at 25  C, and the plants were transplanted to fresh Hoagland solution containing various concentrations of phosphorus (CK: 1 mM KH2PO4, EP: 10 mM KH2PO4, and LP: 0.1 mM KH2PO4). The phenotypes and growth status were observed and analyzed statistically 10 days after initiation of treatment. (A) Phenotypic responses of wild-type (WT) and transgenic tobacco plants (B) Number of lateral roots. T3-1, T3-8, and T3-10 are three individual TaEXPB23 transgenic lines. Each column represents the mean  SE of two biological replicates. * and ** indicate significant differences in comparison to the WT at P < 0.05 and P < 0.01, respectively.

4.3. Increased expansin gene expression may be involved in LPinduced and auxin-induced RSA improvement Changes in mineral nutrient availability and heterogeneous distribution of mineral nutrients in the soil induce various adaptive mechanisms in plants, among which the plasticity of root development is of crucial importance (Hell and Hillebrand, 2001). Phosphate availability also has a strong effect on RSA. Low P availability in the external medium strongly alters RSA, leading to

an increased ratio of root surface to explored soil volume, which increases the P uptake capacity of the plant. In Arabidopsis, P starvation affects the growth of the primary root and the initiation and elongation of lateral roots (Williamson et al., 2001). Auxin is closely involved in P-starvation-induced root development (Al-Ghazi et al., 2003). Indeed, auxin may play an important role in the effects of P on lateral roots, as proposed by studies indicating that the effects of P on lateral root density and elongation are significantly altered in both auxin mutants and auxin-treated

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Fig. 8. Changes in photosynthetic gas exchange parameters in WT and transgenic plants under different P conditions. Three-month-old tobacco plants were irrigated with Hoagland nutrition solution containing different concentrations of P (CK: 1 mM KH2PO4, EP: 10 mM KH2PO4, and LP: 0.1 mM KH2PO4). (A) Pn, (B) E, and (C) Gs were detected one month after the initiation of treatment. T3-1 and T3-10 are two individual transgenic lines. Each column represents the mean  SE of three biological replicates. * and ** indicate significant differences in comparison to the WT at P < 0.05 and P < 0.01, respectively.

WT plants (López-Bucio et al., 2005; Al-Ghazi et al., 2003). Moreover, auxin is an important regulator of various plant growth processes, and notably, this phytohormone contributes to the regulation of cell elongation (Fleming, 2006). Therefore, what is the target of auxin, which induces changes in RSA under conditions of low P availability? Previous studies have demonstrated that auxin induces expansin activation and mediates the expression of expansin genes (Cosgrove, 2012; Zhao et al., 2011). And some expansins play a role in RSA (Guo et al., 2011; Lin et al., 2011; Yu et al., 2011). We previously observed that the expression level of TaEXPB23 was obviously up-regulated by IAA (Han et al., 2012), and in the current study, overexpression of TaEXPB23 improved the RSA of transgenic tobacco plants (Fig. 7). Therefore, perhaps expansins play a role in the response of root development to P limitation, and auxin may serve as an intermediary or signaling molecule in this response. Taken together, the results suggest that expansins may be involved in the improvement of phosphorus availability by regulating the growth and development of plant roots. Overexpression of TaEXPB23 resulted in an improved phenotype in transgenic tobacco plants under EP and LP conditions, which particularly exhibited improved RSA, as indicated by an increased number of lateral roots.

Acknowledgments We are very grateful for Dr Yong Wang for his help. This work was supported by National Natural Science Foundation of China (No. 31370304) and by the Opening Foundation of the State Key Laboratory of Crop Biology (2013KF01).

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.02.016. Contributions The work presented here was carried out in collaboration between all authors. Wang W. defined the research theme. Han Y.-Y. designed most of the methods and experiments, and analyzed the data. Han Y.-Y. and Zhou S. carried out the laboratory experiments. Chen Y.-H. and Kong X.-Z. participated the prepare of materials of the experiment. Xu Y. provided assistant of the experimental instrument. Han Y.-Y. wrote the paper. All authors have contributed to, seen and approved the manuscript. References Al-Ghazi, Y., Muller, B., Pinloche, S., Tranbarger, T., Nacry, P., Rossignol, M., Tardieu, F., Doumas, P., 2003. Temporal responses of Arabidopsis root architecture to phosphate starvation: evidence for the involvement of auxin signalling. Plant, Cell. Environ. 26, 1053e1066. Bates, T., Lynch, J., 1996. Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability. Plant, Cell. Environ. 19, 529e538. Coker, J.S., Davies, E., 2003. Selection of candidate housekeeping controls in tomato plants using EST data. Biotech 35, 740e749. Cosgrove, D.J., 2012. Molecular mechanisms of plant cell Wall loosening: expansin structure & function. In: Research Meeting, vol. 715, p. 26. Dai, F., Zhang, C., Jiang, X., Kang, M., Yin, X., Lü, P., Zhang, X., Zheng, Y., Gao, J., 2012. RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose Petals. Plant Physiol. 160, 2064e2082. Fang, S., Yan, X., Liao, H., 2009. 3D reconstruction and dynamic modeling of root architecture in situ and its application to crop phosphorus research. Plant J. 60, 1096e1108.

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The involvement of expansins in responses to phosphorus availability in wheat, and its potentials in improving phosphorus efficiency of plants.

Phosphorus (P) is a critical macronutrient required for numerous functions in plants and is one of the limiting factors for plant growth. Phosphate av...
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