GmEXPB2, a Cell Wall b-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development1[OPEN] Xinxin Li, Jing Zhao, Zhiyuan Tan, Rensen Zeng, and Hong Liao* Haixia Institute of Science and Technology, Root Biology Center (X.L., H.L.) and College of Life Sciences (R.Z.), Fujian Agriculture and Forestry University, Fuzhou 350002, China; and College of Agriculture, South China Agricultural University, Guangzhou 510642, China (X.L., J.Z., Z.T., H.L.) ORCID ID: 0000-0002-6435-3054 (R.Z.).

Nodulation is an essential process for biological nitrogen (N2) fixation in legumes, but its regulation remains poorly understood. Here, a b-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. b-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N2 fixation and growth of soybean.

Legumes not only provide abundant protein and oil for human and animal diets, they are also the most important nitrogen (N) source in agroecosystems. Most legumes can form symbiotic associations with rhizobia as nodules to fix atmospheric N2 into ammonia, which is in turn converted into various fixation products, such as ureides, allantoin, and allantoic acids (Collier and Tegeder, 2012). The amount of symbiotically fixed N2 1

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB15030202), the National Key Basic Research Special Funds of China (2011CB100301), and the National Natural Science Foundation of China (U1301212 and 31470109). * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hong Liao ([email protected]). H.L. and X.L. conceived the research plans; H.L. and J.Z. supervised the experiments; X.L. performed most of the experiments; J.Z. and Z.T. provided technical assistance to X.L.; H.L. and X.L. designed the experiments and analyzed the data; H.L. conceived the project and wrote the article with contributions of all the authors; H.L. and R.Z. supervised and complemented the writing. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.01029 2640

totals roughly 20 million tons each year (Herridge et al., 2008), which makes this natural process of vital importance in world agriculture. Nodule formation has been outlined as a three-step process (Oldroyd and Downie, 2008; Oldroyd et al., 2011). The first step is a molecular dialogue between leguminous plants and rhizobial bacteria that is established through recognition of bacterial Nod factors by legume Nod factor receptors. Secondly, rhizobial bacteria attach to root hairs and induce root hair curling that entraps attached bacteria and proceeds to infection thread (IT) formation. The third step includes nodule organogenesis from nodule primordia to development of mature nodules with the ability to fix N2. Important progress has been made in understanding the initial symbiotic stage associated with Nod factor perception. Typically, this involves signaling pathways triggered by a series of receptor-like kinases, such as NFR1 (a Nod-factor receptor kinase gene) and NFR5 in Lotus japonicus and LysM domain-containing receptor-like kinase in Medicago truncatula (Limpens and Bisseling, 2003; Limpens et al., 2003; Madsen et al., 2003; Radutoiu et al., 2003; Oldroyd and Downie, 2004; Spaink, 2004; Popp and Ott, 2011; Singh and Parniske, 2012). In addition, genetic studies exhibited that a number of genes required for rhizobial IT development has been identified

Plant PhysiologyÒ, December 2015, Vol. 169, pp. 2640–2653, www.plantphysiol.org Ó 2015 American Society of Plant Biologists. All Rights Reserved.

Expansin Affects Root Architecture and Nodulation

through analysis of nodulation-deficient mutants (Kouchi et al., 2010). LjNPL, a L. japonicus nodulation pectatelyase gene, for instance, is required for rhizobia to penetrate the cell wall and initiate formation of ITs (Xie et al., 2012). On the other hand, little has been uncovered in regards to the detail of molecular mechanisms controlling nodule growth and development during nodule symbiosis. After IT formation, nodule organogenesis becomes a crucial process for mature nodule formation and differentiation of rhizobia into intracellular N-fixing bacteroids (Oke and Long, 1999). Although nodule formation generally benefits host plants in the acquisition of N, nodulation also consumes substantial amounts of energy (Tjepkema and Winship, 1980). This leads to the hypothesis that legumes tightly control the number of nodules so that sufficient N is acquired and energy expenditures are limited. To maintain an appropriate number of nodules, host plants possess both positive and negative regulatory systems to both facilitate nodule formation and avoid hypernodulation. A number of genes from many legumes contribute to positive regulation of nodule numbers. Examples include symbiosis receptor kinase-interacting E3 Ubiquitin ligase from L. japonicus (Yuan et al., 2012) and MtSymSCL1 (a SCARECROW-like gene) from M. truncatula (Kim and Nam, 2013). On the other hand, legumes have developed a systemic negative feedback regulatory pathway, known as autoregulation of nodulation, to control nodule numbers by long-distance signaling (Oka-Kira and Kawaguchi, 2006; Magori and Kawaguchi, 2009; Reid et al., 2011). Specific genes with reported functions in negative control (CK) of nodule numbers include a calcium-dependent protein kinase isoform in M. truncatula and a trehalase gene in common bean (Phaseolus vulgaris; Gargantini et al., 2006; Barraza et al., 2013). Recently, several genes have been characterized with relation to soybean nodulation. Overexpression (OX) of GmNFR1a, soybean (Glycine max) nodulation factor receptor kinase 1a, leads to increased nodule number and N content in soybean transgenic composite plants (Indrasumunar et al., 2011). Another gene that has been identified as the soybean homolog of tomato (Lycopersicon esculentum) fruit weight2.2 (FW2.2), soybean FW2.2-like1, plays an essential role in soybean nodule organogenesis (Libault et al., 2010). Others, when suppressed by RNA interference of soybean b-carotene hydroxylases, including GmBCH1, GmBCH2, and GmBCH3, yield plants with severely impaired N2 fixation, decreased nodule numbers, and reduced nodule weights (Kim et al., 2013). Legumes form both indeterminate and determinate types of nodules (Lauridsen et al., 1993). Indeterminate nodules, such as in Medicago spp., pea (Pisum sativum), and alfalfa (Medicago sativa), initiate cell division in the pericycle followed by inner cortical cells and then develop a nodule primordium with the presence of a persistent meristem. This leads to nodules with ovoid zonation demarcated as a meristem zone (I), infection Plant Physiol. Vol. 169, 2015

zone (II), interzone (II-III), fixation zone (III), and senescence zone (IV). Determinate nodules, as found in soybean and bean, derived from cell divisions in root outer cortical cells, and meristematic activity disappears at very early stage of nodule development (Crespi and Gálvez, 2000; Popp and Ott, 2011). In this case, cell expansion and cell wall extension might play key roles in nodule organogenesis. Expansins have been identified as mediators of pH-dependent cell wall loosening in plants (McQueenMason et al., 1992). These proteins anchor on cellulose, where they disrupt the bonding of glycans to the microfibril surface or weaken the noncovalent binding between cell wall polysaccharides, which allow polymer chain movement and stress relaxation and thereby may accommodate cell wall surface expansion and cell enlargement (McQueen-Mason and Cosgrove, 1994; Cosgrove, 2000). Expansins are encoded by a super family of genes that encompasses four main types, including a-expansins and b-expansins (EXPBs) and expansin-like A and expansin-like B proteins (Choi et al., 2006). The sum of many studies reveals a wide range of biological roles for expansions in multiple aspects of plant growth and development, such as root hair formation and elongation (Kwasniewski and Szarejko, 2006; Won et al., 2010; Lin et al., 2011), floral organ development (Li et al., 2003), fruit softening (Gaete-Eastman et al., 2009; Karaaslan and Hrazdina, 2010), and responses to abiotic stresses (Xu et al., 2007; Geilfus et al., 2010; Zhao et al., 2011). To date, a small number of expansin genes have been identified as putatively functioning in nodulation. Among 256 clones derived from M. truncatula noduleexpressed sequence tags, two represented an expansin and an expansin-like gene (Györgyey et al., 2000). In roots of white sweet clover (Melilotus alba), the mRNA accumulation of a sweetclover a-expansin gene (MaEXP1) was enhanced within hours of rhizobial inoculation, and the transcript of MaEXP1 was also present in nodule cortex and meristem cells, the invasion zone, and interzone II-III (Giordano and Hirsch, 2004). In pea, western-blot analysis revealed that an expansinlike protein, PsEXP1, was localized in nodule apoplasts and IT walls, suggesting that PsEXP1 might be required for nodule cell and IT growth (Sujkowska et al., 2007). Although transcriptional analysis of genes involved in soybean nodulation have been carried out (Carvalho et al., 2013), details of gene functions during nodulation remain poorly understood. Expression patterns of soybean b-expansin family members responding to rhizobial symbiosis indicate that most of these expansins are expressed in nodules and suggest that the differentially expressed b-expansins fulfill certain functions during soybean nodule formation and development (Li et al., 2014). However, none of the expansins associated with nodulation to date has yet been functionally analyzed. Detailed observations of expansin family members during nodulation will further clarify functions of these genes and contribute to current understanding of the nodulation process. 2641

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Phosphorus (P) is the second most important macronutrient after N, which plays key roles in plant organ growth and development. Phosphorus is especially critical for legume growth and development because the process of N2 fixation in nodules demands abundant P for energy transfer/consumption and protein as well as lipid synthesis (Mullen et al., 1988; Gentili and Huss-Danell, 2003). It has been found that phosphate (Pi) starvation-induced P transporters in rhizobia are required for symbiotic N2 fixation (Bardin et al., 1996). In soybean, maintaining Pi homeostasis in nodules through enhanced P transporters is very important for N2 fixation efficiency (Qin et al., 2012). Moreover, several genes such as phosphoenol pyruvate phosphatase in common bean (Bargaz, et al., 2012) and purple acid phosphatase in soybean (Li et al., 2012) are highly upregulated in nodules at low P level, implying that these genes might be involved in the adaptation of symbiosis to Pi starvation. Previously, a soybean b-expansin gene, GmEXPB2, has been cloned from a Pi starvation-induced soybean complementary DNA library, and has been identified as being intrinsically involved in soybean root system architecture responding to Pi starvation as well as some other abiotic stresses (Guo et al., 2011). In this study, GmEXPB2 was found to be highly expressed in young nodules under both low- and high-P conditions. Overexpressing GmEXPB2 was found to remarkably enhance soybean root growth and increase the rhizobial infection events (more bacteria adhesion and ITs formation) and facilitate the nodule primordium initiation as well. Moreover, GmEXPB2 was observed to participate in the development of the nodule vascular trace (NVT) and bundles and subsequently affect development of nodule infection zone, number, and nitrogenase activity in transgenic plants and thus promoted the soybean N/P nutrient efficiency. RESULTS Preferential Expression of GmEXPB2 in Nodules

Expression profiles of GmEXPB2 in different organs at various growth stages of soybean development were analyzed using quantitative reverse transcription (qRT)-PCR. The results showed that the transcripts of GmEXPB2 were most abundant in nodules, followed by roots. Except for very low levels of GmEXPB2 expressed in pods, no GmEXPB2 could be detected in shoot tissues, including stems, leaves, flowers, and seeds (Fig. 1A). These suggested that in addition to being involved in root growth and development as reported previously (Guo et al., 2011), GmEXPB2 might also participate in nodule growth and development. Further analysis revealed the level of GmEXPB2 transcript accumulation in nodules at different days after inoculation (dai) with rhizobia. The mRNA accumulation of GmEXPB2 was enhanced in the roots at 4 dai, peaked in the nodules at 7 dai, and then decreased with the increasing growth of root nodules. At 21 dai, the expression of GmEXPB2 in nodules dropped to the same level as that in noninoculated roots, and no 2642

Figure 1. Expression pattern and histochemical localization of GmEXPB2 in soybean. A, GmEXPB2 expression in various plant tissues, including root, nodule, stem, leaf, flower, pod, and seed. B, GmEXPB2 expression in noninoculated roots (N-root) and inoculated roots at 4 dai with rhizobia (4d-root) and nodules at 7, 14, 21, 30, and 40 dai. The relative expression value was calculated by the ratio of the expression value of GmEXPB2 to that of soybean housekeeping gene TefS1 (accession no. X56856). Each bar represents the mean of four biological replicates with SE. C, Histochemical detection of the GUS expression when fused to the GmEXPB2 promoter in roots and nodules of soybean transgenic composite plants. I to VIII and XII, Histochemical localization analysis of GmEXPB2 in the longitudinal sections (I–IV) and cross sections (V–VIII and XII) of transgenic soybean nodules at different developmental stages. IX to XI, Expression of the CK vector with CaMV35S promoter in longitudinal section (IX) and cross sections (X and XI) of nodules. Soybean transgenic composite plants harboring ProGmEXPB2::GUS were grown in low-N nutrient solution for 4 d (I and V), 7 d (II and VI), 14 d (III and VII), 21 d (XII), and 30 d (IV and VIII), while CaMV35S::GUS expression analyzed in transgenic nodules at 14 (IX and X) and 21 (XI) d after rhizobia inoculation was used as CK. NVB, Nodule vascular bundle; Nc, nodule cortex. Bars = 20 mm (A and E), 50 mm (B, C, F, G, I, and J), and 100 mm (all other images).

GmEXPB2 transcripts could be detected in nodules at 30 dai (Fig. 1B). We also found that P level did not affect GmEXPB2 expression in nodules, as supported by the expression level of GmEXPB2 in nodules at 7 and 14 dai under both low- and high-P conditions (Supplemental Fig. S1A). These results together indicate that GmEXPB2 is involved in nodule development during early stages (before 14 dai in this study) of organogenesis in soybean regardless of P supply. Plant Physiol. Vol. 169, 2015

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Histochemical Detection of GmEXPB2 Promoter Activity in Transgenic Soybean Nodules

To more precisely determine the tissue localization of GmEXPB2 transcripts, transgenic composite plants harboring ProGmEXPB2::GUS or Cauliflower mosaic virus 35s promoter::GUS as CK were inoculated with rhizobia and harvested at various periods to obtain a spectrum of nodule growth stages for GUS staining. CK nodules showed clear and strong GUS staining throughout all nodules (Supplemental Fig. S1B). By contrast, GUS staining in nodules carrying the ProGmEXPB2::GUS construct was strongest during early development at 7 and 14 dai and was significantly reduced as nodules matured, until it was nearly undetectable at 40 dai (Supplemental Fig. S1B). Nodule longitudinal sections showed that ProGmEXPB2:: GUS expression changed with nodule growth (Fig. 1C, I–IV). The GmEXPB2 promoter-driven expression initiated in the root stele, gradually extended to the conjunction area between roots and nodules, and then became localized at the NVT, where root vascular tissues connect with nodule vascular bundles (Lotocka et al., 2012). GUS staining of cross sections further showed that GmEXPB2 expression started upon initiation of nodule primordia (Fig. 1C, V), which was followed by expression in NVT and nodule vascular bundle tissues during early nodule development and then extended to the nodule cortex (Cheng et al., 2011) and parenchymatous cells (Pas) in elder nodules (Fig. 1C, VI and VII). Interestingly, GmEXPB2 expression was restricted to the cortex and Pas in the 2-dai nodules (Fig. 1C, XII) yet hardly detected in the 30-dai nodules (Fig. 1C, IV and VIII). Furthermore, light microscopy and 30 mL water, 80 g chloral hydrate, and 10 mL 100% glycerol (HCG) transparency analysis of ProGmEXPB2::GUS staining in nodules demonstrated that GmEXPB2 expression was localized to the nodule vasculature (Supplemental Fig. S1C, II–VII), while CaMV35S::GUS expression was detected in all nodule tissues (Fig. 1C, IX–XI; Supplemental Fig. S1C, I). Taken together, these results suggest that GmEXPB2 might play important roles in nodule formation and development, especially at the early developmental stages (before 14 dai) of nodule organogenesis.

composite plants had more than 90% transgenic roots, while single transgenic hairy root per plant was used for further study (Supplemental Figs S2 and S3A). The qRTPCR showed that the transcription of GmEXPB2 was 57.1 and 446.2 times higher in OX and 19.4 and 5.8 times lower in RNAi plants than that in CK lines under low- and highP conditions, respectively (Supplemental Fig. S3B). GmEXPB2 expression significantly affected soybean nodulation in transgenic composite plants. As shown in Figure 2A, nodules were more numerous and bigger in OX lines and less numerous and smaller in RNAi lines, especially for the big nodule group, when compared to nodulation in CK lines under both low- and high-P conditions. The total number of nodules increased by 24.5% and 23.1% in OX lines and decreased by 13.7% and 30.4% in RNAi lines, while the total weight of nodules increased by 62.1% and 35.6% in OX lines and decreased by 12.4% and 43.3% in RNAi lines compared with CK lines in low- and high-P, respectively (Fig. 2, B and C). In addition, altering GmEXPB2 significantly influenced growth and nitrogenase activity of the big

Effects of GmEXPB2 Expression and P Supply on Growth and Nodulation in Soybean Transgenic Composite Plants

To illuminate possible roles that GmEXPB2 plays in nodule formation and development, effects of OX and RNA interference (RNAi) of GmEXPB2 on soybean growth and nodulation were evaluated using transgenic composite plants at 30 or 50 dai with rhizobia. The quality of gene transformation in transgenic hairy roots was checked through GUS staining assay for CK to test the infection rate, qualitative PCR, and qRT-PCR analysis for all the transgenic hairy roots (OX, RNAi, and CK lines were included) to determine the target gene expression. The results showed that all the transgenic hairy roots harbored the target gene and each soybean transgenic Plant Physiol. Vol. 169, 2015

Figure 2. Effects of OX and knockdown (RNAi) lines of GmEXPB2 on nodulation of soybean transgenic composite plants under LP or HP conditions. A, Growth performance of nodules. B, Nodule number. C, Nodule dry weight. D, Average nodule size. E, Nitrogenase activity. Soybean transgenic composite plants inoculated with rhizobia were grown in sand culture under LP (10 mM KH2PO4) or HP (500 mM KH2PO4) conditions for 30 d. Nodules were classified into two groups according to their diameter (D): big ($2 mm) and small (,2 mm) nodule groups. CK refers to transgenic nodules harboring empty vector. Each bar represents the mean of four biological replicates with SE. Asterisks represent significant differences between OX or RNAi lines and CK plants for the same trait at the same P value in Student’s t tests (*, 0.01 , P # 0.05; **, 0.001 , P # 0.01; and ***, P # 0.001). ns, Not significant at 0.05 value. 2643

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nodules, as indicated by a 12.1% and 13.0% and 38.1% and 64.0% increase in OX lines, while RNAi lines had a 21.2% and 12.1% and 43.7% and 28.4% decrease in nodule size and nitrogenase activity compared with CK lines under low- and high-P conditions, respectively (Fig. 2, D and E). However, no significant changes of nodule size and nitrogenase activity were found in the small nodule group (Fig. 2, D and E). We also harvested transgenic nodules for slicing analysis (Fig. 3; Supplemental Fig. S4). Compared with CK, OX nodules displayed larger infection zone and more infection cells, but RNAi nodules showed smaller infection zone and less infection cells under both low- and high-P conditions, suggesting that GmEXPB2 is closely involved in soybean nodule development. Soybean growth was significantly enhanced in OX lines while inhibited in RNAi lines compared with CK lines after inoculation with rhizobia under both lowand high-P conditions (Supplemental Fig. S3C). As seen in Supplemental Figure S5, OX of GmEXPB2 led to increases of 45.7% and 58.6%, 82.4% and 78.0%, and 44.9% and 50.0% in dry weight, N content, and P content at low- and high-P conditions, respectively. On the other hand, suppression of GmEXPB2 resulted in inhibition of plant dry weight by 33.5% and 22.7%, of N content by 32.6% and 28.5%, and of P content by 23.7% and 41.0% under low- and high-P conditions, respectively. These results, taken together, suggest that the expression of GmEXPB2 in soybean nodules directly affects nodule formation and development and subsequently influences plant growth, along with N and P nutrient status. In addition to GmEXPB2 expression, P supply also significantly affected growth and nodulation in soybean transgenic composite plants, as indicated by the results from two-way ANOVA (Supplemental Table S1). Except for the average size of the small nodule group, sufficient P supply dramatically increased number, weight, size, and nitrogenase activity of nodules, demonstrating that P supply is critical for nodule formation and development as well as N 2 fixation as reported previously (Mullen et al., 1988; Gentili and Huss-Danell, 2003).

Root Architecture Is Modified by OX of GmEXPB2 in Soybean Whole Transgenic Plants

OX of GmEXPB2 significantly modified soybean root architecture, as reflected by longer primary roots with both elongated root hair and nonroot hair zone compared with wild-type plants (Fig. 4; Supplemental Fig. S6). The root hair zones in OX1-3 lines were 43.1%, 49.5%, and 49.5% and 29.2%, 36.6%, and 36.7% longer than those in 2- and 3-d-old seedlings of wild-type plants; meanwhile, the three GmEXPB2-overexpressing lines showed a 22.5%, 33.8%, and 31.3% and 23.7%, 22.3%, and 32.0% increase in nonroot hair zone compared with 2- and 3-dold seedlings of wild-type plants, respectively (Fig. 4B). GmEXPB2 OX lines also had denser root hairs, as 2644

indicated by 32.6% more root hairs per cross section and 22.4% higher percentages of epidermal cells for developing root hairs in OX than wild-type plants (Fig. 4, C–E; Supplemental Fig. S6C), implying that the changed root architecture, especially on the root hair zone as well as root hair density by OX of GmEXPB2, might affect rhizobial infection. Overexpressing GmEXPB2 dramatically increased the size of both meristematic and elongation zones of root apex in 2-d-old soybean seedlings (Fig. 5). Compared to the wild type, the three OX lines showed 35.5%, 51.4%, and 54.1%, as well as 51.7%, 89.6%, and 79.4% increases in the size of the meristematic and elongation zones, respectively (Fig. 5B). In the meristematic zone, overexpressing GmEXPB2 promoted cell division but not elongation, as indicated by a 35.9%, 41.8%, and 33.1% increase in the number but no significant changes of the average length of cortex cells in the three OX lines compared with the wild-type line (Fig. 5, C and D; Supplemental Fig. S6B). However, in the elongation zone, OX of GmEXPB2 could promote both cell division and elongation, as indicated by a 17.6%, 23.9%, and 13.1% as well as a 29.9%, 53.3%, and 58.3% increase in the number and the average length of cortex cells in the three OX lines compared with the wild type, respectively (Fig. 5, C and D; Supplemental Fig. S6B). These findings further demonstrate that GmEXPB2 is involved in regulating root architecture through modifying cell division and elongation of root tips as reported previously (Guo et al., 2011).

Involvement of GmEXPB2 in the Process of Rhizobial Infection

Because the expression level of GmEXPB2 was closely related to nodulation in soybean transgenic composite plants, we further examined the infection events and formation of nodule primordia by inoculation with the GFP-labeled USDA110 rhizobium strain. The results showed that the infection events, including the sums of microcolonies in root hair tips and ITs formed in curled root hairs, were significantly increased in OX lines compared with wild-type plants at 2 and 3 dai (Fig. 6, A–L). Compared with wild-type plants, GmEXPB2 OX lines displayed a 63.0% and 54.8% increase in microcolonies and IT number at 2 and 3 dai, respectively (Fig. 6, S and T). Furthermore, at 4 dai, only ITs were observed in the cortical cells of wild-type plants, while nodule primordia were found in OX lines (Fig. 6, M–R). This implies that the expression of GmEXPB2 might affect soybean nodulation through increasing infection events and facilitating nodule organogenesis.

Promotion of Nodulation and Biomass in GmEXPB2Overexpressing Soybean Whole Transgenic Plants

To further evaluate the effects of overexpressing GmEXPB2 on nodulation, plant growth, and N and P Plant Physiol. Vol. 169, 2015

Expansin Affects Root Architecture and Nodulation

Figure 3. Toluidine blue-stained nodule cross sections of OX and knockdown lines of GmEXPB2. Soybean transgenic composite plants inoculated with rhizobia were grown in sand culture under LP (10 mM KH2PO4, A–F) or HP (500 mM KH2PO4, G–L) conditions for 30 d. D to F was amplified image for A to C and J to L was amplified image for G to I showing the infection zone. A, D, G, and J, GmEXPB2-overexpressing soybean nodules. C, F, I, and L, GmEXPB2-suppressing soybean nodules. B, E, H, and K, Transgenic soybean nodules carrying empty vector. Bars = 200 mm (A–F) and 100 mm (G–L).

efficiency at the whole-plant level, three T3 OX lines of GmEXPB2 and wild-type plants were inoculated with rhizobia, BXYD3, under low-N solution with LP (5 mM KH2PO4) and HP (500 mM KH2PO4) conditions for 25 d. The transcript level of GmEXPB2 in nodules was first Plant Physiol. Vol. 169, 2015

monitored using qRT-PCR (Supplemental Fig. S7). As shown in Figure 7A, overexpressing GmEXPB2 significantly promoted soybean nodulation in both low- and high-P conditions. Compared with wild-type lines, the three OX lines showed 104.3%, 80.1%, and 55.3% 2645

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Figure 8A, the three OX lines grew much better than wild-type plants, and this growth improvement in OX1 to OX3 lines was seen not only at low P, but also at high P. Compared with wild-type plants, the OX1 to OX3 lines showed 52.5%, 35.2%, and 40.1% increases in dry weight at low P and 42.6%, 25.7%, and 41.6% increases at high P (Fig. 8B). The three GmEXPB2 OX lines led to increases, relative to wild-type plants, of 20.8%, 24.6%, and 43.9% and 36.2%, 17.0%, and 30.3% in N content and 26.0%, 28.1%, and 43.2% and 43.9%, 25.6%, and 62.1% in P content in low- and high-P treatments, respectively (Fig. 8, C and D). These findings further prove that OX of GmEXPB2 can boost soybean growth and N and P nutrient efficiency through promotion of nodulation. In addition to clarifying roles of GmEXPB2 in both nodulation and root growth, total root length was also measured for OX1 to OX3 and wild-type soybean plants 25 d after growth with or without rhizobia under high- and low-P conditions, respectively (Supplemental Fig. S8). Without rhizobia inoculation, the three OX lines significantly promoted root elongation, as indicated by 43.7%, 43.8%, and 96.5% and 50.5%, 40.7%, and 37.1% longer roots than wild-type roots in low- and high-P treatments, respectively (Supplemental Fig. S8A). However, no such promotion effects of overexpressing GmEXPB2 on root elongation could be found when roots were inoculated with rhizobia (Supplemental Fig. S8B). These results suggest that GmEXPB2 plays different roles in root elongation and nodulation. Figure 4. Effects of overexpressing GmEXPB2 on root architecture of soybean whole transgenic lines. A, Root hair growth of 2-d-old soybean seedlings. The red box represents the amplifying region of root hair zone for OX or wild-type (WT) soybean plants, respectively. B, Primary root length including root hair and nonroot hair zone of 2- and 3-d-old seedlings. C, Cross-sectional images of soybean root hairs. D, Root hair number per whole cross section. E, Percentage of root hairs to epidermal cells per section. OX refers to soybean whole transgenic lines overexpressing GmEXPB2. Each bar was the mean of 10 biological replicates with SE. Asterisks represent significant differences between OX and wild-type plants for different parameters at the same growth period in Student’s t tests (*, 0.01 , P # 0.05; **, 0.001 , P # 0.01; and ***, P # 0.001). Bars = 100 mm.

increases in nodule numbers at low P and 47.1%, 29.8%, and 58.6% increases at high P, while nodule dry weight increased by 125.5%, 113.8%, and 117.0% at low P and 40.0%, 38.6%, and 40.7% at high P at 25 dai (Fig. 7, B and C). However, the nitrogenase activity increased by 86.4%, 158.0%, and 375.0% in OX1 to OX3 lines at highP conditions; no such increase was seen at low P, except only one OX line showed a significant enhancement compared with the wild type (Fig. 7D). These results further demonstrated that OX of GmEXPB2 facilitates nodule formation and development, as well as N2 fixation in soybean. Moreover, OX of GmEXPB2 in soybean significantly promoted plant growth as well as N and P nutrient status after rhizobia inoculation for 25 d. As shown in 2646

DISCUSSION

Nodulation in legumes is accomplished through interactive processes involving rhizobium infection via ITs and nodule organogenesis resulting from cortical cell division and proliferation (Oldroyd and Downie, 2008; Oldroyd et al., 2011). Although recent genetic studies demonstrate that a number of genes are involved in nodule symbiosis (Kouchi et al., 2004, 2010; Hayashi et al., 2012), very few host genes have been well characterized, and the molecular mechanisms underlying nodule initiation and development still need to be further clarified. Because meristematic activity of determinate nodules is lost after initiation, correspondingly, nodule growth likely is primarily dependent on cell expansion instead of cell division (Crespi and Gálvez, 2000; Popp and Ott, 2011). Therefore, the process of rapid cell expansion should be particularly important in the organogenesis of determinate nodules, such as soybean nodule. As cell wall-loosening proteins, expansins can induce cell wall extension in vitro and cell expansion in vivo and thus are widely involved in plant organ growth and morphogenesis (Cosgrove, 1998, 2000; Choi et al., 2006). In this study, a soybean b-expansin GmEXPB2 was preferentially expressed in the early growth of nodules but hardly detected in mature nodules (Fig. 1B). This Plant Physiol. Vol. 169, 2015

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Based on these results, we developed a schematic representation for how GmEXPB2 is involved in soybean nodulation (Fig. 9). In this representation, we propose that this expansin, which presumably mediates root cell wall loosening, is both directly and indirectly involved in promoting nodule formation and development. The indirect pathway involved GmEXPB2 in modified root architecture, resulting in an enlarged root hair zone with increased root hair density, which will increase the frequency of rhizobial bacterial attachments to root hairs (Figs. 4 and 6). It has been reported that root growth and development are sustained by root apical meristem and elongation (Perilli et al., 2012; Alarcón et al., 2014), accompanied by an increase in the plasma

Figure 5. Effects of overexpressing GmEXPB2 on root cell division and elongation of soybean whole transgenic lines. A, Root apex. The distance between black arrows in each root represents the elongation zone, and the red arrow pointed the demarcation between meristematic and elongation zone of root apex. B, Sizes of the meristematic and elongation zones. C, Number of cortex cells in the meristematic and elongation zones. D, Average length of cortex cells in the meristematic and elongation zones. Wild-type (WT) and three transgenic soybean lines overexpressing GmEXPB2 (OX1–OX3) were grown on paper culture system for 2 d. Each bar was the mean of 10 biological replicates with SE. Asterisks represent significant differences between OX and wild-type lines for the same trait in Student’s t tests (*, 0.01 , P # 0.05; **, 0.001 , P # 0.01; and ***, P # 0.001). ns, Not significant at 0.05 value. Bars = 100 mm.

expression pattern of GmEXPB2 was further confirmed through analysis of GUS activity driven by the GmEXPB2 promoter in transgenic nodules (Fig. 1C; Supplemental Fig. S1, B and C). Together, these results provide compelling evidence for concluding that GmEXPB2 is involved in the formation of nodule primordia and rapid nodule cell enlargement and expansion. Our results clearly show that OX of GmEXPB2 in both soybean transgenic composite plants and stably transformed soybean plants leads to significant increases in nodule number and nodule weight (Figs. 2 and 7). Plant Physiol. Vol. 169, 2015

Figure 6. Confocal microscopy of root hairs of soybean seedlings inoculated with rhizobia USDA110 carrying GFP. A to F, Root hairs in the preinfection period at 2 dai. G to L, Infected root hairs formed ITs at 3 dai. M to R, IT growth and reaching to the cortical root cells at 4 dai. A to C, G to I, and M to O, Wild-type (WT) plants. D to F, J to L, and P to R, Whole transgenic soybean lines overexpressing GmEXPB2. S, Green fluorescent area per image represents bacteria adhesion to root hairs. T, IT number per 2-mm length of roots. Each bar represents the mean of 12 biological replicates with SE. Asterisks represent significant differences between OX and wild-type CK lines for the same trait in Student’s t tests (*, 0.01 , P # 0.05; and ***, P # 0.001). Bars = 100 mm (A–F) and 50 mm (all other images). 2647

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Figure 7. Effects of overexpressing GmEXPB2 (OX) on nodulation of soybean whole transgenic lines. A, Photographs showing soybean nodules of 25-d-old seedlings under both LP and HP conditions. B, Nodule number per plant. C, Nodule dry weight per plant. D, Nitrogenase activity. OX1 to OX3 refer to three independent soybean whole transgenic lines overexpressing GmEXPB2. WT1 and WT2 refer to wildtype soybean plants. LP and HP were 5 and 500 mM KH2PO4, respectively. Each bar represents the mean of four biological replicates with SE. Asterisks represent significant differences between OX and wild-type lines for the same trait at the same P value in Student’s t tests (*, 0.01 , P # 0.05; **, 0.001 , P # 0.01; and ***, P # 0.001). ns, Not significant at 0.05 value.

membrane and cell wall surface area. Here, we found that GmEXPB2 could enhance cell division and elongation in the root apex as revealed by the increased cortex cell number and length and especially the increase in the average length of cortex cells in the elongation zone (Fig. 5; Supplemental Fig. S6B). These changes could explain why OX of GmEXPB2 could facilitate greater root growth and thus increased the size of the root hair zone, which increases the root region for interaction with rhizobial cells. The direct pathway would involve expansin loosening the root cell wall in the OX lines that could enhance rhizobium infection. This is supported by the observation that GmEXPB2 OX leads to increased numbers of infection events, including a higher rate of rhizobial adhesion to the root hair surface, enhanced IT growth, and more rapid development of nodule primordia in OX lines (Fig. 6). These 2648

all demonstrate that expression of the cell wallloosening gene GmEXPB2 increases the frequency of infection events by rhizobial bacteria through modifying root architecture and altering (loosening) root cell walls. Although expansin activity is often associated with cell wall loosening or extension, the precise mode of action remains a mystery. One of the hypothesized actions of expansin is the weakening of noncovalent binding between cellulose fibers, and it is supported by the finding of expansin weakening the hydrogen bonding of pure cellulose paper (McQueen-Mason and Cosgrove, 1994). Meanwhile, many studies have reported that expansins have no hydrolytic or other enzymatic activities (McQueen-Mason et al., 1993; Li and Cosgrove, 2001; Tabuchi et al., 2011). In addition to that, Cosgrove et al. (1998) also mentioned that although expansin lacks hydrolytic activity itself, it does enhance the hydrolysis of crystalline cellulose by cellulases. Therefore, we propose that GmEXPB2 might weaken the hydrogen bonding of crystalline cellulose to make cell wall expansion/extension during nodule growth and development. It has been documented that bacteria within nodules require an abundant supply of carbohydrates and related substances generated from host plants for nodule growth and N2 fixation (Streeter, 1980; Reich et al., 2006; Aleman et al., 2010). The nodule vascular system is important for importing these compounds into nodules. Factors that affect development of the NVT may result in abnormal nodules. For example, boron deficiency inhibits development of the NVT and thus causes undeveloped nodules in Vicia faba (Brenchley and Thornton, 1925). In addition, a pleiotropic pea mutant R50 (sym16) with abnormal vascular strands produces few nodules (Pepper et al., 2007). In this study, GUS staining and HCG analysis using transgenic soybean nodules revealed that GmEXPB2 is mainly localized in the NVT and nodule vascular bundles in young nodules (Fig. 1C; Supplemental Fig. S1). This implies that GmEXPB2 might play a vital role in development of nodule vascular strands, which accommodate the import of carbohydrates and other substances from the host plant to support nodule organogenesis during growth. This is further supported by results from our work with soybean transgenic plants in which OX of GmEXPB2 significantly increases nodule number, nodule weight, and nodule size (Figs. 2 and 7). Furthermore, GmEXPB2 is also expressed in Pas of nodules during early nodule development (before 14 dai) and diminished in mature nodules (Fig. 1C). This expression pattern together with the increase/decrease in nodule size of the big nodule group in GmEXPB2-OX/ RNAi lines (Fig. 2D), suggests that GmEXPB2 might also affect nodule formation and development during enlargement and expansion of Pas in nodules. Root growth is closely related to the formation of symbiotic nodules in legumes. In general, plant roots supply carbohydrates and nutrients other than N to nodules, while nodules reciprocally return fixed N to Plant Physiol. Vol. 169, 2015

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changes of root growth in response to low P stress (Guo et al., 2011). Our results demonstrate that overexpression of GmEXPB2 dramatically increased total root length in 1-month-old soybean plants regardless of P supply with low N under noninoculation conditions (Supplemental Fig. S8A) and, together with the increased size and cortex cell number in both meristematic and elongation zones in GmEXPB2-overexpressing lines (Fig. 5; Supplemental Fig. S6), confirm that GmEXPB2 could affect root growth and change soybean root architecture, especially at low P level as reported before (Guo et al., 2011). However, the enhancement of root elongation precipitated by overexpressing GmEXPB2 was diminished after inoculating with rhizobia (Supplemental Fig. S8B), and the expression of GmEXPB2 was not affected by P level (Supplemental Fig. S1A), while OX or suppression of GmEXPB2 still appears to affect soybean nodulation. A possible explanation is that there might be a competition between root growth and nodule development, probably for carbohydrates. Before rhizobium infection, GmEXPB2 mainly functions in modifying root architecture for increasing rhizobial infection events and results in elongated roots with a longer root hair zone and denser root hairs as discussed above. After infected by rhizobia, GmEXPB2 mainly functions in nodule formation and

Figure 8. Plant growth and N and P content as affected by overexpressing GmEXPB2 (OX) in soybean whole plant transgenic lines. A, Photographs showing soybean growth performance. B, Dry weight. C, N content. D, P content. OX1 to OX3 refer to three independent soybean transgenic lines overexpressing GmEXPB2. LP and HP were 5 and 500 mM KH2PO4, respectively, which were applied for 25 dai. Each bar represents the mean of four biological replicates with SE. Asterisks represent significant differences between OX and wild-type (WT) plants for the same trait at the same P value in Student’s t tests (*, 0.01 , P # 0.05; **, 0.001 , P # 0.01; and ***, P # 0.001).

host plants (Djordjevic, 2004; Aleman et al., 2010). Therefore, better root growth can facilitate nodulation and vice versa. Genes involved in nodulation might also regulate root growth in host plants. Two examples are Nodule root/Cochleata from M. truncatula/pea and non-symbiotic retarded root growth in L. japonicus, which play essential roles in the development of both roots and nodules (Buzas and Gresshoff, 2007; Couzigou et al., 2012). Furthermore, P, an essential macronutrient, critically regulates root growth as well as nodulation (Bardin et al., 1996; Bonser et al., 1996; Wu and Wang, 2008; Bargaz et al., 2012; Niu et al., 2013). GmEXPB2 has been previously identified to function in adaptive Plant Physiol. Vol. 169, 2015

Figure 9. Schematic comparison of GmEXPB2 expression involved in soybean nodulation, including indirect pathway through root architecture modification and direct pathway through promotion of nodule formation and development between wild-type (WT) and overexpressing GmEXPB2 (OX) soybean plants. Compared with the wild type, OX plants modified root architecture with a longer root hair zone and denser root hairs, which might increase the rhizobium infection events. Overexpressing GmEXPB2 might also directly facilitate the growth of nodule primordia; thereby, OX of GmEXPB2 in either soybean hairy roots or whole transgenic plants resulted in increased nodule number, nodule mass, and thus elevated plant N content as well as biomass. 2649

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development, as indicated by the highest expression of GmEXPB2 being found in nodules, particularly in young nodules, but not in roots (Fig. 1B), and thus OX lines significantly increase nodule number and nodule weight (Figs. 2 and 7). In addition to the promotion of nodule formation and development, the OX lines also significantly promoted the nitrogenase activity (Figs. 2E and 7D). Hence, the plant dry weight and N and P content were significantly increased in OX lines compared with wild-type plants (Fig. 8; Supplemental Fig. S5). All of these results showed that more carbon may be allocated to nodules for increasing N2 fixation in N-limiting conditions. Therefore, GmEXPB2 might indirectly affect carbon allocation between roots and nodules. When P is limiting, more carbon is allocated to roots for promoting root growth for better P uptake. When P is sufficient, more carbon is distributed to nodules for nodule growth and development for efficient N2 fixation. Based on all of the findings presented here, it is reasonable to conclude that the soybean b-expansin gene GmEXPB2 plays an important role in regulation of soybean nodulation through modification of the root cell wall, leading to altered root architecture and promoting greater interactions between the root and rhizobia as well as enhancement of nodule infection, formation, and development. These interactions, in turn, increase soybean nodule number and weight, which enhances biological N2 fixation and growth of soybean. To the best of our knowledge, this is the first report demonstrating that a cell wall b-expansin gene critically functions in nodule symbiosis.

MATERIALS AND METHODS Plant Growth Conditions Soybean (Glycine max) genotype HN66 and rhizobium strain Bradyrhizobium sp. BXYD3 were employed throughout the study (Cheng et al., 2009). For spatial expression analysis of GmEXPB2, sterilized soybean seeds were geminated in sand, and after 5 d, seedlings were first inoculated with BXYD3 by immersing roots in a rhizobial suspension for 1 h and then transplanted into a low-N nutrient solution containing 500 mM N, including KNO3, Ca (NO3)2$4H2O, NH4NO3, and (NH4)2SO4 (15:12:4:1.5), and 1,200 mM CaCl2, 1,000 mM K2SO4, 500 mM MgSO4$7H2O, 25 mM MgCl2, 2.5 mM NaB4O7$10H2O, 1.5 mM MnSO4$H2O, 1.5 mM ZnSO4$7H2O, 0.5 mM CuSO4$5H2O, 0.15 mM (NH4)6Mo7O24$4H2O, 40 mM Fe-Na-EDTA, and 500 mM KH2PO4. Nodules, roots, stems, leaves, and flowers were harvested separately at different growth stages. Young pods and seeds were harvested at 39 d after transplanting. All tissues were stored at –80°C for RNA extraction and qRT-PCR analysis. Because soybean nodules belong to the determinate type, the size of nodules could represent the age of nodules. Therefore, we harvested the roots at 4 dai as 4d root and harvested the biggest nodules at 7, 14, 21, 30, and 40 dai to represent the nodules with the age of 7, 14, 21, 30, and 40 d, respectively, and noninoculated 14-d roots were used as CK. Moreover, the soybean nodules grown at both low P (5 mM P, added as KH2PO4) and high P (500 mM P, added as KH2PO4) at 7 and 14 dai were also harvested for analyzing P effects on GmEXPB2 expression.

RNA Extraction and qRT-PCR Analysis Total RNA was isolated using RNAiso Plus reagent (TaKaRa) according to the manufacturer’s instructions. qRT-PCR was performed using a Rotor-Gene RG-3000 (Corbett Research) with 20-mL volumes containing 2 mL of 1:50 diluted 2650

reverse transcription product, 0.6 mL of specific primers, 6.8 mL of distilled, deionized water, and 10 mL of SYBR Premix EX Taq (TaKaRa). Primers for qRTPCR analysis of GmEXPB2 and TefS1, a soybean housekeeping gene encoding the elongation factor EF-1a (accession no. X56856), are listed in Supplemental Table S2.

Vector Constructs for the GmEXPB2 Promoter Region For promoter analysis, a 2,742-bp fragment upstream of the ATG start codon of GmEXPB2 was designated as the promoter region (ProGmEXPB2). This fragment was amplified and digested by PstI and NcoI and then fused to the pCAMBIA 3301 vector (CAMBIA) with a GUS reporter gene. Expression driven by a CaMV35S promoter in the pCAMBIA 3301vector was used as the CK. Vectors for OX and suppression of GmEXPB2 were constructed according to Guo et al. (2011).

Histochemical GUS Staining of Tissue Sections Soybean transgenic hairy roots and nodules harboring ProGmEXPB2::GUS or CaMV35S::GUS were generated using the hypocotyl injection method as described previously (Qin et al., 2012). Roots and nodules were harvested at 7, 14, 21, 30, and 40 dai with BXYD3. Transgenic roots and nodules with a GUS reporter gene driven by a CaMV35S promoter were included as CK. Transgenic nodules were incubated in GUS staining solution containing 50 mM Pi-buffered saline (Na2HPO4-NaH2PO4 buffer, pH 7.2), 0.1% (v/v) Triton X-100, 2 mM K3Fe (CN)6, 2 mM K4[Fe(CN)6]$3H2O, 10 mM EDTA-2Na, and 2 mM 5-bromo-4chloro-3- indolyl-b-D-GlcA at 37°C for 12 h and then washed three times with 70% ethanol. After GUS staining, a portion of roots and nodules was immersed in HCG solution (30 mL of water, 80 g of chloral hydrate, and 10 mL of 100% glycerol) overnight to increase transparency and then transferred to a slide with one drop of HCG and covered with a cover slip for observation. Other nodules for section analysis were embedded in paraffin and then sectioned transversely to a thickness of 7 mm with a microtome (CUT 5062) for GUS activity observation with a light microscope (LEICA DM5000B) as described previously (Qin et al., 2012).

Transgenic Soybean Growth and Analysis of Root Traits For observation of root traits, seeds of three overexpressing GmEXPB2 soybean transgenic lines (OX1–OX3) and wild-type plants were surface sterilized, well soaked in one-quarter-strength modified soybean nutrient solution as described previously (Li et al., 2012), and then germinated on paper culture system under 16 h of day/8 h of night at 25°C for 3 d. For primary root analysis, the length of both soybean root hair and nonroot hair zones was quantified under stereo microscope (LEICA M165C) conditions after 2 and 3 d growth, respectively. For microscopic observation, 2-d-old soybean root tips and root hair segments were longitudinally divided into two parts through free-hand cutting, and then one of them was separately immerged in HCG solution overnight for transparency. After transparency, the root apex and root hair zone were visualized using differential interference contrast optics, and then the numbers of the third-layer cortex cells in division and elongation zones were separately counted. The demarcation between two zones was pointed in Figure 5A. The length of division and elongation zones was separately measured using ImageJ software (http://rsb.info.nih.gov/ij/). For quantifying root hair numbers, the root hair zone of 2-d soybean plants was sectioned transversely after paraffin embedding as described above and then dewaxed before being stained with 0.5% Fast Green for 2 min. The photos taken by the microscope were used to count root hair number and percentages of epidermal cells for developing root hairs in both OX and wild-type lines.

Observation of Infection Events For GFP-marked rhizobium strain USDA110 construction, the triparental mating method was used in this study. Briefly, the plasmid pMP2444 harboring GFP was transformed into Escherichia coli S17-1 as a donor; then with the helper plasmid of E. coli pRK2013, the recipient strain USDA110 was mixed at a ratio of 1:1:1 (v/v), and we performed triparental conjugation by using a filter-mating technique (Simon et al., 1983) at 28°C for 36 h on a tryptone, yeast extract, and CaCl2 plate (Wang et al., 2006). Bacterial colonies were exposed to UV light to check the expression of GFP, and the fluorescent bacteria were selected for further analysis. Plant Physiol. Vol. 169, 2015

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For infection procedure observation, the OX and wild-type soybean seeds as described above were surface sterilized and then germinated on paper for 4 d. The uniform seedlings were selected and inoculated with bacteria strain USDA110-GFP by immersing roots in a rhizobial suspension for 1 h and then transplanted into a sand culture system irrigated with N-free nutrient solution. The infection events including bacteria attachment and IT formation as well as nodule primordium were separately observed at 2, 3, and 4 dai with a confocal laser scanning microscope (ZEISS LSCM 7DUO 780&7 Live). To represent the bacteria adhesion to root hairs, the green fluorescent area was further analyzed using the image showing rhizobium attachments through Photoshop software on the basis of pixel transformation. Furthermore, 2-mm length of root segments from OX and wild-type plants at 3 dai were used to count IT numbers. The mean of IT numbers of the five root segments from each plant was considered as one biological replicate, and there are 12 biological replicates. GFP fluorescence was viewed with 488-nm laser light for excitation and a 515- to 545-nm filter for detection.

Characterization of Nodule Organogenesis Using Soybean Whole and Composite Transgenic Plants To explore effects of GmEXPB2 on nodule organogenesis, soybean transgenic composites and whole transgenic plants were generated according to Guo et al. (2011) and Zhou et al. (2014), respectively. The transgenic composite plants included GmEXPB2 OX, suppression (RNAi), and empty vector (CK) lines. When the emerged hairy roots from hypocotyl were approximately 10 cm long, the main root was removed, and each individual hairy root was first checked for quality of transformation using qualitative PCR by analyzing the expression of a hygromycin resistance gene, which was on the vector carrying the target gene sequence, or by GUS staining assay (Supplemental Figs. S2 and S3). The selected soybean transgenic hairy root was inoculated with rhizobium BXYD3 for 1 h and then transplanted into sand culture irrigated with 500 mM low-N nutrient solution with two P treatments (LP, 10 mM KH2PO4, and HP, 500 mM KH2PO4). Nodules at 30 dai were collected for RNA extraction and qRT-PCR analysis. The specific primers used are listed in Supplemental Table S2. Four transgenic plants representing four biological replicates were analyzed for each treatment. To better evaluate how GmEXPB2 expression affected nodule growth and development, 30-d transgenic nodules from composite plants were separately embedded in paraffin for membrane-enclosed bacteroid observation and then stained with toluidine blue after dewaxing and examined by light microscopy. In addition, transgenic nodules were classified into two different groups based on diameter, including a big ($2 mm) and small (,2 mm) group. After that, nodule numbers, weight, size, and nitrogenase activity were separately analyzed in different groups. Nodule size was calculated as the average nodule dry weight. Fifty dai, plants were harvested to determine dry weight and N and P content. In addition to the soybean transgenic hairy roots, the soybean whole-plant transformation lines were further studied, including three overexpressing lines (OX1–OX3) and wild-type lines. Seeds were germinated and inoculated as described above. Seedlings were grown in low-N nutrient solution as described above, and treated with 5 mM (LP) or 500 mM (HP) P added as KH2PO4. Shoots, roots, and nodules were separately harvested for dry weight, total root length, N and P content, nodule number, nodule dry weight, and nodule size measurements at 25 dai. Roots were scanned as digital images using a specialized color scanner (Epson Expression 800). Root length was quantified using WinRHIZO Pro (Regent Instrument). Nodules were harvested at 7 dai for the qRT-PCR analysis to check GmEXPB2 expression levels.

Measurement of N and P Content Plant N and P content were analyzed using a continuous flow analyzer (SAN++). Following the manufacturer’s protocol, about 0.2 g of each sample was digested and reacted in the flow analysis machine. Signals were output to a computer, and results were analyzed in FlowAccess software (SAN++ FlowAccess V3 data acquisition Windows software package).

Data Analysis Data from qRT-PCR results were normalized in each experiment. All data were analyzed statistically using Microsoft Excel 2007 for calculating mean and SE. Comparisons between groups were performed using Student’s t test in Microsoft Excel 2007 or univariate analysis in SPSS (version 16.0). Plant Physiol. Vol. 169, 2015

Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Expression and histochemical localization analysis of GmEXPB2. Supplemental Figure S2. Process of generating soybean transgenic composite hairy roots. Supplemental Figure S3. Evaluation of soybean transgenic composite hairy roots. Supplemental Figure S4. Effects of OX and knockdown of GmEXPB2 on nodule development. Supplemental Figure S5. Effects of OX and knockdown (RNAi) lines of GmEXPB2 on growth of soybean transgenic composite plants under LP or HP conditions. Supplemental Figure S6. Effects of overexpressing GmEXPB2 on root architecture of soybean whole transgenic plants. Supplemental Figure S7. Relative expression value of GmEXPB2 in transgenic soybean nodules. Supplemental Figure S8. Effects of overexpressing GmEXPB2 on total root length of soybean whole transgenic plants. Supplemental Table S1. F-value and significance level from ANOVA of nodulation and growth traits as affected by P level and GmEXPB2 expression in soybean transgenic composite plants. Supplemental Table S2. Gene-specific primers used for qRT-PCR analysis and GmEXPB2 promoter amplification.

ACKNOWLEDGMENTS We thank Dr. Xiurong Wang for providing the seeds of overexpressing GmEXPB2 soybean whole transgenic lines, the members of Root Biology Center for assistance with harvesting plants, and Dr. Thomas Walk for review of the article. Received July 7, 2015; accepted October 1, 2015; published October 2, 2015.

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