Spatial Divergence of <i>PHR-PHT1</i> Modules Maintains Phosphorus Homeostasis in Soybean Nodules

Abstract

Legume nodules provide agriculture and ecosystems with nitrogen from the ammonium ion (NH4 +) through N2 fixation. Phosphorus (Pi) is utilized as a macronutrient in plants and affects nodule initiation, development, and N2 fixation (Tang et al., 2001; Valentine et al., 2017). Pi deficiency directly impairs many cellular functions not only by reducing the available ATP necessary for enzymatic activity, membrane transport, utilization of inorganic N sources, and photosynthesis but also by limiting the biosynthesis of macromolecules (proteins, lipids, and nucleic acids; Bosse and Kock, 1998). In root nodules, the role of Pi is vital in the metabolic energy processes driving symbiotic N2 fixation into NH3, and nodules act as strong sinks for P even when the supply of P is adequate (Israel, 1987; Drevon and Hartwig, 1997). Therefore, the availability of Pi is pivotal to nodule morphogenesis and the efficiency of N2 fixation (Drevon and Hartwig, 1997; Tang et al., 2001; Sulieman et al., 2013). The effect of Pi on nodule initiation is nodule-specific and independent of plant growth, indicating that Pi has dual functions as a critical mineral element in cells and as a signal when Pi is deficient in nodules (Gentili and Huss-Danell, 2003). A phosphate transporter is responsible for intercellular and intracellular Pi transport (Bardin et al., 1996; Qin et al., 2012a). The PHOSPHATE TRANSPORTER1 (PHT1) genes encode a family of plasma membrane phosphate transporters (Nussaume et al., 2011). In the soybean (Glycine max) genome, there are at least 14 members (GmPHT1;1–GmPHT1;14) in this family with different names in different labs (Qin et al., 2012a; Tamura et al., 2012; Fan et al., 2013). These GmPHT1s have different affinities to Pi (Fan et al., 2013). Among them, only GmPHT1;1 (Chen et al., 2019) and GmPHT1;4 (Qin et al., 2012b) are expressed in nonfixing regions of nodules, and they also play a role in nodulation and Pi homeostasis in nodules. GmPHT1;11, GmPHT1;12, and GmPHT1;13 are arbuscular mycorrhiza-inducible phosphate transporter genes of soybean (Tamura et al., 2012), and GmPHT1;11, an early divergent gene from other GmPHT1s (Fan et al., 2013), is related to arbuscular development and leaf senescence (Inoue et al., 2014). PHT1 is regulated by many genes at different levels (Puga et al., 2017). When Pi is deficient, PHOSPHATE STARVATION RESPONSE1 (PHR1), a constitutively expressed MYB-domain transcription factor (Rubio et al., 2001; Puga et al., 2017), promotes Pi uptake by directly inducing the expression of PHT1. PHR1 not only functions in controlling Pi transport but also plays a role in directly repressing plant defenses (Castrillo et al., 2017) by activating jasmonate biosynthesis (Khan et al., 2016). PHR1 proteins bind to specific cis-elements (P1BS, GnATATnC) of Pi starvation-inducible genes, including PHT1, phosphatase, and RNase genes (Rubio et al., 2001; Schünmann et al., 2004; Nilsson et al., 2007; Guo et al., 2015). Therefore, the PHR-PHT1 module, a regulatory module conserved across phylogenetically distant plant species, plays an important role in maintaining Pi homeostasis (Puga et al., 2017). However, there are no reports elucidating the functions of PHR-PHT1 modules in soybean nodules. In this study, we identified PHR and PHT1 genes in soybean nodules and constructed multiple PHR-PHT1 modules. These modules formed a network and contributed to maintaining Pi homeostasis in nodules, which controls nodule development and functions in soybean. Our results show that in nodules, one GmPHR has several target GmPHT1s, and one GmPHT1 is under the control of several GmPHRs. Therefore, the divergence of GmPHR-GmPHT1 modules in a tissue-specific mode endows nodules with special characteristics during Pi signaling in soybean. The PHT gene family is responsible for transport of Pi, and PHT1 is a subgroup of the PHT family, which mainly functions in cellular phosphate transport. In the soybean genome, 14 PHT1 homologous genes (GmPHT1;1–GmPHT1;14) have been identified in our laboratory and other laboratories, and at least four PHT1s (GmPHT1;1, GmPHT1;4, GmPHT1;11, and GmPHT1;14) are expressed in roots (Qin et al., 2012a; Fan et al., 2013). Among these four GmPHT1s, GmPHT1;1 and GmPHT1;4 were reported to function in nodules (Qin et al., 2012b; Chen et al., 2019). It has been reported that GmPHT1;11 functions in the absorption of fungus-derived phosphate and in arbuscular development (Inoue et al., 2014). We postulated that if a gene was related to nodule function, it may be expressed in roots. Therefore, GmPHT1;1, GmPHT1;11, and GmPHT1;14, working together with GmPHT1;4, may synergistically function in nodules. These GmPHT1 proteins were all found to localize on the cytoplasmic membrane (Supplemental Fig. S1), as described in a previous report (Fan et al., 2013). One of the direct regulators of PHT1 is PHR1, a MYB transcription factor that can bind to a cis-element with an imperfect palindromic sequence (GnATATnC; [P1BS]) in its target genes and regulate its expression (Rubio et al., 2001; Bari et al., 2006; Bustos et al., 2010). Hence, the PHR1-PHT1 module plays pivotal roles in regulating Pi signaling and maintaining Pi homeostasis. In the soybean genome, it has been reported that there are 35 PHR1 genes encoding potential transcriptional factors (Xue et al., 2017), but no detailed functions have been revealed. Using the protein sequence of Arabidopsis (Arabidopsis thaliana) PHR1 as a query, we obtained four highly conserved PHR1 homologous genes in the soybean genome, named GmPHR1 to GmPHR4 here (Supplemental Fig. S2). The GmPHR1 to GmPHR4 proteins share two conserved functional domains, the Myb coiled-coil motif (a potential dimerization motif) and the Myb DNA-binding motif (Supplemental Fig. S2), indicating potential binding activity for their target genes (Rubio et al., 2001). To investigate the subcellular localization of GmPHR1 to GmPHR4 proteins, we amplified their coding sequences from soybean ‘Tainlong1’ and constructed the fusion genes 35S:GmPHR1:GFP, 35S:GmPHR2:GFP, 35S:GmPHR3:GFP, and 35S:GmPHR4:GFP. Then, the corresponding vectors were infiltrated into Nicotiana benthamiana leaves mediated by Agrobacterium tumefaciens. Similar to PHR proteins in other plants (Rubio et al., 2001; Ren et al., 2012; Li et al., 2014), GmPHR1:GFP, GmPHR2:GFP, GmPHR3:GFP, and GmPHR4:GFP proteins mainly target the nucleus (Fig. 1). Localization of GmPHR proteins. GmPHR1:GFP, GmPHR2:GFP, GmPHR3:GFP, and GmPHR4:GFP fusion genes were coinfiltrated with the mRFP:AHL22 nuclear marker in N. benthamiana leaves. All GmPHR:GFP proteins mainly localized in the nuclei. All experiments were performed with at least three independent biological repeats, and all gave similar results. A single representative result is shown. Bars = 20 μm. The soybean genome at least three of during its which in multiple of a gene et al., 2010). To including divergence in tissue-specific expression and 2004; et al., 2012; et al., 2013). We on the GmPHT1;1, GmPHT1;4, GmPHT1;11, and genes in our study, to their expression in roots (Qin et al., 2012; Fan et al., Chen et al., which that they may function in nodules. To the expression of PHR-PHT1 modules in nodules, we the sequence of the of these genes and to the The vectors were into soybean roots by Agrobacterium The roots were with to nodule nodules and their were by to the tissue-specific expression of the of Pi in nodules, there were no in nodules (Supplemental Fig. GmPHR1 was expressed in the of nodules, GmPHR4 expression was to regions (Fig. A and were independent of the of available Pi in the even Pi levels GmPHR gene Similar to the phosphate transporter GmPHT1s also tissue-specific of gene GmPHT1;1 was an with a previous report (Chen et al., GmPHT1;11 was only expressed in regions (Fig. and indicating that GmPHT1;11 was not expressed in the was expressed in the root no signal was in the nodule (Supplemental Fig. A previous report that GmPHT1;4 is expressed in nonfixing regions of nodules (Qin et al., expression of GmPHR-GmPHT1 modules in soybean nodules. GmPHR1 GmPHR4 GmPHT1;1 and GmPHT1;11 were to a and the vectors were into soybean roots that were under Pi were with and nodule were with a were from of different nodules from at least two independent biological A single representative result is shown. nodule nodule and root Bars = and To the gene expression we in in nodules from roots of soybean plants in there were no found on all by the of the of GmPHT1;1, and GmPHT1;11 in soybean nodules (Fig. with the activity of their the marker (Fig. these results that the expression of different GmPHR GmPHT1 in different nodule the in of GmPHR-GmPHT1 modules in soybean nodules. on soybean roots at 14 were for in and and were for of the of GmPHR1 GmPHR4 GmPHT1;1 and GmPHT1;11 and show of and GmPHR1 and GmPHT1;1 were expressed in nodule and and GmPHR4 and GmPHT1;11 were expressed in nodule and GmPHT1;1, and GmPHT1;11 were expressed in root and were found for to nodules were for nodule nodule and root was μm. Bars = and and and different GmPHT1s have highly conserved sequences their family, it is of to the and GmPHT1s to in soybean nodules. we performed an to if these GmPHR proteins bind to GmPHT1 GmPHR1 and GmPHR4 directly to cis-element in the of GmPHT1;1, GmPHT1;11, and GmPHT1;4 (Fig. in are four potential in the GmPHT1;1 (Fig. only two of be the of GmPHR1 and GmPHR4 proteins (Fig. Fig. that the sequence of other factors the PHR proteins and the availability of Pi not in there was no binding in the of Pi in the GmPHR1 and GmPHR4 proteins bind directly to cis-elements in the of GmPHT1 in of genes GmPHT1;1, GmPHT1;11, and The of the are in of the the corresponding for experiments and the corresponding regions for Fig. the of the the regions the for and the corresponding for in are the show that GmPHR1 and GmPHR4 proteins bind to of GmPHT1;1 GmPHT1;11 and GmPHT1;4 independent of Pi in The of and are in of the of with All experiments were performed with at least three biological repeats, which similar results. A single representative result is shown. To the the was to roots the fusion The results that GmPHR1 GmPHR4 protein in special of GmPHT1;1 (Fig. GmPHT1;11 (Fig. and GmPHT1;4 (Fig. However, the binding was for a GmPHR with different as and GmPHT1;1 (Fig. the of the sequences of as and with the characteristics of transcription factors et al., 2013). it was that the of available Pi have an on GmPHR binding in to were for GmPHR1 and GmPHR4 binding to their in indicating that the and were Pi GmPHR1 and GmPHR4 proteins in the of GmPHT1 in roots. that GmPHR1 and GmPHR4 in the GmPHT1;1 and in the GmPHT1;11 and in the GmPHT1;4 in of the of Pi in the and GmPHR1 has GmPHR4 The of the on the genes are as in All show from at least three biological was performed and To the effect of GmPHR on GmPHT1 transcription activity, we transcriptional experiments N. benthamiana leaves. We constructed the fusion genes GmPHT1;1 GmPHT1;4 and GmPHT1;11 which were with as a into N. benthamiana mediated by tumefaciens. Then, the transcription activity of the GmPHT1 was through the by The results show that GmPHR1 and GmPHR4 on the of in different GmPHR1 and GmPHR4 the activity of GmPHT1;1 (Fig. and GmPHT1;4 (Fig. but the activity of GmPHT1;11 (Fig. GmPHR1 and GmPHR4 genes regulate the activity of GmPHT1 GmPHR1 and GmPHR4 genes the activity of the GmPHT1;1 and GmPHT1;4 but the activity of the GmPHT1;11 in N. benthamiana leaves. All are as from at least three biological was performed we the effect of in GmPHR expression on GmPHT1 gene expression in We of of GmPHR genes these genes by we roots GmPHR1 GmPHR4 the expression levels were that in as by transcription (Supplemental Fig. A and Then, we constructed roots with GmPHR1 GmPHR4 (Supplemental gene These not proteins the coding of their genes in early even they expression of the GmPHR1 GmPHR4 (Supplemental Fig. A and expression were independent of the availability of Pi (Supplemental Fig. A and these was to the in PHT1 expression levels by the of the GmPHR1 GmPHR4 gene under different Pi a effect of GmPHR1 and GmPHR4 on GmPHT1;1 expression (Fig. was with the results of the activity (Fig. GmPHR1 and GmPHR4 genes regulate GmPHT1 gene expression in soybean nodules. GmPHR1 gene GmPHT1;1 expression under GmPHR4 gene GmPHT1;1 expression independent of the availability of GmPHR1 GmPHR4 GmPHT1;11 expression in soybean nodules independent of the availability of from of GmPHR1 GmPHR4 was with from these genes the GmPHR1 and GmPHR4 genes regulate GmPHT1;4 expression in soybean nodules on the availability of All show from at least three biological with was performed and of the was performed the expression of the control under as a GmPHR4 GmPHT1;11 of the Pi (Fig. which also with the results of the transcription activity (Fig. However, as for GmPHR1 and GmPHT1;11, the results (Fig. were to the results of the transcription activity (Fig. GmPHR1 GmPHT1;11 expression under and in nodules (Fig. results that there is of GmPHR1 function on In N. GmPHR1 GmPHT1;11 in soybean nodules, GmPHR1 GmPHT1;11 In other the effect of GmPHR1 on GmPHT1;11 expression was on The expression of GmPHT1;4 was on Pi When the of Pi was the GmPHR4 gene GmPHT1;4 expression (Fig. However, when the of Pi was GmPHR1 GmPHT1;4 of GmPHR4 GmPHT1;4 and GmPHR4 not GmPHT1;4 expression (Fig. the nodule was for it is to that regulate the expression of GmPHT1s in a tissue-specific mode in nodules. To investigate the function of GmPHR-GmPHT1 in Pi homeostasis in nodules, we the Pi in different nodules similar GmPHR1 GmPHR4 the of Pi in nodules in a (Fig. levels of GmPHT1;11 also Pi in nodules (Fig. These results that and GmPHT1;11 all in maintaining Pi homeostasis in nodules. GmPHR-GmPHT1 modules play important roles in maintaining Pi homeostasis in nodules and in regulating nodule initiation and GmPHR1 and GmPHR4 genes, nodules from roots were for GmPHT1;11, nodules from roots of plants were Pi homeostasis in nodules. and GmPHT1;11 Pi in nodules. and The of and GmPHT1;11 on nodule initiation and development All show from at least three biological was performed and we the function of PHR-PHT1 modules in nodulation through of the nodule and of nodules as in Our results show that and GmPHT1s functions the of nodules (Fig. GmPHR1 nodule initiation, and GmPHR1 levels in an of nodules at Pi expression of GmPHR4 the of nodules in the it was that the effect of GmPHT1;11 on nodule was independent of Pi nodule the results were to the of nodules in to Pi signaling (Fig. and GmPHT1;11 the of nodules, and GmPHR1 on nodule GmPHR4 in the of nodule In and GmPHT1;11 not the of nodules (Supplemental Fig. To that GmPHT1;11 functions in we constructed roots (Supplemental and and The nodulation that of GmPHT1;11 the of nodules root and nodule but nodule These results are with the results of the soybean (Fig. and were GmPHR1 and GmPHR4 GmPHT1;4 and in GmPHR1 expression GmPHR4 expression under and and (Supplemental Fig. and However, of GmPHT1;11 GmPHT1;4 of GmPHT1;11 no effect on the expression of GmPHT1;4 (Supplemental Fig. The effect of GmPHR-GmPHT1 modules on nodule was a which on the different GmPHR-GmPHT1 and availability of These results that there a potential GmPHR-GmPHT1 similar to previous reports et al., et al., reports that the expression of GmPHT1;1 and GmPHT1;4 was related to activity and the of N in soybean plants (Qin et al., 2012b; Chen et al., 2019). We also the activity in nodules of GmPHT1;11 plants to an described et al., of the GmPHT1;11 gene the activity of of GmPHT1;11 GmPHT1;11 expression to the function of nodules in soybean. activity is in of and GmPHT1;11 with their under and and on the of the the different for the and was on with plants and were three with similar results. A single representative is the other two are in that Pi plays important roles in (Israel, Tang et al., 2001; and Huss-Danell, et al., 2006; Sulieman et al., et al., 2014). Phosphorus not only controls the energy of N2 fixation et al., but also nodule and development (Israel, through specific and not a a plant effect (Gentili and Huss-Danell, 2003). can Pi utilization the nodules to Pi deficiency et al., and the Pi in nodules not under et al., 2014), that Pi homeostasis in nodules is The PHR-PHT1 module is one of the regulators for Pi transport in plant cells (Bardin et al., 1996; et al., 2001; Qin et al., 2012a; Puga et al., 2017). In our study, we the role of PHR-PHT1 modules in maintaining Pi homeostasis in soybean nodules. Our identified and their target transporter genes in soybean nodules. proteins directly to on and regulated their their were independent of Pi availability in a of available Pi in that GmPHR-GmPHT1 signaling in nodules was a to GmPHR1 and GmPHR4 the expression of GmPHT1;1 and GmPHT1;4 but GmPHT1;11 expression in N. benthamiana GmPHR1 GmPHT1;11 expression in indicating of the GmPHR1 proteins and the GmPHT1;11 We postulated that there are other regulators controlling GmPHR1 activity that may be specific to soybean nodules. are a reports that proteins PHR binding to its in Arabidopsis (Puga et al., and et al., 2014). is that the of gene may play an important role in the of GmPHR our that different and GmPHT1s tissue-specific expression Therefore, the of regulating GmPHT1 expression is in soybean nodules. In all previous were reported as of PHT1 expression (Rubio et al., 2001; Bari et al., 2006; et al., Bustos et al., 2010). However, our results that GmPHR be an of PHT1 expression under special on its target genes, the availability of expression in nodules was for different GmPHR GmPHT1 GmPHR1 and GmPHT1;1 were expressed in nodule GmPHR4 and GmPHT1;11 were expressed in in nodules. It was reported that the expression of GmPHT1;4 was also in in nodules (Qin et al., 2012b) and that GmPHT1;1 was expressed in nodules (Chen et al., 2019). on our of expression and and in to previous reports (Qin et al., 2012b; Chen et al., we an for GmPHR-GmPHT1 modules in nodule (Fig. In GmPHR1 not only GmPHT1;1 expression but also However, our results show that GmPHR1 GmPHT1;4 expression in N. benthamiana and no signal was for the in regions of nodules, even the activity of the GmPHR1 was in these Therefore, the transcriptional of GmPHT1;4 in regions may be contributed by transcription factors other A working of GmPHR-GmPHT1 modules in soybean nodules. the from the The and the results in our study, factors and their in our factors the for In GmPHR1 and GmPHR4 the expression of GmPHT1;1 and to the effect of GmPHR1 and GmPHR4 on GmPHT1;11 in N. benthamiana other factors be in the of GmPHT1;11 transcription in It is in our that there were several GmPHR-GmPHT1 modules in nodules that the The effect of on GmPHT1s be on their but independent of the availability of Pi The module different characteristics in different GmPHR1 GmPHT1;11 in regions but may not GmPHT1;11 expression in A previous the of for the phosphate transporter expression in root cells arbuscular et al., Our results show that the may be also important for PHT1 These modules other the expression of one PHR the expression of other and a similar for the in GmPHT1;11 expression GmPHT1;4 One GmPHR several GmPHT1 and one GmPHT1 several regulating which the network with GmPHR and protein of transcriptional factors cells in nodules, the regulatory network may be divergence in expression to the of a regulatory network of Pi homeostasis in nodules. are in which is show nitrogen fixation efficiency even under et al., et al., et al., Valentine et al., 2017). The PHR-PHT1 module network described may Pi homeostasis in soybean nodules. genes in the module network different on nodule and Therefore, expression of one of the in this network Pi homeostasis in nodules, in in Pi and nodule initiation, development, and Our results also that nodule initiation and may be two processes by PHR-PHT1 that the factors to nodule initiation nodule growth, and GmPHT1;11 nodule but nodule of GmPHR1 GmPHR4 the of nodules, but the nodules in when to levels of GmPHR GmPHT1 N2 fixation GmPHT1;1 activity in soybean plants (Chen et al., 2019). In our study, we that the expression of GmPHT1;11 was related to the activity of of the effect of and GmPHT1s on nodules to the nitrogen fixation in soybean. All were constructed with soybean (Glycine mediated by Agrobacterium for Agrobacterium for root All soybean plants were in a under of of at with Nicotiana benthamiana plants were in a at with a was by with from to for soybean to for N. on the the plants and the Using the Arabidopsis (Arabidopsis thaliana) PHR1 protein sequence as a to its in the soybean genome in four GmPHR members and with were identified in soybean and named as GmPHR1 to and GmPHR from to with from to Similar to and GmPHR4 proteins also two conserved Myb and coiled-coil of two of sequences was All GmPHT1;1, GmPHT1;4, GmPHT1;11, and genes were identified in our previous (Fan et al., 2013). the of the transcription of GmPHR1 and GmPHR4 were into the et al., with and Then, the were and to the in a to the GmPHR of the of GmPHT1;1 GmPHT1;11 and were into the with and The were into a with and to GmPHT1 genes, the coding regions of GmPHR1 and GmPHR4 were into the et al., with and and to the gene in the these were with a et al., and a in our to by All expression vectors were into for soybean root et al., GmPHT1;11 the coding of GmPHT1;11 was into et al., with and to a Then, the fusion gene was into with and the a of the GmPHT1;11 coding sequence was amplified and into the et al., in and and vectors of GmPHT1;11 were into for soybean All sequences were by and are to the corresponding genes in genome in we constructed two vectors to and A gene et al., by the was into with and The et al., two and the were into with and The was to two by of the two and the with and To of target genes, we the et al., target sequences were and into with and and were into a in our to vectors for gene The vectors were into for root and the roots with et al., were for functional We also a similar to a previous report et al., to that the in in which the target of and GmPHT1;11 were amplified by with three of (Supplemental In this study, two target for gene were to (Supplemental and A detailed for and nodules is in and the roots of the for of roots to roots and with the marker et al., 2011). nodule and nodules on roots and the nodules named roots. roots and roots and nodules. on the from roots for of nodule and all nodules. the of Pi in nodules. gene expression in nodules. All genes in and proteins. to 14 of the a single with the in the the are and for and However, we not nodules for and gene all nodules were was as the and as the mediated by was to a to plants root mediated by was performed previous et al., 2007; et al., as a marker and an et al., as a A nodulation was performed to a previous report and the soybean and plants were with a of in et al., 2012), to were with the and soybean nodules were for nodule nodule and Pi was as the of a single Pi was as described et al., of activity of nodules was performed a previous report et al., roots with nodules were in a and was into the The was for in Then, was by The for was to the of to the of was to the activity of nodules as at were for of The was with to and to the The was to with in a and of the was for with the on the at of at and at was to and The were by All are in was at least three for nodules by we roots by a with an marker et al., 2011). roots were and were The with roots were to and with for nodule of nodules were of roots was through with a of two target The were on an and the with at least two (Supplemental Fig. were and into the for into independent were to the efficiency (Supplemental Fig. from the corresponding nodules was and for gene expression (Fig. the nodule of corresponding nodules were for of nodule and (Fig. The coding regions of and GmPHR4 were into the (Chen et al., The were into et al., to vectors by The expression were into which was infiltrated into the of leaves of to N. benthamiana the signal was a in et al., was as a control and as a nuclear marker et al., of GmPHT1 protein localization in cells was as in our previous report (Fan et al., 2013). of expression was performed mainly to a previous report et al., with The roots with were for The roots and nodules least independent were and in with infiltrated with phosphate and and at for Then, were through an and and with a root with nodules were and for and were with a in was a previous et al., 2013). with soybean plants in for 14 and nodules were in and for at and in in were with a and (Supplemental were by in transcription the The were by and with were with a The coding sequences of GmPHR1 and GmPHR4 genes were into the and and the vectors were into The proteins were The of the were by the and its was performed to the of the of binding binding of protein 20 of target and to of target A of Pi was to binding for and was for All are in were performed as described and 2014). was from the roots The was with and by The were by and to The are in The regions of the of GmPHT1;1 GmPHT1;4 and GmPHT1;11 were into the (Chen et al., Then, the were to the gene on the et al., by The expression vectors of and were for were the were into the of leaves of to N. benthamiana of the infiltrated leaves were and with and and the was a plant The were by et al., was as a were at least three biological for The to coding regions of and regions of GmPHT1s are in All experiments in our were with at least three biological and at least three independent all of which similar results. the show only representative results of at least in all from three independent All were the to P and P for this can be found in with the gene GmPHR1 GmPHR4 GmPHT1;1 GmPHT1;4 GmPHT1;11 and The are localization of GmPHT1;11, GmPHT1;1, GmPHT1;4, and proteins. PHR proteins with their in Arabidopsis and expression of the of and GmPHR1 and GmPHR4 proteins not bind to two of the gene The gene GmPHR-GmPHT1 modules by of gene of of GmPHR1 by in soybean roots. of GmPHT1;11 by the but the of nodules in soybean roots. A for and roots. The of of nodules from gene roots. of GmPHT1;11 by the but the of nodules in soybean roots. in our activity The sequences of the and the coding regions of We Chen and for with all and in but are not in the and for its during the of the

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