The PHOSPHATE1 genes participate in salt and Pi signaling pathways and play adaptive roles during soybean evolution
The PHOSPHATE1 (PHO1) gene family plays diverse roles in inorganic phosphate (Pi) transfer and signal transduction, and plant development. However, the functions and diversification of soybean PHO1 family are poorly understood.
Cultivated soybean (Glycine max) was domesticated from wild soybean (Glycine soja). To illuminate their roles in this evolutionary process, we comparatively investigated the G. max PHO1 genes (GmPHO1) in Suinong 14 (SN14) and G. soja PHO1 genes (GsPHO1) in ZYD00006 (ZYD6). The sequences of the orthologous Gm-GsPHO1 pairs were grouped into two Classes. The expression of Class I in both SN14 and ZYD6 was widely but relatively high in developing fruits, whereas Class II was predominantly expressed in the roots. The whole family displayed diverse response patterns to salt stresses and Pi-starvation in roots. Between SN14 and ZYD6, most PHO1 genes responded similarly to salinity stresses, and half had sharp contrasts in response to Pi-starvation, which corroborated the differential response capacities to salinity and low-Pi stress between SN14 and ZYD6. Furthermore, in transgenic Arabidopsis plants, most Class II members and GmPHO1;H9 from Class I could enhance salt tolerance, while only two Class II genes (GmPHO1;H4 and GmPHO1;H8) differently altered sensitivity to Pi-starvation. The expression of critical genes was accordingly altered in either salt or Pi signaling pathways in transgenic Arabidopsis plants.
Our work identifies some PHO1 genes as promising genetic materials for soybean improvement, and suggests that expression variation is decisive to functional divergence of the orthologous Gm-GsPHO1 pairs, which plays an adaptive role during soybean evolution.
KeywordsAbiotic stress Adaptive evolution Functional diversification Gene expression Orthologous gene PHO1 gene family Soybean
open reading frame
reverse transcription-polymerase chain reaction
Phosphorus is one of the most important macroelements for plants. Inorganic phosphate (Pi), the predominant form of phosphorus that is absorbed and utilized by plants, is limited in many soils. Under Pi-deficiency conditions, plants can remodel their root architecture to inhibit primary root growth and promote lateral root growth and root hair formation to improve phosphorus acquisition [1, 2]. Various Pi-starvation inducible genes involved in the Pi signaling pathway trigger different molecular processes to improve plant survival under Pi-starvation conditions , including AT4, INDUCED BY PHOSPHATE STARVATION1 (IPS1), PHOSPHATE TRANSPORTER (PHT1;4), and PHOSPHATE1 (PHO1;H1) in Arabidopsis [4, 5, 6]. Arabidopsis PHO1 is the first gene that has been reported to be involved in the transfer of Pi from the roots to the shoots [7, 8], and in the signal transduction of Pi-deficiency response [9, 10, 11]. The Arabidopsis pho1 mutant has low leaf Pi levels and shows severe shoot Pi-deficiency due to its defectiveness in loading Pi into the xylem . PHO1 is predominantly expressed in root stelar cells and participates in loading inorganic Pi into the xylem of roots . The PHO1-like gene is involved in Pi transport in yeast (Saccharomyces cerevisiae) , suggesting a conserved role of PHO1 in Pi transport and homeostasis.
The Arabidopsis genome contains 10 additional homologs of PHO1 (identified as PHO1;H1 to PHO1;H10), and their putatively encoded proteins contain SPX (named after SYG1, PHOSPHATASE 81, and XPR1) and EXS (named after ER RETENTION DEFECTIVE1, XPR1, and SYG1) domains [11, 12]. However, only PHO1 and PHO1;H1 are involved in Pi transport from the roots to the shoots . Three PHO1 homologs (OsPHO1;1, OsPHO1;2, and OsPHO1;3) have been identified in the rice genome, but only OsPHO1;2 has been shown to play a role in loading Pi into the xylem . These observations suggest that this gene family plays diverse roles. In corroboration with this notion, members of the PHO1 gene family in Arabidopsis were also found to participate in signal transduction, as well as plant growth and development. SHORT HYPOCOTYL UNDER BLUE1 (SHB1, also named as PHO1;H4) is involved in blue light signaling responses, flowering, and seed development [14, 15, 16], whereas PHO1;H10 responds to various abiotic and biotic stresses and participates in the abscisic acid (ABA)-mediated signal transduction pathway . Interestingly, Arabidopsis PHO1 has also been reported to play an important role in stomatal responses to ABA , suggesting a possible interaction among different signal transduction pathways in plants such as drought and salinity stresses.
Salt stress largely influences global agriculture. Excessive soluble salts in soil can reduce plant growth, thereby severely affecting crop yield [19, 20]. Salt tolerance mechanisms are highly complex and can be controlled by the ionic signaling pathway, osmotic pressure, and the ABA-mediated pathway . The salt overly sensitive (SOS) pathway controls the expression and activity of ion transporters by the SOS3-SOS2 protein kinase complex, which can be activated by salt stress-elicited calcium signals [21, 22]. The delta1-pyrroline-5-carboxylate synthetase gene (P5CS1), which encodes a rate-limiting enzyme involved in proline synthesis, can increase salt tolerance in plants by increasing osmotic pressure [23, 24]. Some other genes that are related to the ABA-mediated pathway such as the alcohol dehydrogenase gene (ADH) and FIERY1 (FRY1) gene, are also involved in salt tolerance [25, 26]. These genes are often employed as marker genes in salt signaling pathways, and variations in the expression of these genes serve as diagnostic parameters in evaluating plant salt tolerance [21, 27].
Cultivated soybean (Glycine max) is thought to have been domesticated from wild soybean (Glycine soja) in China as early as 5000–9000 years ago [28, 29]. Various abiotic stresses largely limit soybean yield. The northeast region of China is a major soybean planting area, with a total of 3.73 × 106 hm2 of sodic land . Salt stress inhibits soybean development from germination to reproductive stage [31, 32] and sharply reduces grain yield to only 38.9% of that under normal conditions [33, 34]. Moreover, low availability of soluble Pi in the soil affects approximately half of the cultivated land around the world  and about 70% of the cultivated land is Pi-deficiency in China . Pi-starvation significantly reduces grain yield by 40% in soybean, whereas Pi supply increases grain yield and pod number by 68 and 61%, respectively . Several quantitative trait loci (QTLs) related to salinity tolerance and Pi-deficiency have been identified in soybean [38, 39, 40, 41, 42]. However, the mechanisms underlying the responses of soybean to these abiotic stresses remain unclear. Wild soybeans with wider geographic distributions and more genetic variations have adapted stress tolerance features compared to cultivated soybeans during evolution [43, 44]. Therefore, wild soybeans may serve as a rich germplasm for the improvement of cultivated soybeans. We previously identified 12 members of the PHO1 gene family in cultivated soybeans and assessed their expression patterns in response to various stresses , which indicated their functions in abiotic stress tolerance. However, the functions of the PHO1 gene family in soybean and functional diversification between wild and cultivated soybeans remain unclear. The present study compared the orthologous PHO1 gene pairs between wild and cultivated soybeans in terms of sequence, tissue-specific expression, and responses to various abiotic stresses, including salinity and Pi-starvation conditions, followed by transgenic Arabidopsis analyses. This is the first investigation on the evolution and function of PHO1 gene family in wild and cultivated soybeans. This study elucidates the adaptive roles of the PHO1 gene family in soybean evolution, as well as reveals a potentially converged role of these genes in Pi and salinity signal pathways, thereby providing novel genetic material and ideas for crop improvement.
Sequence comparison of the GmPHO1 and GsPHO1 cDNAs
Organ-specific expression of soybean PHO1-like genes
Assessment of expression diversity has been utilized to establish functional divergence among genes. To investigate the tissue-specific expression patterns of the PHO1-like genes, the total RNAs from the roots, leaves, stems, flowers, and developing fruits (1, 3, 5, and 15 days after pollination, shortened as fruit1, fruit3, fruit5, and fruit15) in SN14 and ZYD6 were subjected to qRT-PCR. Due to high sequence identity, common but specific primers were designed for paralogous gene pairs PHO1;H1/H4, PHO1;H9/H10, and PHO1;H12/H14. The results showed that the PHO1 genes belonging to Classes I and II exhibited different organ-specific expression patterns (Additional file 1: Figure S3). Most soybean PHO1 genes in Class Ι were differentially expressed among the vegetable and reproductive organs (Additional file 1: Figure S3a-e). A few extreme variations in expression were observed. PHO1;H7 was active in most developing organs but hardly detected in roots (Additional file 1: Figure S3d), whereas PHO1;H9/H10 were predominantly expressed in the roots (Additional file 1: Figure S3e). However, the expression patterns for genes in Class II were strikingly different from those in Class I. Soybean genes in Class II had similar expression patterns and were predominantly expressed in vegetable tissues, particularly the roots and stems (Additional file 1: Figure S3f-i). Moreover, the expression of these genes in fruits was relatively low (Additional file 1: Figure S3f-i).
Comparison of the orthologous Gm-GsPHO1 pairs indicated that Class I genes displayed diverse expression patterns in various organs between SN14 and ZYD6. GmPHO1;H7 had higher expression than GsPHO1;H7 in all assessed organs (Additional file 1: Fig. S3d), whereas GsPHO1;H3 had a relatively higher expression in flowers and early developing fruits (fruit1) than GmPHO1;H3 in SN14 (Additional file 1: Figure S3b). Nevertheless, some genes such as PHO1;H2 and PHO1;H6 showed complex expression patterns in different tissues between SN14 and ZYD6. GmPHO1;H2 exhibited lower expression levels in the roots, leaves, and flowers, but higher expression in other organs than GsPHO1;H2 (Additional file 1: Figure S3a). Similarly, PHO1;H6 showed lower expression levels in the roots and leaves but higher expression in the developing fruits in SN14 than in ZYD6 (Additional file 1: Figure S3c). Despite the complex expression patterns of genes in Class I, these showed relatively higher expression levels in developing fruits (fruit3-fruit15) in SN14 than in ZYD6. However, Class II genes were highly expressed in the vegetative plant parts in ZYD6 than in SN14 as well as in the roots, stem, or leaves (Additional file 1: Figure S3f-i).
Soybean PHO1-like gene expression under various stresses
Class II PHO1 genes (PHO1;H1/4, PHO1;H5, PHO1;H8, and PHO1;H12/14) showed similar expression profiles during root and leaf development under normal conditions (red and blue solid lines, Fig. 4f-i). With Pi-deficiency, these were similarly upregulated in the roots between ZYD6 and SN14 and reached a maximum at 3 weeks (red dashed lines in Fig. 4f-i). These genes were upregulated in the leaves of ZYD6, whereas their expression in the leaves of SN14 did not change (dashed blue lines in Fig. 4f-i).
These findings suggest that the PHO1 gene family may play roles in soybean adaptation and morphological divergence. Moreover, the expression variation is likely decisive evolutionary event between the orthologous pairs of PHO1 genes, which occurred during the divergence of wild and cultivated soybeans. We therefore functionally tested these assumptions by overexpressing the cDNA of these genes from cultivated soybean in transgenic Arabidopsis.
Overexpressing GmPHO1 genes in transgenic Arabidopsis
Due to the highly identical sequences within paralog pairs GmPHO1;H9/H10 and GmPHO1;H12/H14 (Additional file 2: Table S1), the GmPHO1;H10 and GmPHO1;H14 were excluded, and thus altogether 10 GmPHO1 genes from the SN14 (cultivar) were included in the transgenic Arabidopsis assays. Three T3 lines of each transgene that were confirmed via RT-PCR (Additional file 1: Figure S5) were further investigated, and all obtained GmPHO1 transgenic plants showed no obvious phenotypic variations compared to wild-type (WT) Arabidopsis, including flowering time, seed size, plant height, and germination rate under normal growth conditions (Additional file 2: Table S3), indicating that overexpressing GmPHO1 did not affect plant development.
GmPHO1 altered the salt tolerance of transgenic Arabidopsis plants
No significant difference in the green rate of seedlings was observed under 125 mM NaCl conditions; however, differences were observed under 175 mM NaCl conditions (Additional file 1: Figure S6a). Only 35S::GmPHO1;H9 from Class I transgenic plants showed significantly higher seedling green rate than WT under NaCl stress (Fig. 5a, Additional file 1: Figure S6a). However, the 35S::GmPHO1;H1, 35S::GmPHO1;H5, 35S::GmPHO1;H8, and 35S::GmPHO1;H12 transgenic lines of Class II exhibited a higher seedling green rate than WT under 175 mM NaCl stress (Additional file 1: Figure S6a). These observations show that GmPHO1 enhances the salt tolerance, however, the function is obviously diverged between Class I and Class II since only one gene in Class I and many in Class II could confer salt tolerance to transgenic Arabidopsis plants.
Transgenic plants of 35S::GmPHO1;H6, 35S::GmPHO1;H8, and 35S::GmPHO1;H9 were selected for the evaluation of salt tolerance in the soil. The four-week old seedlings were supplied with 250 mM NaCl to induce salt stress. After 15 days of treatment, the WT and transgenic lines harboring GmPHO1;H6 showed extensive bleaching and wilting, whereas the 35S::GmPHO1;H8 and 35S::GmPHO1;H9 transgenic lines all exhibited intense green color and were more vigorous than the WT (Additional file 1: Figure S6b-e). Furthermore, 35S::GmPHO1;H8 and 35S::GmPHO1;H9 transgenic plants all showed significantly higher chlorophyll content than the WT and the 35S::GmPHO1;H6 transgenic lines (Additional file 1: Figure S6f). Moreover, the aboveground biomass of 35S::GmPHO1;H8 and 35S::GmPHO1;H9 transgenic plants after one-month treatment were much higher than the WT and 35S::GmPHO1;H6 lines (Additional file 1: Figure S6 g). These observations indicate that overexpressing soybean PHO1 genes (GmPHO1;H1, GmPHO1;H5, GmPHO1;H8, GmPHO1;H9, and GmPHO1;H12) improves salt tolerance in Arabidopsis.
GmPHO1 affects the salt tolerance pathways in transgenic Arabidopsis
The SOS2, SOS3, ADH, P5CS1, and FRY1 genes are essentially involved in salt tolerance pathways in plants [21, 22, 23, 24, 25, 26]. To elucidate the possible connection of GmPHO1 with salt stress pathways, we checked the expression of these salt tolerance pathway genes in transgenic plants. The seven-day-old seedlings of WT, 35S::GmPHO1;H8, 35S::GmPHO1;H9, and 35S::GmPHO1;H6 transgenic Arabidopsis plants were transferred to a medium with 175 mM NaCl for 24 h, and total RNA from the whole seedlings was subjected to qRT-PCR. Under normal growth (0 mM NaCl) conditions, we found that each transgene could differentially affect the expression of these marker genes compared to the WT (highlighted by the black stars above the open color columns in Fig. 5b-d). Furthermore, we observed that ADH, P5CS1, SOS3, and FRY1 were upregulated by NaCl treatment, whereas SOS2 expression was not significantly affected in the WT background (highlighted by the black star above the blocked gray columns compared to the corresponding open gray columns in Fig. 5b-d). Moreover, these salt pathway marker genes were differentially expressed in the transgenic plants compared to the WT under salt stress conditions (highlighted by the red stars above the blocked color columns relative to the blocked gray column in Fig. 5b-d).
In the 35S::GmPHO1;H8 transgenic plants, except for the P5CS1 downregulation, the SOS2, SOS3, ADH, and FRY1 genes were upregulated under 0 mM NaCl. However, compared to the WT under salt treatments, only the expression of ADH sharply increased, whereas the other genes were downregulated (Fig. 5b). Nonetheless, relative to the corresponding transgenic lines under 0 mM NaCl, both ADH and P5CS1 were upregulated, and the other genes were downregulated under salinity stress (Fig. 5b). Compared to the WT, in 35S::GmPHO1;H9 transgenic Arabidopsis, SOS2, SOS3, ADH and P5CS1 were downregulated, whereas FRY1 was upregulated under 0 mM NaCl conditions (Fig. 5c). However, when treated with NaCl, the SOS3 and ADH genes were upregulated in these transgenic lines, and SOS2, FRY and P5CS1 were downregulated (Fig. 5c). Relative to the corresponding transgenic lines without NaCl supplementation, SOS3, ADH and P5CS1 were upregulated and the other genes were downregulated with NaCl treatments (Fig. 5c). Interestingly, in the 35S::GmPHO1;H6 transgenic lines, changes in the expression of the salt pathway marker genes were observed under both normal and salt stress conditions (Fig. 5d); however, no changes in salt tolerance were detected in the transgenic plants. Compared to the changes in gene expression patterns in the 35S::GmPHO1;H6 transgenic lines (Fig. 5d), the upregulation of the ADH gene in both 35S::GmPHO1;H9 and 35S::GmPHO1;H8 (Fig. 5b, c) might play an essential role in enhancing salt tolerance in the two transgenic analyses. Nonetheless, changes in the expression of the marker genes in the salt-tolerance pathways in overexpressing GmPHO1;H6 are suggestive of the inherent nature of PHO1 genes in salt response.
GmPHO1 is involved in the responses to Pi-starvation in transgenic Arabidopsis
GmPHO1 affects the Pi signaling pathway in transgenic Arabidopsis
IPS1, AT4, PHT1;4, and PHO1;H1 are upregulated in Arabidopsis during Pi-deficiency [4, 5, 6] and are considered to be marker genes in the Pi signal pathway. To further evaluate the possible roles of the GmPHO1 genes in the Pi pathway, we investigated the expression levels of these marker genes in transgenic Arabidopsis lines. The 35S::GmPHO1;H8, 35S::GmPHO1;H4, and 35S::GmPHO1;H6 plants were evaluated and compared to the WT. Under normal conditions, IPS1 and PHT1;4 were upregulated in the 35S::GmPHO1;H4 transgenic lines, whereas PHO;H1 was downregulated, and AT4 expression did not change (Fig. 6e-h). PHT1;4 expression did not change, whereas PHO;H1, IPS1, and AT4 were downregulated in the 35S::GmPHO1;H8 transgenic lines compared to the WT (Fig. 6i-l). The variations in the expression of these marker genes in the 35S::GmPHO1;H6 plants was similar to that in the 35S::GmPHO1;H8 plants (Fig. 6m-p). These results indicate that overexpression of GmPHO1 could affect the expression of genes that are involved in the Pi pathway.
However, under Pi-starvation conditions, we found that IPS1, AT4, and PHT1;4 were indeed upregulated by Pi-deficiency in WT Arabidopsis, whereas PHO1;H1 was significantly downregulated (comparisons between blue columns under Pi normal and deficiency conditions in Fig. 6e-p). Moreover, the expression levels of these marker genes in the 35S::GmPHO1;H4 transgenic lines decreased compared to the WT, indicating their insensitivity to Pi-deficiency (Fig. 6e-h), whereas these were all upregulated in the 35S::GmPHO1;H8 transgenic lines, showing hypersensitivity to Pi-deficiency (Fig. 6i-l). However, changes in the expression of these marker genes in the 35S::GmPHO1;H6 transgenic plants in response to Pi-starvation were similar to those of the WT (Fig. 6m-p). Therefore, in the GmPHO1 family, GmPHO1;H4 or GmPHO1;H8 might participate in the Pi pathway by affecting the expression of genes that are homologous to Arabidopsis PHO;H1, IPS1, AT4, and PHT1;4.
Responses of SN14 and ZYD6 to salinity stresses and Pi-starvation
Based on the results of transgenic Arabidopsis analysis, we then checked the expression of soybean genes that were homologous to the Arabidopsis Pi pathway marker genes. Searching in the NCBI and Phytozome databases did not identify any homologs of Arabidopsis IPS1 and AT4 in soybean, whereas soybean PHT1;4-like genes were found. Phylogenetic reconstruction of the PHT1;4 genes depicted the putative orthology of the soybean genes (Glyma.03G162800, Glyma.10G036800, and Glyma.19G164300) and Arabidopsis PHT1;4 (Additional file 1: Figure S10). The total RNAs from the roots were subjected to qRT-PCR analysis, which revealed that soybean PHT1;4-like genes showed similar expression profiles during root development in both ZYD6 and SN14, but had different expression levels in roots. In particular, Glyma.19G164300 showed trace levels of expression under normal conditions (Fig. 7i-k). Moreover, these were all upregulated in ZYD6 and SN14 under Pi-deficiency (Fig. 7i-k). Nevertheless, the increase in the level of gene expression in response to Pi-starvation in ZYD6 was significantly higher than that in SN14.
These results suggest the differential response capacities of wild and cultivated soybeans to salinity and Pi-starvation at both phenotypic and molecular levels, which was linked with the PHO1 gene family since the salt and Pi signaling pathways could be affected by the expression of some GmPHO1 genes.
Soybean PHO1 genes are involved in salinity or phosphate pathways
Analysis of the expression and the divergence pattern of soybean PHO1 genes have suggested the role of this gene family in plant and seed development. However, overexpression of soybean PHO1 genes in transgenic Arabidopsis did not reveal any variations in seed germination, flowering time, seedling morphology, plant height, and seed size. These findings were discordant from those of Arabidopsis PHO1 genes [15, 16], thereby suggesting that the PHO1 genes might have functionally diverged in plant development. The expression of soybean genes in response to salinity and phosphate deficiency suggests that these may play roles in these processes, and transgenic plants harboring specific soybean PHO1 genes have revealed alterations in tolerance to either salinity stress or Pi-deficiency.
Overexpressing the GmPHO1 genes in transgenic Arabidopsis plants has facilitated in the identification of five members of this gene family (GmPHO1;H1, GmPHO1;H5, GmPHO1;H8, GmPHO1;H9, and GmPHO1;H12) that improve salt tolerance in Arabidopsis. The germination rate, the green rate, and growth under salt stresses either was not affected or even much better than the WT. We further investigated the expression of the SOS2, SOS3, ADH, P5CS1, and FRY1 genes, which are involved in salt tolerance pathways [21, 22, 23, 24, 25, 26]. GmPHO1 genes apparently have the capability to affect the expression of these genes using various mechanisms, thus influencing salt tolerance in the transgenic Arabidopsis plants. The molecular interaction between the GmPHO1 family and salt tolerance pathways requires further investigation. However, not all genes confer salt tolerance to the transgenic Arabidopsis plants, possibly due to variations in the molecular interactions during evolution. Nevertheless, the phenotype could likely depend on how the transgenes affect ADH expression. The increase in ADH expression seems to be essential to conferring strong salt tolerance to the transgenic Arabidopsis plants.
Overexpressing GmPHO1 in transgenic Arabidopsis plants demonstrated that GmPHO1;H4 and GmPHO1;H8 could also affect tolerance to Pi-deficiency. These observations agree with the findings in various plants. In Arabidopsis, PHO1 and PHO1;H1 are involved in phosphate transfer from the roots to the shoots of the xylem [6, 8, 48]. The PHO1 genes have also been shown to participate in Pi transfer in rice and potato [13, 49]. Various genes such as IPS1, AT4, and PHT1;4 are considered as marker genes in Arabidopsis in response to Pi-starvation [4, 5], and pho1 shows all the hallmarks associated with Pi-deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes [2, 11]. PHO1 expression in response to low Pi is regulated by the transcription factor WRKY6, which is degraded by a ubiquitin E3 ligase in Arabidopsis [9, 48]. This regulatory mechanism requires further investigation in soybean. However, we found that the expression of the abovementioned marker genes of the Pi signaling pathways in GmPHO1 transgenic plants were differentially affected, which in turn supports notion on the diversity of tolerance to Pi-starvation. Compared to the WT under low-Pi conditions, the expression of all the four marker genes of the Pi signal pathways increased in the 35S::GmPHO1;H8 Arabidopsis plants, indicating their hypersensitivity to low-Pi conditions, which was accompanied by shortening of the roots and suggesting that root development is inhibited in 35S::GmPHO1;H8 transgenic Arabidopsis plants with Pi-starvation. However, the opposite was observed in 35S::GmPHO1;H4 plants. The expression patterns of these marker genes in 35S::GmPHO1;H6 plants were similar to those of 35S::GmPHO1;H8 plants under normal conditions. However, relative to the WT, no changes in the expression of these marker genes were observed in the 35S::GmPHO1;H6 plants when challenged with low-Pi conditions, and no phenotypic variations were detected. These results indicate the complexity of the regulatory mechanism underlying the response to low-Pi levels. Nevertheless, the homologs of Arabidopsis PHT1;4 in soybeans were differentially expressed between SN14 and ZYD6, thus further corroborating the distinct responses to low-Pi treatment between the two soybean species/accessions.
Transgenic analysis also showed that some transgenic plants harboring a few GmPHO1 genes such as GmPHO1;H8 showed strong tolerance to salinity stress and elevated sensitivity to Pi-deficiency, suggesting a possible interaction between the two abiotic signaling pathways. However, the molecular details of this possible crosstalk and how GmPHO1 genes affect plant fitness (including yields, biomass accumulation etc.) in transgenic Arabidopsis plants require further investigations. Nevertheless, these results indicate that soybean PHO1 genes are involved in either salinity or Pi pathways, which are conserved among plant species [8, 9, 13, 48], further suggesting the functional divergence of the soybean PHO1 paralogs in response to abiotic stresses.
The evolutionary implication of soybean PHO1 genes
Cultivated soybeans were domesticated from wild soybeans at least 5000 years ago [28, 29]. Variations in either coding sequence or expression of the domesticated genes are the main causative forces affecting phenotypes during plant domestication [50, 51, 52, 53, 54, 55]. Differential expression of PHO1 genes in developing fruits was detected between the wild and cultivated soybeans; however, this was not functionally linked to any fruit-related domesticated trait. Wild soybean is endowed with some excellent agronomic traits such as stress tolerance due to the wide geographic distributions and rich genetic variations [43, 44]. However, unlike seed size, the adaptive variation does not seem to be a domestication trait, but a result of diverse challenges in the environments. Elite allelic variations in certain genes involved in the related pathways in either wild or cultivated accessions could confer tolerance to certain stresses. We observed that cultivated soybean SN14 and wild soybean ZYD6 showed distinct sensitivities to low Pi, although no obvious difference in tolerance to salinity was detected. A comparative study of the PHO1 genes between the two accessions would provide insights to the observed differences. This gene family evolved from the soybean common ancestors and showed overall similar variation patterns during soybean evolution (Fig. 8a-c). The soybean PHO1 genes from both G. max and G. soja were divided into two classes, namely, Class I and Class II. Fourteen members of the PHO1 family were predicted in soybean ; however, in the present study, we confirmed that 12 members are expressed and thus, pseudogenization may have occurred in Class I of both G. max and G. soja. The actively expressed genes in Class I were basically present in all evaluated organs, whereas the genes from Class II predominantly accumulated in the roots, thereby suggesting functional divergence of the two classes. The restricted expression of Class II PHO1 genes in the roots could limit their role in root development and adaptation. This notion is further supported by the observations of gene expression variations in soybean roots of these PHO1 genes in response to salinity stress and Pi-starvation.
Most PHO1 genes showed similar expression tendency in roots between SN14 and ZYD6 (Fig. 8b, c), thus conferring a similar salt tolerance between the two accessions, while the interactions among PHO1 genes, particularly two genes (GmPHO1;H4 and GmPHO1;H8) from Class II seemed to display an essential role in conferring soybean tolerance to low Pi. Moreover, transgenic Arabidopsis analysis further substantiated these notions and revealed that manipulating the PHO1 family genes could alter the plant tolerance to salinity stresses or low-Pi treatments (Fig. 8c). These observations suggested that PHO1 gene family is involved in determining the tolerance to salinity and low-Pi stresses. Particularly, as confirmed by transgenic Arabidopsis analysis, GmPHO1;H4 and GmPHO1;H8 were potentially functional members conferring soybean sensitivity to low Pi (Fig. 8c), which might be fulfilled by affecting the expression of PHT1;4 genes, since the Arabidopsis PHT1;4 gene, which plays essential role in Pi acquisition , was significantly affected in the transgenic Arabidopsis plants. Moreover, the expression of PHT1;4 genes in response to low Pi were significantly higher in ZYD6 than that in SN14, correlating to differential response capacities to Pi-deficiency between the two accessions that ZYD6 was insensitive than SN14. The relationship between PHO1 and PHT1;4 genes in soybean needs further investigations, however, our results suggest the functions and the divergence of the soybean PHO1 gene family in relation to plant adaptation during evolution. Nevertheless, not all GmPHO1 members altered the tolerance to salinity and low Pi in transgenic Arabidopsis plants (Fig. 8c), suggesting the functional divergence between soybean paralogs, which might be mainly due to variations in the coding sequences after gene duplications.
The PHO1 orthologous genes in G. max and G. soja had arisen from common soybean ancestors (Fig. 8a). We found that the 12 active PHO1 orthologous gene pairs between wild and cultivated soybeans show limited sequence divergence, and all the detected variations were predicated to be neutral, suggesting conserved functions of Gm-GsPHO1 orthologous gene pairs between the wild and cultivated soybeans. However, we have also revealed that the Gm-GsPHO1 orthologous gene pairs display a diverse tissue-specific expression patterns during fruit developments and gene expression patterns in response to various abiotic stresses, including salinity and Pi-deficiency, suggesting that domestication affects their expression. Interestingly, the level of PHO1 gene expression in developing fruits of G. max was overall higher than that of G. soja, whereas the PHO1 expression in roots of G. soja was generally higher than that in G. max, indicating the functional divergence of these genes during soybean domestication. In particular, the expression of Class I PHO1 genes has been broadly extended to various tissues and it showed differential expression between wild and cultivated soybeans. This differential expression patterns may be involved in multiple developmental roles and be possibly associated with phenotypic divergence during soybean domestication because Class I gene AtPHO1;H4 (SHB1) plays developmental roles in seed size and flowering time in Arabidopsis [12, 15, 16]. However, transgenic Arabidopsis analysis of soybean PHO1 genes did not support this notion (Fig. 8c). The roles of soybean genes in response to salinity and low-Pi stresses were well evidenced in transgenic Arabidopsis studies, which is in line with the observation that the expression of soybean PHO1 genes diversely responded to stresses, and the overall four patterns of gene expression occurred in response to salinity (S1-S4) or low Pi (P1-P4) conditions in soybeans, where some Gm-GsPHO1 orthologous pairs had similar response patterns with different extent under either salinity stress or Pi-deficiency. However, others had quite different expression variations both in expression trend and strength, even a contrary tendency (Fig. 8b). PHO1 genes differentially responded to salinity and low-Pi stresses in the two soybean accessions; however, only differential responses to low Pi were observed, suggesting that PHO1 genes are primarily involved in the Pi pathways, whereas the role of these genes in salinity stresses, which was reflected in transgenic Arabidopsis, could be masked by variations in other soybean genes. The molecular basis underlying the responses to salinity and low Pi in soybean might be highly complex. However, the divergence of expression and its response to stresses of PHO1 gene family in the roots of SN14 and ZYD6, if this could be generalized between G. max and G. soja, could play a role in differentiating the capability of stress signal perception and transduction, hence tolerance to certain stresses between the two species during the evolution.
Functional diversification of the plant PHO1 genes
The PHO1 gene family plays multiple roles during plant development and evolution. Arabidopsis SHB1 (PHO1;H4), which is involved in flowering time and seed size control [12, 15, 16], belongs to Class I. Transgenic Arabidopsis analysis did not show any evidence supporting such a role for PHO1-like genes in soybean. However, such a role was not excluded because the expression of Class I genes had diverged between G. max and G. soja during fruit development. Alternatively, its role in seed size might be specific to the Brassica species or only in Arabidopsis. Class I genes show broad and diverse expression patterns, which is indicative of multiple developmental roles. The role of Class II members in low Pi is particularly conserved in various plants, including Arabidopsis [6, 8], rice , cultivated soybean , and potato , suggesting a specific role for Class II PHO1 genes in the Pi signaling pathways. This observation to a certain extent corroborates the different responses of SN14 and ZYD6 to low-Pi treatments. We also have elucidated the role of soybean PHO1 genes in responses to salinity stresses, and in transgenic Arabidopsis, most genes from Class II alter tolerance to salinity stresses. However, only one gene GmPHO1;H9 from Class I did, further supporting functional divergence between the two classes. However, differential expression of these PHO1 genes apparently could not confer differential salinity tolerance to SN14 and ZYD6, further reflecting the complexity of the molecular mechanism underlying plant responses to abiotic stresses. The plant PHO1 gene family has diverse roles, which were determined by expression divergence (Fig. 8b, c), although the molecular variations underlying the expression divergence needs further investigation. The expression of all soybean genes in the whole family in response to various abiotic stresses seems to be diversely active, implying that the PHO1-like genes are primarily involved in the adaptive evolution of plants.
Taken together, significant variations in gene expression among different tissues, particularly in developing fruits and roots, and its variations in roots in response to low-Pi and salinity stresses largely underlies the functional divergence of the orthologous gene pairs of the PHO1 family between wild and cultivated soybeans. No direct functional evidence was observed to support their role in soybean fruit development, however, the expression variation in roots matters in determining the response capacities in different soybean accessions. The modes of functional evolution of the soybean PHO1 genes are overall unclear, but transgenic Arabidopsis analyses have revealed functional divergence between members of this gene family in response to salinity and low-Pi stresses, thus endorsing that the PHO1 genes play an adaptive role in soybean evolution. The mechanisms of the soybean PHO1 genes in relation to various stresses, their crosstalk, and roles of adaptive evolution need further investigations in the native hosts. This study also provides promising genetic materials for crop improvement; i.e., genetically manipulating GmPHO4, GmPHO8, and GmPHO9 could improve soybean performance under salinity stresses or low-Pi conditions.
Plant materials and growth conditions
The cultivated soybean Suinong14 (SN14) and the wild accession ZYD00006 (ZYD6)  were grown in a greenhouse of the Institute of Botany (Beijing, China) under long-day conditions (16 h light/8 h dark at 23 °C–25 °C). Roots, leaves, and stems of three-week-old seedlings were collected, and mature flowers and 1-, 3-, 5-, or 15-day post-fertilization fruits were gathered. Biological samples from four additional wild (Y1, Y2, Y3, and Y4) and cultivated (Hefeng48, Nenfeng16, Heinong35, and Dongnong53) soybeans  were included for tissue-specific expression profiling. The soybean seeds are available upon request for research only.
Salinity stresses and low-Pi treatment of soybeans
For salt stresses, seeds of SN14 and ZYD6 were germinated in vermiculite in a chamber with 1/5 Hoagland solution for 2 weeks and then supplemented with 1/5 Hoagland solution with different NaCl concentrations (100 mM, 150 mM, 200 mM, and 250 mM). Soybean seedlings without salinity treatment were used as controls. The roots were harvested after treatments for 4 h for gene expression study.
For low-Pi treatments, seeds of SN14 and ZYD6 were germinated and cultivated in a chamber with 1/5 Hoagland solution with normal concentration Pi (1.25 mM) or without Pi for 3 weeks. The roots and leaves were respectively harvested after one, two, and 3 weeks of treatments for gene expression profiling.
Each treatment consisted of three replicates. For phenotypic observations, the same solution was supplemented every 2 days in salinity stresses or refreshed every week in low-Pi treatments until the observed time as indicated.
Quantitative RT-PCR (qRT-PCR) analysis
Total RNA was extracted using SV Total RNA Isolation System (Promega, Madison, WI, USA), and subsequently treated with DNase I (Promega) to digest the residual DNA. The cDNA was synthesized with oligo (dT)18 primers following the instructions of the M-MLV cDNA synthesis kit (Invitrogen, Carlsbad, USA). qRT-PCR was performed on an Mx3000P QPCR system (Agilent, CA, USA) using SYBR Premix Ex Taq (TaKaRa, Dalian, China). Soybean ACTIN gene (Glyma18g52780) was used as internal control. Each experiment was performed using three independent biological samples, and means ± standard deviations are presented. The gene-specific primers for PHO1 family were designed as previously described . Primers for the Arabidopsis marker genes of the Pi signaling pathways and the salt tolerance pathway genes were respectively synthesized as described in the previous studies [3, 56]. All primer sequences designed in this work are presented in Additional file 2: Table S7.
Transgenic Arabidopsis analysis
The open reading frames (ORF) of the GmPHO1 genes were inserted into a pCAMBIA1300 vector that was driven by a cauliflower mosaic virus 35S promoter. Each construct was transformed into Arabidopsis thaliana as mediated by Agrobacterium tumefaciens strain GV3101 using the floral dipping method . The transgenic plants were selected on Murashige & Skoog (MS) medium containing 35 mg/L hygromycin (Roche, Basel, Switzerland) and confirmed by RT-PCR. Arabidopsis plants were grown in a growth chamber under long-day conditions (16 h light/8 h darkness at 23 °C–25 °C).
Seeds of the T3 transgenic Arabidopsis plants were sterilized using 70% ethanol (v/v) for 3 min and in 15% NaClO (v/v) for 10 min. These were then washed four to five times with sterile ddH2O and stratified for 3 days at 4 °C before transfer to a growth chamber. The seeds were germinated on 1/2 MS medium for 1 week and then transferred to the soil for observing the phenotype of transgenic plants. Germination rates were also checked at 4 days. The number of rosette leaves at bolting was recorded for the flowering time. The height of seven-week-old seedlings was measured. The seeds were weighted using a laboratory balance (Mettler-Toledo, Zurich, Switzerland) and photographed under a stereomicroscope (Nikon, Tokyo, Japan).
Salt treatments of transgenic Arabidopsis lines
For the observation of green rate, seeds were plated on 1/2 MS medium containing two concentrations of NaCl (125 mM and 175 mM) for 6 weeks. Germination rate was recorded at two, four, and six days after plating. To further observe salinity tolerance of transgenic lines, seedlings were grown on 1/2 MS medium for 1 week, and then transferred to the soil for 3 weeks. These plants were treated with 250 mM NaCl (once every 5 days). Approximately 15 days after treatment, the bleaching degree of plants and the content of chlorophyll were measured. Then, the aboveground biomass at the 30th day of treatment was also measured. One-week-old seedlings grown on 1/2 MS were transferred to a medium with 175 mM NaCl for 24 h, and the whole seedlings were harvested to investigate the expression of salt tolerance pathway genes.
Low-Pi treatments of transgenic Arabidopsis plants
Seeds were germinated on 1/2 MS with low-Pi and normal-Pi medium for 2 weeks. Low (0 mM KH2PO4) and normal (1.25 mM KH2PO4) Pi medium were modified based on Murashige and Skoog medium with pH 5.7, 0.5% (w/v) sucrose, and 0.4% (w/v) phytagel. The basic medium was prepared as previously described .
Determination of chlorophyll content
Aerial parts of the Arabidopsis plants were collected and crushed in absolute alcohol. The mixture was rapidly shaken and left to stand in the dark for 16 h. The absorbance of supernatant was recorded at wavelengths of 663 nm and 645 nm using a UV-visible spectrophotometer (ChemitoSpectrascan, UV 2600). Chlorophyll content was estimated according to Arnon (1949) .
Soybean morphological analysis
After salinity stresses and low-Pi treatments, the seedlings were photographed using a camera (Nikon, Tokyo, Japan). In particular, after low-Pi treatments, root length was measured using a WinRHIZO root analysis system (WinRHIZO, Regent, Canada), and the number of roots was manually counted. The fresh and dry biomass was recorded using laboratory balance (Mettler-Toledo, Zurich, Switzerland).
Each experiment/measurement was performed using three independent biological replicates or repeated three times unless stated otherwise. A student’s two-tailed t-test was used for statistical analysis, which was performed by using IBM SPSS Statistics for Windows, Version 24.0 (IBM Corp, New York, NY, USA).
The PHO1-like genes in Williams 82 was characterized in our previous work . The gene-specific primers were designed to obtain the full cDNA sequences of SN14 (GmPHO1) and ZYD6 (GsPHO1). The unrooted neighbor-joining (NJ) phylogenetic tree was constructed based on amino acid sequences by using MEGA5 . Sequence divergence between Gm-GsPHO1 orthologous pairs, including the substitution and in-frame insertions and deletions, was predicted using SNAP and PROVEAN [62, 63]. Sequencing was conducted by Taihe Biotechnology Company (Beijing, China). Primers used in the present study (Additional file 2: Table S7) were commercially synthesized in Taihe Biotechnology Company.
We appreciate Prof. Yongcai Lai for offering the soybean seeds. We gratefully thank the anonymous reviewers for the constructive comments and suggestions on revising the manuscript.
CYH designed the study and conceived the experiments. YW, HHG and LLH isolated the sequences and investigated gene expression. LXY participated in stress treatment and gene expression in soybeans. LLH generated transgenic Arabidopsis lines. YW, HHG and WWZ participated in characterizing transgenic Arabidopsis. YW, HHG, WWZ, LXY, QSC and CYH analyzed data. YW and CYH drafted the manuscript. All authors read and approved the manuscript. CYH agrees to serve as the author responsible for contact and ensures communication.
This work was supported by the grants of the National Natural Science Foundation of China (31525003) and the Chinese Academy of Sciences (ZDRW-ZS-2019-2 and XDA08010105) to CYH. The funders have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
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The authors declare that they have no competing interests.
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