Evolution of the SPX gene family in plants and its role in the response mechanism to phosphorus stress

Molecular and genomic studies have shown the presence of a large number of SPX gene family members in plants, some of which have been proved to act in P signalling and homeostasis. In this study, the molecular and evolutionary characteristics of the SPX gene family in plants were comprehensively analysed, and the mechanisms underlying the function of SPX genes in P signalling and homeostasis in the model plant species Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and in important crops, including wheat (Triticum aestivum), soya beans (Glycine max) and rapeseed (Brassica napus), were described. Emerging findings on the involvement of SPX genes in other important processes (i.e. disease resistance, iron deficiency response, low oxygen response and phytochrome-mediated light signalling) were also highlighted. The available data suggest that SPX genes are important regulators in the P signalling network, and may be valuable targets for enhancing crop tolerance to low P stress. Further studies on SPX proteins should include more diverse members, which may reveal SPX proteins as important regulatory hubs for multiple processes including P signalling and homeostasis in plants.


Introduction
The SPX family was named after SYG1, PHO81 and Xpr1, the first three SPX gene members identified [1]. SYG1 and PHO81 encode yeast gpa1 suppressor and cyclin-dependent kinase, respectively, while Xpr1 codes for the xenotropic and polytropic retrovirus receptor 1 in humans. More and more studies have shown that SPX genes are involved in phosphorus (P) signalling and homeostasis and are prevalent in plants, and phosphate transport is impaired if the SPX domain is mutated [2]. P is an indispensable macroelement required for normal plant growth and development, and P content is quite high in plant tissues. P is not only an important component of membranes and nucleic acids, but also plays important roles in diverse physiological processes, including photosynthesis, enzyme activity regulation, respiration, signal transduction, oxidationreduction reactions, energy metabolism and carbon metabolism [3][4][5][6]. Plants absorb P mainly from the soil through their roots [7,8]. Soil P exists primarily in the forms of calcium, iron and aluminium salts, and organic molecules, which are difficult for the roots to absorb [9]. This decreases the bioavailability of P, leading to an available P content in the soil that is far lower than that required for normal plant growth [10][11][12]. In plant cells, P concentration in the cytosol is approximately 60 -80 mM [13], which is much higher than the concentration of available P in the soil (less than 10 mM) [14]. Thus, plants are usually under low P conditions. The phenotypic symptoms of P deficiency are mainly dark-green leaf colour, reduced elongation rate of shoot and decreased leaf size [15]. To  large quantity of P fertilizers is often applied to solve the problem of P deficiency. However, this approach can cause not only water eutrophication but also overexploitation and consumption of phosphate ore, which is non-renewable. Solving this problem is critical to environmental protection and sustainable development. To adapt to P-deficient environments, plants undergo phenotypic changes in the root system to increase the absorption of P from the soil [16 -19]. A series of P transport mechanisms at the molecular level are gradually established to overcome P starvation and to maintain P homeostasis, in which phosphate transporter is the basic effector involved in P uptake, transfer and storage [20,21]. These mechanisms are controlled by complex and sophisticated molecular regulatory networks. Recently, many genes have been identified and functionally linked to these molecular regulatory networks, including various protein-encoding genes and non-coding RNA genes [22]. Hence, the discovery of genes conferring low P tolerance has great importance.
In this study, the structural and evolutionary characteristics of the SPX gene family in plants were systematically analysed, the latest research progress on SPX gene functions was summarized and the mode of action of these genes in the P regulatory signalling network was discussed.

Structural characteristics of SPX genes and proteins in plants and their relationships with phosphorus metabolism
The SPX domain found in eukaryotic proteins is rather conserved and has hydrophilic properties [23]. It is often located at the N-terminus of eukaryotic proteins [23]. SPX domain has an average length of 165 amino acids and can be divided into three subdomains with 30 -40 amino acids in each (figure 1). They are separated from each other by low similarity regions [24]. In plants, SPX domains can be grouped into several distinct subfamilies: SPX proteins carrying only SPX domain, SPX -EXS proteins containing SPX and an EXS (ERD1, XPR1 and SYG1) domain, SPX -MFS proteins with SPX and the major facility superfamily (MFS) domain and SPX-RING proteins containing SPX and the RING-type zinc finger domain [25]. Many studies have shown that the SPX domain is closely related to P signalling and plays an important role in maintaining P homeostasis [22,[26][27][28]. The SPX domain can indicate the phosphate status in fungal, plant and human cells. SPX domain-containing proteins are indispensable for the absorption, transport, storage and signal transduction of inorganic P in eukaryotes. Wild et al.
[2] studied the ligand of the SPX domain and suggested that the domain provided a binding surface for small molecules (inositol polyphosphate signalling molecules, InsPs). In this way, the balance of P in plant cells can be regulated by the binding of different InsPs to SPX. In phosphatedeficient plant cells, InsPs bind to SPX domains, being able to interact with several other proteins involved in the regulation of P signalling in plants [2,29]. If the SPX domain is mutated, then phosphate transport capacity is impaired [2], highlighting the unique importance of the SPX domain in P metabolism. P exists in different molecular forms in plants to serve their needs at different times [30]. One form is inorganic polyphosphate (polyP). PolyP includes hundreds of types of phosphoric anhydrides, which can be hydrolysed to meet the needs of various molecular processes [31][32][33]. In yeast, the vacuolar transporter chaperone (VTC) complex can synthesize polyP [34]. The VTC is a fairly large protein complex (Vtc1 -5) located on the vacuole membrane [35,36]. Approximately 80% of VTC proteins contain an SPX domain, which may contribute to P homeostasis in the cells [5]. Additionally, an important role for the phosphate starvation response 1 (PHR1) protein has been identified in the P signalling network. This MYB-like transcription factor is homologous to phosphorus starvation response 1 (PSR1), which participates in the P sensing process in Chlamydomonas reinhardtii [37,38]. PHR1 regulates the expression of AtACP5, AtIPS1, PHT1.1 and RNS1 [39,40], as well as the expression of several PSR genes, including microRNA399 and the SPX genes [38], by binding to their promoters through the ciselement PHR1-binding sequence (P1BS; GNATATNC) [40][41][42].

Evolutionary analysis of SPX gene family in plants
Through analysis of existing plant genomic sequences, 20 SPX gene family members have been identified in Arabidopsis, including four SPX genes whose deduced proteins contain only the SPX domain [26]. Meanwhile, 15 SPX gene family members are identified in rice, including six SPX genes whose products carry only the SPX domain [24]. Numerous SPX gene family members also exist in the genomes of legumes and other important crops [43,44]. A phylogenetic tree was constructed for some of the important SPX genes using the multi-sequence alignment generated by CLUSTALW and the neighbour-joining method with 1000 bootstrap replicates in MEGA software (figure 2). The results showed evolutionary divergence in SPX genes, and the compared genes were clustered into five types of sub-structures (numbered I, II, III, IV and V). Most of the SPX genes fell into types I and IV, whereas no more than five genes fell into each of the other three types. GmSPX8, GmSPX5, OsSPX4, OsSPX6 and TaSPX129 genes exhibited the most rapid evolution for each type. The paralogues of these genes included AtSPX1/AtSPX2, GmSPX2/GmSPX4, GmSPX5/ GmSPX9, OsSPX1/OsSPX2, GmSPX1/GmSPX10 and OsSPX5/OsSPX6. Each gene may evolve under different evolutionary pressure and may possibly acquire new function during the course of evolution. A multiple sequence alignment of the SPX domains was analysed ( figure 3). The result showed a high degree of similarity in the SPX domains in Arabidopsis, rice, common beans, soya beans, wheat and Brachypodium. The SPX domains in TaSPX129 and BdSPX129 were of SPX-MFS type, and have an increased length, whereas the remaining SPX domains ranged from 200 to 400 amino acids. Amino acid point mutations are the main sources of variation, which may affect the role of SPX genes in P regulatory networks in plants.
Notably, the SPX domains in TaSPX129 and BdSPX129 had many insertions (figure 3); whether this is caused by the presence of an additional MFS domain is a matter for further study.

Research progress on the functional analysis of SPX genes in plants
A large number of SPX domain-containing proteins have been identified in plants [11]. Here, SPX proteins refer to the proteins that contain only the SPX domain. Owing to their presence in different subcellular structures, they may have different functions in the P signalling network. Here, we summarize the studies on the genes whose proteins contain only the SPX domain, including SPX1 -4 in Arabidopsis, SPX1 -6 in rice, SPX1 -10 in soya beans, SPX1-3 in common bean and TaSPX129 in wheat (table 1).

Functional analysis of SPX genes in Arabidopsis
Twenty SPX domain-containing proteins were found in Arabidopsis, among which four proteins contained only the SPX domain (AtSPX1-AtSPX4) [26]. AtSPX1, localized in the nucleus, is a P-dependent suppressor of AtPHR1 in Arabidopsis [53]. AtPHR1 overexpression results in an increase in P concentration in the shoot and induces the expression of a series of Pi starvation-induced (PSI) genes that encode phosphate transporter, phosphatase or RNase [54,55]. Coimmunoprecipitation experiment showed that the AtSPX1/ AtPHR1 interaction was strongly dependent on P level. AtSPX1 is a competitive suppressor that binds AtPHR1 through its recognition sequence. The working model in figure 4 depicts the interactions of AtSPX with AtPHR1 in response to cellular Pi concentration for PSI transcription. Under high P conditions, AtSPX1 has a high binding affinity for AtPHR1, and thus the process by which AtPHR1 regulates PSI genes through the P1BS is inhibited, resulting in a decrease in PSR gene expression. Under P deficiency conditions, the AtSPX1/AtPHR1 interaction weakens, thus facilitating the binding of AtPHR1 to the P1BS to regulate PSR gene expression [49].
In Arabidopsis, no significant phenotypic differences have been found among the single-gene knockout mutants of Atspx1, Atspx2 and Atspx4 under P-sufficient or -starvation conditions. However, in plants with AtSPX1 overexpression, the expression levels of some PSI genes (i.e. ACP5, PAP2 and RNS1) are significantly increased regardless of P concentration, thus suggesting that AtSPX1 may function in the transcriptional regulation of P starvation. Additionally, inhibition of AtSPX3 through RNAi can change the phenotypes and gene expression levels under P-starvation conditions, rendering an increase in P concentration in the shoot tissues and a reduced P concentration in the roots [26,43]. The expression levels of AtPHT1-4, AtPHT1 -5, AtACP5, AtRNS and AtAT4 in spx3 deletion mutants are increased irrespective of P concentration [43], indicating that AtSPX3 is a negative regulator of the signalling process of P starvation. Collectively, these results indicate that SPX proteins have functional redundancy with one another and can serve as an important role in regulating Pi signalling and homeostasis in plants.

Functional analysis of SPX genes in rice
P is an important nutrient element that limits the yield of rice. Studies of P relevant genes in rice, especially SPX genes, can potentially aid rice yield improvement. A total of 15 SPX domain-containing proteins have been identified in rice, of which only six are SPX proteins (OsSPX1-OsSPX6) [5]. OsSPX1 inhibits P uptake and P-starvation signalling through negative feedback regulation [27,55]. OsSPX1 is induced by P starvation in the roots, and inhibition of OsSPX1 by RNAi leads to an excessive accumulation of P and thus induces severe toxicity. This phenotype is similar to that observed in the plants overexpressing OsPHR2 and the pho2 mutant. OsPHR2 overexpression leads to increased PSI gene expression, including IPS1 and PT2, which promotes excessive P absorption and accumulation and results in leaf necrosis. Quantitative polymerase chain reaction (qRT-PCR) assay showed that OsSPX1 expression was strongly induced in the plants with OsPHR2 overexpression and the pho2 mutant, suggesting that OsSPX1 may function downstream from PHO2 and OsPHR2. Wang et al. [27] analysed the expression levels of 10 genes involved in the rice P-starvation signalling pathway. OsPT2 and OsPT8 were significantly induced in OsSPX1 RNAi plants, pointing to increased P transport and accumulation. By contrast, OsSPX1 overexpression inhibited the expression of 10 phosphate starvationmediated genes, including IPS1, IPS2, OsPAP1, OsSQD2 (sulfo quinovosyl diacylglycerol 2), miR399d, miR399j, OsPT2, OsPT3, OsPT6 and OsPT8. However, in the double mutant plants with overexpression of OsPHR2 and OsSPX1, P concentration and PSR gene expression levels were basically the same as those in wild-type plants, which indicated that rsob.royalsocietypublishing.org Open Biol. 8: 170231 OsSPX1 was a negative regulator of OsPHR2-mediated signal transduction. Previous studies found that overexpression of OsSPX1, OsSPX2, OsSPX3, OsSPX4 and OsSPX5 attenuated the phenotype of OsPHR2 overexpression [45][46][47]53]. Thus, OsSPX1, OsSPX2 and OsSPX4 may interact with OsPHR2 and inhibit its binding to the P1BS cis-acting element ( figure 4). The interaction between SPX proteins and PHR1/2 was strongly dependent on P concentration [45,47]. Knockout of OsSPX1, OsSPX2 and OsSPX4 results in P accumulation in the shoot and significant leaf tip necrosis [45,47]. P accumulation and leaf necrosis also occurred in the Osspx3 and Osspx5 double mutant, and the expression levels of PSI genes, including IPS1, miR399, PT2, miR827, PAP10 and SQD2, were significantly upregulated [46]. This observation indicated that OsSPX3 and OsSPX5 were homologous and that they responded to P stress at the transcriptional and post-transcriptional levels. Collectively, the data gathered so far support the function of the studied OsSPX genes in rice tolerance to P deficiency by regulating P acquisition and its transport from roots to leaves.

Functional analysis of SPX genes in legumes
Phylogenetic analysis demonstrated that GmSPXs 1-10 can be divided into three groups [43]. Quantitative PCR assay showed that the expression of these genes was significantly increased under low P conditions and decreased rapidly one day after P supplementation [43]. The expression of these genes was highly sensitive to low P conditions. Overexpression of GmSPX3 led to increased P concentration in both leaf and root tissues, and increased transcriptional levels of seven PSI genes in the root hairs, under high P conditions [44]. Analysis rsob.royalsocietypublishing.org Open Biol. 8: 170231 of GmSPX1 overexpression in Arabidopsis spx3 mutant showed that GmSPX1 negatively regulated many PSR genes, including AtPHT1-4, AtPHT1-5, AtACP5, AtRNS and AtAT4, in a P leveldependent manner [43]. Furthermore, GmSPX1 interacted with a newly identified P starvation-induced transcription factor GmMYB48, and this interaction may represent a potential suppressor module of the P signalling network in soya bean [43]. Three SPX proteins (PvSPX1-PvSPX3) have been found in common bean (Phaseolus vulgaris), and their expression levels were significantly increased in roots and leaves under P-starvation conditions [44]. PvSPX1 is localized in the nucleus and exhibits a more sensitive and rapid response to P starvation. Overexpression of PvSPX1 resulted in an increase in P concentration in root tissues, a configuration change of root hairs, growth inhibition of the main root and an increase in the number of lateral roots, accompanied by upregulated transcription of 10 PSR genes [44]. Further studies showed that PvPHR1 overexpression increased PvSPX1 transcription level, and thus PvSPX1 may act downstream from PvPHR1 [44,50].

Functional analysis of SPX genes in wheat
P deficiency is also a primary factor constraining the yield of wheat [15]. Therefore, identification of P-regulated genes and breeding of low P-tolerant cultivars are of prime importance to increasing global wheat productivity without excessive use of P fertilizers. Owing to the possession of a complex hexaploid genome, functional analysis of P-regulated genes (including SPX members) in common wheat (2n ¼ 6x ¼ 42) is lagging behind that in Arabidopsis and rice. Shang et al. [56] found that TaSPX3 was strongly induced by low P stress, but became significantly downregulated when P supply was restored; the expression profile of TaSPX3 differed among cultivars, indicating that the mechanism of low P stress response may vary among different wheat genotypes. Shukla et al. [57] demonstrated that the relative transcriptional level of TaSPX1 was higher in the aleurone than in the endosperm in developing wheat grains, which paralleled the accumulation of more P in aleurone tissues.

Functional analysis of SPX genes in rapeseed
Du et al. [11] analysed 69 SPX gene family members in rapeseed (Brassica napus) and found that the expression levels of different BnaSPX genes differed under P-starvation conditions. The expression levels of nine genes in the SPX subfamilies were significantly induced by P starvation and rapidly declined upon P supplementation. Analysis of two BnaSPX1 genes (i.e. BnaA2.SPX1 and BnaC3.SPX1) in transgenic Arabidopsis revealed functional difference between them: the transgenic lines of BnaA2.SPX1, but not those of BnaC3.SPX1, showed retarded growth and higher sensitivity to P deficiency when compared with wild-type control [11].
In two other studies, BnSPX3;1 and BnSPX3;2 were found rsob.royalsocietypublishing.org Open Biol. 8: 170231 Table 1. List of the plant SPX genes whose function has been analysed to some extent. N, cell nucleus; M, cell membrane; C, cytoplasm; þ, increase; þ(*), increase (except seeds); þ(**), increase (except flowers and seeds); ¼, Wang et al. [27,28] OsSPX2 N þ OsSPX2 can interact with OsPHR2 and acts as a negative regulator of OsPHR2. PHR2, SPX1 and SPX2 constitute a regulatory feedback loop in P signalling Wang et al. [28,45] OsSPX3 OsSPX3 plays an important role in OsIPS1/miR399-mediated long distance regulation on OsPHO2 and acts as a negative regulator of OsPHR2. OsSPX3 negatively regulates the root-to-shoot transportation of P. Overexpression of OsSPX3 inhibits plant growth, which is more severe under P-deficient conditions Wang et al. [28] Shi et al. [46] OsSPX4 N/C Yao et al. [44] PvSPX3 N/C þ ¼ PvSPX2 participates in P signalling pathway in both shoot and root tissues. PvSPX3 expression is less sensitive to P deficiency compared with that of PvSPX1 and PvSPX2 Yao et al. [44] Triticum aestivum TaSPX129 þ -Fang et al. [51] TaSPX TaSPX participates in high temperature-induced resistance to wheat stripe rust Wei et al. [52] rsob.royalsocietypublishing.org Open Biol. 8: 170231 specifically induced by P deficiency and that the induction was rapid and reversible [58,59]. Unlike P deficiency, the deprivation of other nutrients (N, K, S or Fe) did not affect the transcription of BnSPX3;1 and BnSPX3;2, and thus the two genes may be used as markers for assessing P-starvation status in plants [58,59].

Discussion and prospects
The available studies clearly suggest that SPX proteins occupy a very important position in the P signalling network, which is tightly related to P uptake, transport, storage and homeostasis. Most of the studied SPX genes are low P inducible and can influence the transcription of downstream PSI (PSR) genes by regulating PHR activity, likely via controlling the movement of PHR from the cytoplasm to the nucleus and by decreasing the binding of PHR to the P1BS cis-element [45,47,49]. Not surprisingly, current understandings on SPX genes are largely based on the data from model plants (i.e. Arabidopsis and rice). The results from complex crop plants (e.g. legumes, common wheat and rapeseed) are much less. Nevertheless, they complemented and expanded the insights obtained from model species, and yielded potential clues and targets for enhancing crop tolerance to low P stress. Considering the urgent need in developing P-efficient cultivars [60], more efforts should be devoted to studying SPX gene functions in crop plants. The accumulation of ever more genomic resources [61], as well as the rapid development of gene-editing technologies for diverse plant species [62], will facilitate such efforts.
Past investigations have mainly concerned relatively simple SPX proteins, i.e. those with only one SPX domain. Future studies should cover more complex SPX proteins that carry extra domains in addition to SPX. The presence of extra domains may confer multiple functions to SPX proteins. This possibility may be illustrated by the analysis of Arabidopsis PHO1 (AtPHO1), which carries the EXS domain in addition to SPX. Although originally found required for xylem loading of inorganic phosphate [63,64], recent investigations have indicated the likely involvement of AtPHO1 in the cross talk among P, sucrose and phytohormone signalling pathways [65].
Future studies will also shed new light on the involvement of SPX proteins in other vital plant processes. There is emerging evidence for the participation of SPX domain-containing proteins in disease resistance [52], iron deficiency response [66], low oxygen response [67] and phytochromemediated light signalling [68]. Considering the diverse and fundamental roles of P in cellular organisms, it may not be surprising to find that SPX proteins act as important regulatory hubs for multiple processes including the fine tuning of P signalling and homeostasis in plants.
Data accessibility. This article has no additional data. Author contributions. All authors contributed to the production of this article.  Figure 4. Interaction of SPX1, SPX2 and SPX4 with PHR under high or low P conditions. Under high P conditions, SPX1 and SPX2 in the nucleus and SPX4 in the cytoplasm bind PHR with high affinity, thus inhibiting PHR binding to the P1BS motif in the promoters of PSI genes and leading to repression of the transcription of PSI genes, including SPX1 and SPX2. Under low P conditions, SPX4 in the cytoplasm is degraded via the 26S proteasome pathway, which promotes the targeting of PHR to the nucleus, thereby releasing PHR to activate downstream PSI gene expression. Meanwhile, SPX1 and SPX2 in the nucleus interact with PHR at a low affinity, which facilitates PHR to bind to the P1BS motif in the promoters of PSI genes, further enhancing the expression of PSI genes, including SPX1 and SPX2. The thick arrow denotes enhancement. The dotted lines represent reduced effects. This working model is drawn based on the studies of SPX1, SPX2 and SPX4 genes in Arabidopsis and rice (Lv et al. [47]; Wang et al. [27,28]; Zhou et al. [53]; Puga et al. [49]). rsob.royalsocietypublishing.org Open Biol. 8: 170231