Genome-Wide Identification of SPX Family Genes and Functional Characterization of PeSPX6 and PeSPX-MFS2 in Response to Low Phosphorus in Phyllostachys edulis

Moso bamboo (Phyllostachys edulis) is the most widely distributed bamboo species in the subtropical regions of China. Due to the fast-growing characteristics of P. edulis, its growth requires high nutrients, including phosphorus. Previous studies have shown that SPX proteins play key roles in phosphorus signaling and homeostasis. However, the systematic identification, molecular characterization, and functional characterization of the SPX gene family have rarely been reported in P. edulis. In this study, 23 SPXs were identified and phylogenetic analysis showed that they were classified into three groups and distributed on 13 chromosomes. The analysis of conserved domains indicated that there was a high similarity between PeSPXs among SPX proteins in other species. RNA sequencing and qRT-PCR analysis indicated that PeSPX6 and PeSPX-MFS2, which were highly expressed in roots, were clearly upregulated under low phosphorus. Co-expression network analysis and a dual luciferase experiment in tobacco showed that PeWRKY6 positively regulated the PeSPX6 expression, while PeCIGR1-2, PeMYB20, PeWRKY6, and PeWRKY53 positively regulated the PeSPX-MFS2 expression. Overall, these results provide a basis for the identification of SPX genes in P. edulis and further exploration of their functions in mediating low phosphorus responses.


Introduction
Moso bamboo (Phyllostachys edulis) is the most widely distributed bamboo species in the subtropical regions of China. The planting area is 4,677,800 hectares, accounting for 70% of the total bamboo forest area in China [1][2][3]. P. edulis not only has the highest ecological and economic value, but also has the highest cultural value of all bamboos. Due to the fast-growing characteristics of P. edulis, its growth requires a high amount of nutrients [4]. Thus, an adequate supply of soil nutrients is necessary to ensure the normal growth of P. edulis.
As an essential mineral element during plant growth and development, phosphorus (P) plays key roles in energy transmission, signal transduction, photosynthesis, and respiration [5][6][7]. Plants prefer to obtain inorganic soluble phosphate in the soil through the root system, and cannot directly assimilate the other forms of P, such as organic phosphate (Pi) [8]. However, the content of inorganic phosphorus in soil is very low, and it does not diffuse easily in the soil. As a result, plants have evolved mechanisms to adapt to Pi-deficient conditions [9]. The phosphorus starvation signaling pathway in plants has been well documented [10].
Among them, SPX proteins play very important roles in P signaling and homeostasis [11,12]. The SPX domain is a conservative domain named after SYG1 (suppressor of yeast gpa1), Pho81 (CDK inhibitor in yeast PHO pathway), and XPR1 (xenotropic and polytropic retrovirus receptor). Based on the presence of additional domains, SPX proteins can be further classified into four subfamilies: SPX proteins, which contain only an SPX domain; SPX-EXS proteins, which have an SPX domain and an EXS (ERD1, XPR1, and SYG1) domain; SPX-MFS proteins, which have an SPX domain and a major facility superfamily (MFS) domain; and SPX-RING proteins, which contain an SPX domain and a RING-type zinc finger domain [13][14][15]. SPX proteins have been identified in many plant species, including Arabidopsis, rice, and maize [16]. For example, there are 20 SPX domain-containing proteins in Arabidopsis. AtSPX1 participates in the transcription regulation of Pi-responsive genes, while AtSPX3 functions in the potential negative feedback regulation of the P signal network [17]. 33 SPX gene family members have been found in maize. Xiao et al. [18] found that ZmSPX4.1 and ZmSPX4.2 showed strong responses to low Pi stress and exhibited remarkably different expression patterns in low-Pi sensitive and insensitive cultivars of maize [18]. Currently, the role of SPX genes in response to low phosphorus stress in P. edulis is still poorly understood. Thus, the systematic identification, molecular characterization, and functional characterization of the SPX gene family in response to low Pi in P. edulis are of great importance to improve the absorption and utilization of Pi in P. edulis.
In this study, the whole genome of SPX members in P. edulis was first identified and characterized. The chromosomal mapping, phylogenetic relationships, and conserved motifs of SPX genes were analyzed. Then, the expression levels of SPX members exposed to low Pi were analyzed using RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR). Furthermore, several transcriptional factors were identified that modulated the expression of PeSPX6 and PeSPX-MFS2 in P. edulis.

Identification of SPX-Domain-Containing Proteins in P. edulis
To identify P. edulis SPXs, the conserved PF03105 domain was used as the probe to execute a genome-wide search of candidate genes with the HMMER tool, and a total of 30 putative protein hits were obtained. To verify the HMMER search results, domain analysis was further performed for the 30 putative proteins with CDD and SMART. Among the 30 putative proteins, 23 harboring the conserved SPX domains were identified using both CDD and SMART. These proteins were designated PeSPX1 through PeSPX1-EXS6 and were considered authentic SPX candidates in the P. edulis genome. The length of these protein-coding regions ranged from 738 bp to 2571 bp. These proteins consisted of 245-856 amino acids, and the corresponding molecular weights ranged from 27.97 to 97.55 kDa. The isoelectric point (pI) value of these PeSPX proteins ranged from 5.04 to 9.4 ( Table 1).

Phylogenetic Analysis of the SPX-Domain-Containing Protein Genes in P. edulis and Other Species
To evaluate the evolutionary relationships of SPX-domain-containing protein genes in P. edulis, this work analyzed the sequence features in three different species, including A. thaliana, O. sativa, and P. edulis, and a total of 56 SPX-domain-containing protein genes were used to construct a phylogenetic tree with the neighbor-joining (NJ) method using MEGA (version 7) (Figure 2). The phylogenetic tree indicated that PeSPXs could be divided into three subfamilies (the SPX, SPX-MFS, and SPX-EXS subfamilies). In addition, the numbers of SPX subfamily proteins in the three species were highly asymmetrical. For example, 11 PeSPXs, five OsSPXs, and four AtSPXs were classified in the SPX subfamilies, and six PeSPXs, 11 AtSPXs, and three OsSPXs were included in the SPX-EXS subfamilies. Only one OsSPX and two AtSPXs were classified in the SPX-RING subfamilies. These results indicate that the SPX gene family is highly conserved and diverse in different plants.

Phylogenetic Analysis of the SPX-Domain-Containing Protein Genes in P. edulis and Other Species
To evaluate the evolutionary relationships of SPX-domain-containing protein genes in P. edulis, this work analyzed the sequence features in three different species, including A. thaliana, O. sativa, and P. edulis, and a total of 56 SPX-domain-containing protein genes

Structure Analysis of the SPX-Domain-Containing Genes and Proteins in P. edulis
All SPX-domain-containing proteins contain the SPX domain in the C-terminal portion ( Figure 3). The three subfamilies of SPX-domain-containing proteins (SPX, SPX-EXS, and SPX-MFS) were found in P. edulis. The SPX subfamily possessed 11 members, the SPX-EXS subfamily contained six members, and the SPX-MFS subfamily had six members.

Structure Analysis of the SPX-Domain-Containing Genes and Proteins in P. edulis
All SPX-domain-containing proteins contain the SPX domain in the C-terminal portion ( Figure 3). The three subfamilies of SPX-domain-containing proteins (SPX, SPX-EXS, and SPX-MFS) were found in P. edulis. The SPX subfamily possessed 11 members, the SPX-EXS subfamily contained six members, and the SPX-MFS subfamily had six members.

Expression Analysis of P. edulis SPX-Domain-Containing Protein Genes
To explore the effects of low phosphorus on the growth and development of P. edulis, P. edulis plants were treated with 1/2 Kimura nutrient solution in different Pi levels. Under low phosphorus conditions, the root length was significantly lower than that under the normal Pi supply, but the number of lateral roots was significantly higher than that under the normal Pi supply ( Figure 4A,B). Furthermore, the phosphorus content in the shoots and roots of P. edulis was also measured. As shown in Figure 4C, the phosphorus content in both shoots and roots of P. edulis treated with low phosphorus was much lower compared to those in shoots and roots of P. edulis with a normal phosphorus supply. To further explore the molecular mechanism of P. edulis in response to low phosphorus, RNA-seq was performed (Table S1). The expression data of 23 PeSPXs were clustered and displayed in a  Next, two PeSPXs (PeSPX6 and PeSPX-MFS2) with higher expression levels were selected for qRT-PCR analysis. Consistent with the RNA-seq result, the expression levels of PeSPX6 and PeSPX-MFS2 were clearly upregulated under low phosphorus ( Figure 5A). Furthermore, the expression patterns of PeSPX6 and PeSPX-MFS2 were also analyzed. As shown in Figure 5B, PeSPX6 and PeSPX-MFS2 were more highly expressed in the roots, while the expression was lower in the leaves and stems of P. edulis. Figure 4. Expression profile of PeSPXs exposed to low phosphorus. (A) Primary root length of Phyllostachys edulis seedlings exposed to low phosphorus. (B) Number of lateral roots of P. edulis seedlings exposed to low phosphorus. (C) Phosphorus content in P. edulis roots and shoots exposed to low phosphorus. (D) Heatmap of PeSPXs exposed to low phosphorus via transcriptome analysis. Error bars in (A-C) indicate SD (n = 3). The asterisk shows a significant difference compared to the control using the unpaired Student's t test (* p < 0.05). . Expression profile of PeSPXs exposed to low phosphorus. (A) Primary root length of Phyllostachys edulis seedlings exposed to low phosphorus. (B) Number of lateral roots of P. edulis seedlings exposed to low phosphorus. (C) Phosphorus content in P. edulis roots and shoots exposed to low phosphorus. (D) Heatmap of PeSPXs exposed to low phosphorus via transcriptome analysis. Error bars in (A-C) indicate SD (n = 3). The asterisk shows a significant difference compared to the control using the unpaired Student's t test (* p < 0.05).
Next, two PeSPXs (PeSPX6 and PeSPX-MFS2) with higher expression levels were selected for qRT-PCR analysis. Consistent with the RNA-seq result, the expression levels of PeSPX6 and PeSPX-MFS2 were clearly upregulated under low phosphorus ( Figure 5A). Furthermore, the expression patterns of PeSPX6 and PeSPX-MFS2 were also analyzed. As shown in Figure 5B, PeSPX6 and PeSPX-MFS2 were more highly expressed in the roots, while the expression was lower in the leaves and stems of P. edulis.

Validation of SPX Gene Network Regulation
Because the expression levels of PeSPX6 and PeSPX-MFS2 were increased by low phosphorus, this study then investigated what transcriptional regulation of PeSPX6 and PeSPX-MFS2 might exist in response to low phosphorus. Pearson's correlations were performed between the expression levels of PeSPX6 and PeSPX-MFS2 and transcription factors induced by low phosphorus (Figure 6, Table S3). Eight transcription factors with high expression levels under low phosphorus stress were selected to explore their function in regulating the expression levels of PeSPX6 and PeSPX-MFS2 using a dual-luciferase analysis (Table S4). The promoters of PeSPX6 (2000 bp) and PeSPX-MFS2 (2000 bp) were separately cloned and fused to the firefly luciferase protein (Fluc) at the N-terminus, which also had a Renilla luciferase (Rluc). The Fluc/Rluc ratio represents the ability of transcription factors to transcriptionally activate the PeSPX6 or PeSPX-MFS2 promoter. As shown in Figure 6, PeWRKY6 enhanced the activity of luciferase driven by the PeSPX6 promoter, while PeCIGR1-2, PeMYB20, PeWRKY6, and PeWRKY53 enhanced the activity of luciferase driven by the PeSPX-MFS2 promoter.

Validation of SPX Gene Network Regulation
Because the expression levels of PeSPX6 and PeSPX-MFS2 were increased by low phosphorus, this study then investigated what transcriptional regulation of PeSPX6 and PeSPX-MFS2 might exist in response to low phosphorus. Pearson's correlations were performed between the expression levels of PeSPX6 and PeSPX-MFS2 and transcription factors induced by low phosphorus (Figure 6, Table S3). Eight transcription factors with high expression levels under low phosphorus stress were selected to explore their function in regulating the expression levels of PeSPX6 and PeSPX-MFS2 using a dual-luciferase analysis (Table S4). The promoters of PeSPX6 (2000 bp) and PeSPX-MFS2 (2000 bp) were separately cloned and fused to the firefly luciferase protein (Fluc) at the N-terminus, which also had a Renilla luciferase (Rluc). The Fluc/Rluc ratio represents the ability of transcription factors to transcriptionally activate the PeSPX6 or PeSPX-MFS2 promoter. As shown in Figure 6, PeWRKY6 enhanced the activity of luciferase driven by the PeSPX6 promoter, while PeCIGR1-2, PeMYB20, PeWRKY6, and PeWRKY53 enhanced the activity of luciferase driven by the PeSPX-MFS2 promoter.

Discussion
SPX family genes are widely found in eukaryotes, including plants, fungi, and multicellular animals [19,20]. At present, the SPX family genes have been extensively studied in Arabidopsis [17] and rice [21,22]. In rice, there are six SPX subfamily genes. Studies on

Discussion
SPX family genes are widely found in eukaryotes, including plants, fungi, and multicellular animals [19,20]. At present, the SPX family genes have been extensively studied in Arabidopsis [17] and rice [21,22]. In rice, there are six SPX subfamily genes. Studies on OsSPX1 and OsSPX genes in rice have found that these two genes can participate in the negative regulation of PHR2 under the condition of rich phosphorus [21]. The SPX-EXS family, involved in phosphorus transport and signaling from root to terminal bud in rice and Arabidopsis thaliana [23][24][25]. Members of the SPX family, including 20 AtSPXs, 69 BnaSPXs, 9 GmSPXs, 14 OsSPXs, and 46 TaSPXs, have been reported and characterized by bioinformatics analysis [17,19,26,27]. However, the functional analysis of the SPX family in P. edulis has not been reported. In the present study, systematic identification, molecular characterization, and functional characterization of the SPX gene family were performed.
Previous studies have shown that SPX family genes play an important role in the sensing, signal transduction, and transport of inorganic phosphate (Pi) in eukaryotes, such as PHO1 (SPX-EXS) [28], PHT5 (SPX-MFS) [29] and AtNLA (SPX-RING) [30]. AtSPX1 was expressed most rapidly in Pi starvation induction, which indicated that AtSPX1 has a potential transcriptional regulation effect on Pi starvation [19]. In rice, eight SPX genes (OsSPX1, OsPHO2) were significantly increased under exposure to low phosphorus [19,31]. The expression of TaSPX2 was significantly induced in wheat under low P stress [32]. It was found that SPX2 was induced to express in tea plants under low phosphorus stress [33]. Bn SPX2 was continuously induced to express in various tissue parts of Brassica napus under low P stress [27]. In the present study, only five SPX genes were clearly upregulated under low phosphorus. Among them, two genes (PeSPX6 and PeSPX-MFS2) showed higher expression levels compared to the others in the roots. This strongly implies that PeSPX6 and PeSPX-MFS2 might regulate the low phosphorus response in P. edulis. In addition, the tissue-specific expression indicated that PeSPX6 and PeSPX-MFS2 were more highly expressed in roots, which further supported the hypothesis that PeSPX6 and PeSPX-MFS2 were involved in phosphorus nutrient uptake in the roots of P. edulis. In future research, the biological function of these genes in P. edulis needs to be explored.
Increasing evidence shows that proteins containing the SPX domain are key players in a series of processes that control the dynamic balance of phosphorus in plants. In Arabidopsis, the physical interaction between AtSPXs and AtPHRs under sufficient Pi conditions prevents AtPHRs from binding to the promoter of AtPSI, thus inhibiting the PHR transcriptional activity [34][35][36]. AtSPX3 is involved in responses to low Pi stress [23]. In rice, phosphorus starvation induces the accumulation of OsSPX3 to restore the phosphorus balance [37]. PeSPX6 has the highest similarity to OsSPX3 and AtSPX3, suggesting that PeSPX6 might be involved in the response to low phosphorus and play a role in the Pi starvation signal transduction pathway of P. edulis. As a volatile phosphate efflux transporter, OsSPX-MFS3 is involved in maintaining phosphate homeostasis in rice. Phylogenetic analysis showed that PeSPX-MFS2 and OsSPX-MFS3 had high homology. Thus, this study speculates that PeSPX-MFS2 might be a low-affinity Pi transporter that mediates Pi efflux from the vacuole into the cytosol. In general, the plant SPX family can be divided into four subclasses based on the presence of additional protein domains, namely SPX, SPX-EXS, SPX-MFS, and SPX-RING [13]. Among them, SPX-RING is the smallest subfamily in the SPX family. For example, there is only one member of the SPX-RING subfamily in rice and tow in Arabidopsis. Surprisingly, this subfamily was not found in the P. edulis genome. This demonstrates the complexity of the SPX family in different plant species.
Wang et al. [35] showed that OsSPX1 and OsSPX2 inhibited phosphate starvation in rice by interacting with PHR2 in a phosphate-dependent manner. AtSPX4 regulates the PHR1-dependent and independent regulation of stem phosphorus status in Arabidopsis [38]. In this study, several transcription factors, namely PeMYB20, PeMYB30, PeWRKY6, PeWYKY53, PeERF110, PeNAC030, PeCIGR1-1, and PeCIGR1-2, were upregulated up by low phosphorus. Furthermore, the dual luciferase experiment in tobacco showed that PeWRKY6 positively regulated the expression of PeSPX6, while PeCIGR1-2, PeMYB20, PeWRKY6, and PeWRKY53 positively regulated the expression of PeSPX-MFS2. These results imply that these transcription factors might also play a vital role in the adaptation of P. edulis to low-phosphorus environments.
In this study, phylogenetic analysis showed that 23 SPXs were classified into three groups and distributed on 13 chromosomes. The analysis of conserved domains indicated that there was a high similarity between PeSPXs among SPX proteins in other species. Pe-SPX6 and PeSPX-MFS2, which were highly expressed in roots, were obviously upregulated under low phosphorus. PeWRKY6 positively regulated the expression of PeSPX6, while PeCIGR1-2, PeMYB20, PeWRKY6, and PeWRKY53 positively regulated the expression of PeSPX-MFS2. Based on this, it is speculated that these genes play different roles in various biological processes, laying a theoretical foundation for elucidating the functions of the SPX family genes in P. edulis.

Phylogenetic Analysis of SPX Proteins
Multiple amino acid sequence alignments were performed using MAFFT with the default parameters utilizing A. thaliana, O. sativa, and P. edulis sequences to examine the evolutionary relationships and the full-length protein sequences of P. edulis SPXs. Then, using the auto mode in MEGA (version 7), a phylogenetic tree of amino acid sequences based on the neighbor-joining (NJ) was created.

Gene Structure and Chromosomal Location
Conserved motif structures were examined using the MEME online software (meme.nb cr.net/meme (accessed on 12 November 2022)) and the motif function was explored using NCBI-CDD. To display the locations of PeSPXs on chromosomes, TBtools was employed. The genomic sequence information applied to the analysis of both gene structure and chromosomal location was derived from NCBI [39].

Transcriptome Sequencing
High-quality RNA was used to create the cDNA library and the 150-bp paired-end reads that resulted were sequenced using the Illumina NovaSeq 6000 platform. After filtering, the cleaning data were compared to the reference genome of P. edulis through HISAT2, and the gene expression levels were counted as Fragments Per Kilobase per Million (FPKM) using StringTie. Based on p-values < 0.05 and fold changes ≥ 2, the R package Deseq2 identified differentially expressed genes (DEGs) in each sample group.

Determination of Phosphorus Content
The content of phosphorus in each organ of P. edulis was determined using the Mo-Sb colorimetric method [40]. The extracted solution was mixed with 2,4-dinitrophenol, NaOH, and 0.5 mol/L H 2 SO 4 until the yellow was arised. Then, the mixed solution was reacted with Mo-Sb-Vc chromogenic agent at 25 • C for 30 min. The absorbance at 700 nm was measured using an ultraviolet-visible spectrophotometer (V 5600, Shang Hai, China), then the phosphorus content was calculated.

Real-Time RT-PCR Analysis
Utilizing the cDNA reverse transcription kit, whole pure RNA was transformed into cDNA (PrimeScriptTM RT Master Mix, Takara, Kusatsu, Japan). Then, qRT-PCR was carried out using the CFX96 TouchTM Thermal Cycler and the ChamQ SYBR qPCR Master Mix kit from Vazyme (Vazyme, Nanjing, China) (Bio-Rad, Hercules, CA, USA). The 2 −∆∆CT method was utilized to normalize for transcript levels using the housekeeping gene PeTIP41 (Table S2) [41].

Luciferase Assays
The full length of transcription factors were separately cloned into pGreenII 62-SK vector. The promoter of PeSPX6 and PeSPX-MFS2 (2000 bp) was cloned into pGreenII 0800 LUC vector (Table S2). Agrobacterium tumefaciens GV3101 was used to convert the aforementioned vectors into four-week-old Nicotiana benthamiana leaves [42]. After infiltration for 4 days, the activity of LUC and REN were assessed in accordance with the instructions provided in the Dual Luciferase Reporter Gene Assay Kit (Beyotime, Nantong, China).

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12071496/s1, Table S1. Summary of the RNA-Seq data. Table S2. Primers used in this study. Table S3. Transcription factor correlation analysis. Table S4. Transcription factors gene ID.  Neighbor-joining aa: Amino acids; in length MWs: Molecular weights Pi: Isoelectric points