Genome-Wide Detection of SPX Family and Profiling of CoSPX-MFS3 in Regulating Low-Phosphate Stress in Tea-Oil Camellia

Camellia oleifera a member of the family Theaceae, is a phosphorus (P) tolerator native to southern China. The SPX gene family critically regulates plant growth and development and maintains phosphate (Pi) homeostasis. However, the involvement of SPX genes in Pi signaling in Tea-Oil Camellia remains unknown. In this work, 20 SPX genes were identified and categorized into four subgroups. Conserved domains, motifs, gene structure, chromosomal location and gene duplication events were also investigated in the SPX gene family. Defense and stress responsiveness cis-elements were identified in the SPX gene promoters, which participated in low-Pi stress responses. Based on transcriptome data and qRT-PCR results, nine CoSPX genes had similar expression patterns and eight genes (except CoPHO1H3) were up-regulated at 30 days after exposure to low-Pi stress. CoSPX-MFS3 was selected as a key candidate gene by WGCNA analysis. CoSPX-MFS3 was a tonoplast protein. Overexpression of CoSPX-MFS3 in Arabidopsis promoted the accumulation of total P content and decreased the anthocyanin content. Overexpression of CoSPX-MFS3 could enhance low-Pi tolerance by increased biomass and organic acid contents in transgenic Arabidopsis lines. Furthermore, the expression patterns of seven phosphate starvation genes were higher in transgenic Arabidopsis than those in the wild type. These results highlight novel physiological roles of the SPX family genes in C. oleifera under low-Pi stress, and lays the foundation for a deeper knowledge of the response mechanism of C. oleifera to low-Pi stress.


Introduction
Phosphorus (P) is among the necessary nutritional elements for the development and metabolism of all living organisms and plays an essential role in all life activities. As the second most important nutrient element, P is not only a constituent of important compounds (e.g., nucleic acids and proteins) in plants, but also regulates nearly all energy metabolism processes such as photosynthesis, respiration, energy conversion and enzymatic reactions [1][2][3]. Soil P is recognized as an integral part in Al, Fe or Ca salt. P can be taken up by plants in the form of orthophosphate (Pi, H 2 PO 4− /HPO 4 2− ) [4]. The concentration of available Pi is less than 10 µM in most soils [5]. Under different Pi environmental stresses, plants have evolved a sequence of adaptative mechanisms in morphological, physiological and molecular functions [6]. These mechanisms in plants are all controlled by complex and exquisite molecular regulatory networks, including an increasing number of transcription factors and Pi starvation responsive genes, microRNAs and other functional genes in the plant Pi signaling regulatory pathways.
The SPX domain (Pfam PF03105) was coined based on the first three letters of Suppressor of Yeast gpa1 (SYG1), Phosphatase 81 (PHO81) and Xenotropic and Polytropic C. lanceoleosa genome were identified, and their structural characteristics and conserved motifs were analyzed. Moreover, the expression profiles of homologous CoSPX genes in the hexaploidy C. oleifera "Changlin166" roots under low-Pi stress were determined. The findings provide a reference basis for perspective functional experiments on the SPX gene family and a new theoretical framework for identifying novel genes, exploring genetic resources and improving Pi tolerance in tea-oil Camellia.

Detection of C. lanceoleosa SPX Genes
The 20 SPX protein sequences of Arabidopsis were downloaded and utilized as query sequences. To determine putative SPX protein in C. lanceoleosa, the SPX (PF03105) domain was confirmed by Pfam, CDD and SMART (http://smart.embl-heidelberg.de/, accessed on 22 April 2023). There were 20 SPX genes (ClSPXs) identified in the genome of C. lanceoleosa, which were named based on the corresponding Arabidopsis homologs. Similar to wheat (Triticum aestivum), tomato (Solanum lycopersicum) and rice (Oryza sativa), the SPX subfamily contained the largest clade with 12 members, followed by 3 SPX-MFS, 3 SPX-EXS and 2 SPX-RING members ( Table 1). The detailed information of all 20 SPX members, such as the number of gene ID, location, exons, protein properties, molecular weight, predicted isoelectric point values and estimated subcellular location, are presented in Supplemental  Table S1. Further analysis uncovered that the ClSPX proteins consisted of 237-699 amino acids, carrying a molecular weight of 19.6-78.1 kDa. EXPASY analysis indicated that the SPX protein sequences had different isoelectric point (pI) values (range = 4.89-9.27). Subcellular location analysis showed that ClSPX proteins were localized in cell membrane, tonoplast, nucleus and chloroplast. This characteristic implies that different subfamilies may exhibit varying biology functions.

Phylogenetic Analysis of ClSPXs
To illustrate the evolutionary relationship among SPX homologs in different plants, a phylogenetic tree was constructed comprising 20 ClSPXs in C. lanceoleosa, 20 AtSPXs in Arabidopsis and 24 CsSPXs in C. sinensis. The SPXs were clustered into four subclasses, namely, groups SPX, SPX-RING, SPX-MFS and SPX-EXS ( Figure 1). The SPX group contained the most members, whereas the SPX-RING group contained the least, only two members.

ClSPX Protein Domain, Motif Composition and Gene Structure
The 20 ClSPX proteins were classified into four subfamilies according to the supplemental domains in protein structure, including SPX, SPX-RING, SPX-MFS and SPX-EXS ( Figure 2B). To further examine the characteristics of SPX genes in C. lanceoleosa, 25 conserved motifs were identified by MEME analysis ( Figure 2C). The specific information about the 25 putative motifs was listed in Supplementary Table S2. It was found that only the SPX subfamily contained motif 2. Motif 1 was specific to almost all ClSPXs (except SPX-EXS subfamilies), while motif 7 was only unique to the SPX-RING and SPX-MFS subfamilies ( Figure 2C). Next, the structure of ClSPX genes was analyzed based on their exons and introns. Most ClSPXs exhibited similar exon-intron organization. ClSPXs with only the SPX domain have three exons, while ClSPX-RING, ClSPX-MFS2b/2c and ClSPX-MFS2a contain five, ten and nine exons, respectively ( Figure 2D).

ClSPX Protein Domain, Motif Composition and Gene Structure
The 20 ClSPX proteins were classified into four subfamilies according to the supplemental domains in protein structure, including SPX, SPX-RING, SPX-MFS and SPX-EXS ( Figure 2B). To further examine the characteristics of SPX genes in C. lanceoleosa, 25 conserved motifs were identified by MEME analysis ( Figure 2C). The specific information about the 25 putative motifs was listed in Supplementary Table S2. It was found that only the SPX subfamily contained motif 2. Motif 1 was specific to almost all ClSPXs (except SPX-EXS subfamilies), while motif 7 was only unique to the SPX-RING and SPX-MFS subfamilies ( Figure 2C). Next, the structure of ClSPX genes was analyzed based on their exons and introns. Most ClSPXs exhibited similar exon-intron organization. ClSPXs with only the SPX domain have three exons, while ClSPX-RING, ClSPX-MFS2b/2c and ClSPX-MFS2a contain five, ten and nine exons, respectively ( Figure 2D).   and Arabidopsis (At). The phylogenetic tree of the three species was constructed according to the maximum-likelihood method with 1,000 bootstrap replicates. Green stars represent At; yellow squares are Cs; red circles indicate Cl.

ClSPX Protein Domain, Motif Composition and Gene Structure
The 20 ClSPX proteins were classified into four subfamilies according to the supplemental domains in protein structure, including SPX, SPX-RING, SPX-MFS and SPX-EXS ( Figure 2B). To further examine the characteristics of SPX genes in C. lanceoleosa, 25 conserved motifs were identified by MEME analysis ( Figure 2C). The specific information about the 25 putative motifs was listed in Supplementary Table S2. It was found that only the SPX subfamily contained motif 2. Motif 1 was specific to almost all ClSPXs (except SPX-EXS subfamilies), while motif 7 was only unique to the SPX-RING and SPX-MFS subfamilies ( Figure 2C). Next, the structure of ClSPX genes was analyzed based on their exons and introns. Most ClSPXs exhibited similar exon-intron organization. ClSPXs with only the SPX domain have three exons, while ClSPX-RING, ClSPX-MFS2b/2c and ClSPX-MFS2a contain five, ten and nine exons, respectively ( Figure 2D).

Assessment of Cis-Acting Elements in ClSPX Promoters
The promoter regions of the upstream 2000 bp of 20 ClSPXs were assessed with PlantCARE. All cis-regulatory elements, such as TC-rich repeat, MBS, ARE, W-box, CATbox, GCN4-motif, GARE-motif, ABRE, P-box, ERE and TATC-box elements, were assigned to three various categories: hormones, development and stress ( Figure 3A). In addition, we identified 11 cis-regulatory elements, including defense and stress, MeJA stress response, auxin-responsive elements and others ( Figure 3B), suggesting that SPX genes play vital roles in regulating plant abiotic stress [39,40].

Chromosomal Location and Synteny-Based Evaluation of ClSPX Genes
The physical locations of SPX genes were localized to the chromosomes (Chr) of C. lanceoleosa with TBtools [41]. The ClSPX genes were mapped to eight chromosomes, but were unevenly distributed. Non-uniform SPX gene distribution was observed on the chromosomes ( Figure S1). Three SPX genes were found on chromosomes 2, 11 and 13, while six SPX genes were on chromosome 3. However, collinear genes were not present on the same chromosome. In fact, most genes possessed two similar genes on different chromosomes (e.g., ClSPX-MFS2b-ClMFS-MFS2c/ClMFS-MFS2a and ClSPX4b-ClSPX2/ClSPX1b), which formed two collinear gene pairs ( Figure 4A). We found 7 segmental duplication gene pairs on six chromosomes. The Ka/Ks ratios of gene pairs were all <0.5, demonstrating that these genes may experience intense selection during the evolution process (Supplementary Table S3). Thus, assessing the evolutionary relationship of SPX family members may help to determine the functions of ClSPX genes. Syntenic maps of C. sinensis and Arabidopsis were constructed with C. lanceoleosa ( Figure 4B). The results showed that 9 ClSPX genes exhibited syntenic relationships with 8 and 11 genes in C. sinensis and Arabidopsis, respectively (Supplementary Table S4). Moreover, 23 orthologous gene pairs were observed between C. lanceoleosa and Arabidopsis, and 21 orthologous gene pairs between C. lanceoleosa and C. sinensis. Six SPX collinear gene pairs were detected among C. lanceoleosa, Arabidopsis and C. sinensis. Most sites in these genes experienced intense purifying selection (Supplementary Table S4).
The promoter regions of the upstream 2000 bp of 20 ClSPXs were assessed with Plant-CARE. All cis-regulatory elements, such as TC-rich repeat, MBS, ARE, W-box, CAT-box, GCN4-motif, GARE-motif, ABRE, P-box, ERE and TATC-box elements, were assigned to three various categories: hormones, development and stress ( Figure 3A). In addition, we identified 11 cis-regulatory elements, including defense and stress, MeJA stress response, auxin-responsive elements and others ( Figure 3B), suggesting that SPX genes play vital roles in regulating plant abiotic stress [39,40].

Chromosomal Location and Synteny-Based Evaluation of ClSPX Genes
The physical locations of SPX genes were localized to the chromosomes (Chr) of C. lanceoleosa with TBtools [41]. The ClSPX genes were mapped to eight chromosomes, but were unevenly distributed. Non-uniform SPX gene distribution was observed on the chromosomes ( Figure S1). Three SPX genes were found on chromosomes 2, 11 and 13, while six SPX genes were on chromosome 3. However, collinear genes were not present on the same chromosome. In fact, most genes possessed two similar genes on different chromosomes (e.g., ClSPX-MFS2b-ClMFS-MFS2c/ClMFS-MFS2a and ClSPX4b-ClSPX2/ClSPX1b), which formed two collinear gene pairs ( Figure 4A). We found 7 segmental duplication gene pairs on six chromosomes. The Ka/Ks ratios of gene pairs were all <0.5, demonstrating that these genes may experience intense selection during the evolution process (Supplementary Table S3). Thus, assessing the evolutionary relationship of SPX family members may help to determine the functions of ClSPX genes. Syntenic maps of C. sinensis and Arabidopsis were constructed with C. lanceoleosa ( Figure 4B). The results showed that 9 ClSPX genes exhibited syntenic relationships with 8 and 11 genes in C. sinensis and Arabidopsis, respectively (Supplementary Table S4). Moreover, 23 orthologous gene pairs were observed between C. lanceoleosa and Arabidopsis, and 21 orthologous gene pairs between C. lanceoleosa and C. sinensis. Six SPX collinear gene pairs were detected among C. lanceoleosa, Arabidopsis and C. sinensis. Most sites in these genes experienced intense purifying selection (Supplementary Table S4).

The Expression Profiles of CoSPX Genes in C. oleifera Tissues
As previously mentioned, C. lanceoleosa is closely related to polyploid C. oleifera Abel. At present, the main cultivar of C. oleifera is a polyploid plant. To further understand the CoSPX gene functions in polyploid C. oleifera, the expression profiles of all putative genes were analyzed in various tissues ( Figure S2). The results demonstrated that most CoSPX genes have higher expression level in roots and stems. The same subfamily members of CoPHO1H3/CoPHO1H5, CoSPX-MFS1/CoSPX-MFS2 and CoNLA1/CoNLA2 had similar expression patterns.

The Expression Distribution of CoSPXs under Pi-Deficiency
The expression patterns of CoSPX genes were determined according to our previously reported RNA-seq data. Using the Fragments Per Kilobase of transcript sequence per Millions of base pairs sequenced (FPKM) values of our previous RNA-seq data, we found that almost all SPX genes could respond to low-Pi stress ( Figure 5A). The results of transcriptome data and qRT-PCR (quantitative real-time polymerase chain reaction) showed that nine CoSPX genes had the same expression tendency, and eight genes (except CoPHO1H3) were upregulated at 30 days after exposure to low-Pi stress ( Figure 5B).

The Expression Profiles of CoSPX Genes in C. oleifera Tissues
As previously mentioned, C. lanceoleosa is closely related to polyploid C. oleifera Abel. At present, the main cultivar of C. oleifera is a polyploid plant. To further understand the CoSPX gene functions in polyploid C. oleifera, the expression profiles of all putative genes were analyzed in various tissues ( Figure S2). The results demonstrated that most CoSPX genes have higher expression level in roots and stems. The same subfamily members of CoPHO1H3/CoPHO1H5, CoSPX-MFS1/CoSPX-MFS2 and CoNLA1/CoNLA2 had similar expression patterns.

The Expression Distribution of CoSPXs under Pi-Deficiency
The expression patterns of CoSPX genes were determined according to our previously reported RNA-seq data. Using the Fragments Per Kilobase of transcript sequence per Millions of base pairs sequenced (FPKM) values of our previous RNA-seq data, we found that almost all SPX genes could respond to low-Pi stress ( Figure 5A). The results of transcriptome data and qRT-PCR (quantitative real-time polymerase chain reaction) showed that nine CoSPX genes had the same expression tendency, and eight genes (except CoPHO1H3) were upregulated at 30 days after exposure to low-Pi stress ( Figure 5B).

Co-Expression Network of CoSPX Genes
To explore the roles of CoSPX genes in regulating Pi response gene expression, a coexpression modulatory axis was constructed according to the previous transcriptome analysis under low-Pi stress, among which two genes were identified as hub genes ( Figure  5C). The hub gene-related nodes were involved in nucleic acid binding, protein binding, molecular function regulation, as well as transcription factor, catalytic, and transporter

Co-Expression Network of CoSPX Genes
To explore the roles of CoSPX genes in regulating Pi response gene expression, a coexpression modulatory axis was constructed according to the previous transcriptome analysis under low-Pi stress, among which two genes were identified as hub genes ( Figure 5C). The hub gene-related nodes were involved in nucleic acid binding, protein binding, molecular function regulation, as well as transcription factor, catalytic, and transporter activities ( Figure 5C). The frequency of GO terms was visualized using a word cloud ( Figure 5D).

Sequence Alignment of ClSPX-MFS2b Homologous Genes
Based on our transcriptome data, a homologous gene CoSPX-MFS3 in C. oleifera was selected as a key hub gene. Then, through multiple sequence alignment, we found the four closest species of CoSPX-MFS3 proteins with SPX and MFS domain ( Figure 6A). By analyzing the SPX-MFS amino acid sequences of the 19 species, we found that CoSPX-MFS3 was the most closely related to ClSPX-MFS2b ( Figure 6B). Thus, the functions of CoSPX-MFS3 were analyzed by transcriptome combined with C. lanceoleosa diploid genome.

Localization of CoSPX-MFS3 in the Tonoplast
Subcellular localization indicated that CoSPX-MFS3-EGFP fusion proteins were gathered in the tonoplast ( Figure 6C), which is in line with the prediction from Cell-PLoc.

Overexpression of CoSPX-MFS3 Enhances Pi Tolerance in Arabidopsis
To evaluate CoSPX-MFS3 action during reduced Pi stress, Arabidopsis plants with Co-SPX-MFS3 overexpression (OE) were established. After qRT-PCR analysis of CoSPX-MFS3 expression, the homozygous T3 lines were generated for further analysis ( Figure  S3). To examine potential CoSPX-MFS3-mediated regulation of Pi signaling and reduced Pi tolerance, we examined the phenotype of Arabidopsis plants overexpressing CoSPX-MFS3 under Pi-deficiency (LP, 5 µM). The results showed a strong tolerance of Arabidopsis transgenic line with longer roots (Figure 7A,C). The accumulation of anthocyanin content is a marker of low-Pi stress in plants [42][43][44]. The degree of damage in leaves at 21 days after exposure to low-Pi stress was strongly elevated in wild-type (WT) versus CoSPX-MFS3 OE lines ( Figure 7B). The total anthocyanin concentration of transgenic plants was considerably diminished, compared to WT plants ( Figure 7F). The root biomass was clearly distinct between WT and transgenic lines, regardless of low or normal Pi (NP, 1 mm) stress treatment ( Figure 7D,E). The total P content in CoSPX-MFS3 OE lines was increased ( Figure 7G,H). Moreover, the malic and citric acid contents, as well as acid phosphatase activity were significantly higher in roots and leaves of the OE line, relative to WT plants ( Figure 8A-F). All results demonstrated that CoSPX-MFS3 could enhance Pi toler-

Localization of CoSPX-MFS3 in the Tonoplast
Subcellular localization indicated that CoSPX-MFS3-EGFP fusion proteins were gathered in the tonoplast ( Figure 6C), which is in line with the prediction from Cell-PLoc.

Overexpression of CoSPX-MFS3 Enhances Pi Tolerance in Arabidopsis
To evaluate CoSPX-MFS3 action during reduced Pi stress, Arabidopsis plants with CoSPX-MFS3 overexpression (OE) were established. After qRT-PCR analysis of CoSPX-MFS3 expression, the homozygous T3 lines were generated for further analysis ( Figure S3). To examine potential CoSPX-MFS3-mediated regulation of Pi signaling and reduced Pi tolerance, we examined the phenotype of Arabidopsis plants overexpressing CoSPX-MFS3 under Pi-deficiency (LP, 5 µM). The results showed a strong tolerance of Arabidopsis transgenic line with longer roots (Figure 7A,C). The accumulation of anthocyanin content is a marker of low-Pi stress in plants [42][43][44]. The degree of damage in leaves at 21 days after exposure to low-Pi stress was strongly elevated in wild-type (WT) versus CoSPX-MFS3 OE lines ( Figure 7B). The total anthocyanin concentration of transgenic plants was considerably diminished, compared to WT plants ( Figure 7F). The root biomass was clearly distinct between WT and transgenic lines, regardless of low or normal Pi (NP, 1 mm) stress treatment ( Figure 7D,E). The total P content in CoSPX-MFS3 OE lines was increased ( Figure 7G,H). Moreover, the malic and citric acid contents, as well as acid phosphatase activity were significantly higher in roots and leaves of the OE line, relative to WT plants ( Figure 8A-F). All results demonstrated that CoSPX-MFS3 could enhance Pi tolerance by increased biomass and organic acid contents. In addition, seven Pi-responsive homologous genes were selected for expression level verifications in transgenic Arabidopsis. The qRT-PCR results showed that all of them were up-regulated in transgenic Arabidopsis compared to WT (Figure 9). In summary, we conclude that CoSPX-MFS3 may play an important role in mediating Pi tolerance in transgenic Arabidopsis.

Discussion
In response to low-Pi tolerance, plants form a series of biochemical and physiological adaptive mechanisms, which in turn enhance the absorption and utilization of soil Pi. Adaptive changes in plants under low-Pi stress are finely regulated by Pi signaling networks, in which SPX can regulate Pi-signaling networks in plants. Tea-oil Camellia mainly grows in acidic soil with very low P content in the southern area of China. Thus, assessing the biological functions of SPX proteins can elucidate the mechanisms underlying the adaption of C. oleifera to low-Pi pressure.
In our study, the total number of SPX genes in C. lanceoleosa was relatively similar to that in Arabidopsis (20) [11] and Solanum lycopersicum (19) [37], but less compared to wheat (46) [34], maize (33) [13] and Brassica napus (69) [35]. These differences in family members of different species may be due to environmental changes during evolution. The

Discussion
In response to low-Pi tolerance, plants form a series of biochemical and physiological adaptive mechanisms, which in turn enhance the absorption and utilization of soil Pi. Adaptive changes in plants under low-Pi stress are finely regulated by Pi signaling networks, in which SPX can regulate Pi-signaling networks in plants. Tea-oil Camellia mainly grows in acidic soil with very low P content in the southern area of China. Thus, assessing the biological functions of SPX proteins can elucidate the mechanisms underlying the adaption of C. oleifera to low-Pi pressure.
In our study, the total number of SPX genes in C. lanceoleosa was relatively similar to that in Arabidopsis (20) [11] and Solanum lycopersicum (19) [37], but less compared to wheat (46) [34], maize (33) [13] and Brassica napus (69) [35]. These differences in family members of different species may be due to environmental changes during evolution. The relationship between orthologs and paralogs shows that the diversity among the members of this gene family occurred before and after the divergences of monocots and dicots [45]. On the other hand, mutations in regulatory regions such as the promoter region and coding DNA regions probably caused the duplicated genes to have different expression patterns. [46,47]. Gene duplication is a crucial mechanism that can acquire new genes and create genetic novelty in organisms [48]. Eight putative ClSPXs were mapped to the corresponding chromosome according to the localization data of the C. lanceoleosa genome. The findings demonstrated that ClSPXs were non-uniformly distributed on the chromosomes of C. lanceoleosa. Meanwhile, it was found that segmental duplication occurred in C. lanceoleosa for expanding all SPX members during the process of evolution. The Ka/Ks rate is of great significance in reconstructing phylogeny and evolutionary selection [49]. Ka/Ks rates of <1, 1 and >1 represent purifying selection, neutral selection and positive selection, respectively [50]. The mean Ka/Ks value (0.177) of C. lanceoleosa demonstrated a strong purifying selection of SPX genes. In the same way, the Ka/Ks values of syntenic gene pairs between C. lanceoleosa and Arabidopsis or C. sinensis were less than 1. The Ka/Ks results imply that these paralogous gene pairs have experienced intense purifying selection during the evolutionary process.
The SPX conserved domain (SPX domain) plays a crucial role in modulating plant nutrient stress. In Arabidopsis, AtSPX1 interacts with a unique four-stranded coiled-coil domain in AtPHR1 [51]. In rice, SPX proteins (phosphate sensor) bind to OsPHR2 in high-phosphate condition, thus inhibiting OsPHR2 binding to P1BS elements in promoters of OsRAM1, OsPT11, OsWRI5A and OsAMT3;1, thus decreasing mycorrhizal-related gene expression and inhibiting mycorrhizal colonization [52]. In maize, ZmSPX genes (except ZmSPX3) were markedly induced by low-Pi stress [13]. In wheat, five TaSPX genes were upregulated after Pi starvation treatment [34]. In this study, nine CoSPX genes had the same expression tendency, and eight genes (except CoPHO1H3) were up-regulated at 30 days after exposure to low-Pi stress, which was similar to other plants with low-Pi treatment. In general, SPX domain proteins as a low-Pi sensor. Two hub genes (CoPHO1H3 and CoSPX-MFS3) were identified by WGCNA analysis. Meanwhile, co-expression analysis revealed that edge genes were involved in protein binding, nucleic acid binding, molecular function regulation, transporter activity, catalytic activity and transcription factor activity. These results demonstrate that SPX domain proteins serve an essential function in low-Pi stress perception and response.
In rice, OsSPX-MFS3 was the first reported as a vacuolar Pi efflux transporter that modulates Pi homeostasis [18] and OsSPX-MFS1 as a major Pi transporter that regulates leaf Pi homeostasis [38]. In Brassica napus, BnA09PHT5;1b and BnCnPHT5;1b were identified as two vacuolar Pi influx transporters [53]. The previous study revealed that SPX-MFS proteins primarily modulate Pi transport and homeostasis. Herein, we conducted a preliminary assessment of CoSPX-MFS3 by heterologous transformed Arabidopsis. Overexpression of CoSPX-MFS3 in Arabidopsis mainly enhanced low-Pi tolerance by increased biomass, organic acid content and total Pi content. Citric acid is an organic compound found in the roots of various plant species in response to Pi stress [54]. In white lupin, the citric acid content was increased at the later stage of Pi deficiency [55]. Although our results are different from rice and Brassica napus, this may be due to the long-term evolution of woody and herbaceous plants. Furthermore, overexpression of CoSPX-MFS3 in Arabidopsis increased the transcription levels of a series of Pi starvation genes, including PTs, SPX1, SPX2 and SPX3. This result is consistent with the previous findings that AtSPX1 overexpression in Arabidopsis could increase the mRNA levels of AtPAP2, AtACP5 and AtRNS1 [11]. In summary, CoSPX-MFS3 is a positive tolerator that responds to low-Pi stress. However, the detailed molecular mechanism of CoSPX-MFS3 regulation in Pi acquisition and translocation need to be further identified.

Assessment of the SPX Family Genes in C. lanceoleosa
The C. lanceoleosa genome sequence and protein sequence information files were sourced from the NCBI database (https://www.ncbi.nlm.nih.gov/;

Construction of Phylogenetic Trees
Full-length protein sequences from Arabidopsis and Camellia sinensis were acquired from the NCBI protein database to determine their evolution. The definitized amino acid sequences of ClSPXs were subjected to multiple sequence alignment via ClustalW (https://www.genome.jp/tools-bin/clustalw, accessed on 22 April 2023). The maximumlikelihood criteria in MEGA 7.0 were used to establish phylogenetic trees, with Poisson corrections and 1000 bootstrap replicates. The recognized ClSPX genes were assigned to distinct categories based on the AtSPXs stratification. Interactive Tree of Life (iTOL.7) software (https://itol.embl.de/, accessed on 22 April 2023) was employed for visualization and modification of the phylogenetic tress.

Assessment of ClSPX Gene Domain, Structure and Motif Sequences
The intron/exon map structure was drawn using the tools of the Gene Structure Display Server v2.0 (http://gsds.gao-lab.org/, accessed on 22 April 2023) platform. The conserved motifs in ClSPX proteins were determined using the MEME program v5.0.5 (http://meme-suite.org/tools/meme, accessed on 22 April 2023), with a maximum of 25 motifs and optimal motif width range of 6-50 amino acid residues.

Determination of Cis-Acting Elements in ClSPX Promoter
The 2000-bp sequences upstream of the ClSPX translation initiation codon were analyzed through the PlantCARE database in order to determine the cis-regulatory elements.

Chromosomal Location and Synteny-Based Evaluation of ClSPX Genes
The chromosomal location data of ClSPXs were extracted from the C. lanceoleosa genome annotation file. This information was also used to construct chromosomal mapping by TBtools [41]. Gene duplication assessment was conducted via the One-Step MCScanX function in TBtools, and the result was visualized by the CGview tool [41].

CoSPXs Expression Profiles
To determine the expression profiles of CoSPX at different stages (0, 1, 3, 7 and 30 days), the FPKM values of CoSPX genes at five treatment stages of C. oleifera were obtained from our previous RNA-seq data [56]. The expression profiles of CoSPXs at different stress stages were analyzed based on their FPKM values, and a heatmap was produced using the R heatmap function (gplots). Z-score normalization was used to normalize the expression values. Total RNA isolation was employed by the RNA kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) following the manufacturer's instructions. To conduct reverse transcription, PrimeScriptTM RT Master Mix (TaKaRa, Dalian, China) was used. Then, quantitative realtime PCR (qRT-PCR) analysis was conducted on the 7300 Real-Time PCR System (Applied Biosystems, Shanghai, CA, USA) with the help of the TB Green ® Premix Ex Taq™ II (Tli RNaseH Plus, Takara). The "Genes" module in SPDE software (Version 2.0)was used to design gene-specific primers [57]. The GAPDH gene served as the endogenous control for CoSPXs [58]. Actin gene was chosen as an internal reference for AtSPXs. Relative gene expression was computed via the 2 −∆∆Ct formula [59]. The employed primers are detailed in Supplemental Table S5.

Co-Expression Regulatory Network of CoSPX Genes
Among all co-expressed genes, CoSPX genes exhibited the largest interconnection. Thus, they were established as hub genes. Edge genes were annotated by GO terms, and their frequency was visualized in a cloud word (https://www.bioladder.cn/web/#/chart/ 20, accessed on 22 April 2023). Finally, the co-expression regulatory network was performed in Cytoscape 3.8.2.

Multiple Sequence Alignment, Subcellular Localization of CoSPX-MFS3
The SPX-MFS3 amino acid sequences of 19 different species were downloaded from NCBI for evolutionary analysis, and a multiple sequence alignment was constructed. The CoSPX-MFS3 coding sequences were fused with the mGFP-encoding sequences in the pMDC43 expression vector with the CloneExpress II One Step Cloning Kit (Vazyme, Nanjing, China). For transient expression analysis, the recombinant plasmids were incorporated into Agrobacterium tumefaciens EHA105 cells, prior to transfer to Nicotiana benthamiana leaves. The empty vectors were employed as controls. After three days, the transient expression of GFP-fusion proteins was determined using an LSM900 confocal microscope imaging system (Zeiss, Shanghai, China). mCherry-labeled vacuole markers were used to visualize the tonoplast.

Ectopic CoSPX-MFS3 Expression in Arabidopsis and Pi Exposure
The full-length CDS of CoSPX-MFS3 was amplified from C. oleifera and inserted into the CaMV 35S promoter-regulated binary vector pEXT06 employing the ClonExpress II One Step Cloning Kit (Vazyme). Subsequently, A. tumefaciens GV3101-regulated transformation of Arabidopsis was employed. The transgenic Arabidopsis seeds were harvested from individual plants, and positive lines were identified on 1/2 MS medium using hygromycin until homozygous transgenic Arabidopsis lines were obtained. Using 75% ethanol, the homozygous seeds of Arabidopsis underwent surface sterilization three times, prior to a 24-h incubation at 4 • C. After incubation, the seeds were placed on petri dishes containing Pi-sufficient (normal Pi [NP], 1 mm Pi) medium for 5 days, prior to transfer to Pi-deficient (low Pi [LP], 5 µM Pi) medium for 7 days with a 16:8-h light/dark cycle at 22 • C. Wildtype (WT) plants and T3 homozygous transgenic lines were cultivated in 1/2-strength Hoagland solution (Coolaber, China) for one week. Subsequently, the plants were placed in 1/2-strength phosphorus-free solution and grown for three weeks. The fresh roots and leaves were immediately frozen in liquid nitrogen and kept at −80 • C. The roots and leaves were harvested for the determination of biomass.

Measurement of Total Pi Content
For determination total P content, the method described in a previous study [60] were strictly followed.

Determination of Anthocyanin and Organic acid Content
Fresh roots and leaves (0.1 g) of Arabidopsis were crushed in liquid nitrogen and incubated with 1% HCl-methanol for 24 h. The optical density values of anthocyanin were recorded at 530 nm (Tang et al., 2022). Commercial kits (Geruisi-bio, Suzhou, China) were employed for the detection of malic acid (Lot. G0862W48), citric acid (Lot. G0864F) and acid phosphatase (Lot.G0903W) contents. Briefly, Arabidopsis samples (0.1 g) were extracted using the kit's buffer, followed by centrifugation (10,000 rpm, 15 min, 4 • C) [41]. The assays were performed in triplicate.

Conclusions
In summary, 20 SPX genes were identified in the C. lanceoleosa genome, and their conserved domain, motif distribution, gene duplication, cis-acting elements and chromosomal distribution were analyzed. Segmental duplication event was the key factor affecting the evolutionary process of the SPX gene family in C. lanceoleosa based on collinearity analysis. Additionally, the expression distribution of SPX genes in low-Pi stress conditions were analyzed using transcriptome data and qRT-PCR experiment. A hub gene CoSPX-MFS3 induced by low-Pi stress could enhance low-Pi tolerance in transgenic Arabidopsis. Altogether, these findings present a reference basis for the enhanced understanding of the physiological roles of SPX genes in C. oleifera.  Data Availability Statement: The C. lanceoleosa genome sequence and protein sequence information files were sourced from the NCBI database (accession number: PRJNA780224).

Conflicts of Interest:
The authors declare no conflict of interest.