Genome-Wide Analysis of DoSPX Genes and the Function of DoSPX4 in Low Phosphorus Response in Dendrobium officinale

Dendrobium officinale Kimura et Migo is a famous Chinese herb. D. officinale grows on rocks where the available phosphorus is low. The SPX family plays a critical role in maintaining Pi homeostasis in plants. In this paper, 9 SPX family genes were identified in the genome of D. officinale. Bioinformatics and qRT-PCR analysis showed that DoSPXs were involved in response to −Pi stress and had different expression patterns. DoSPX4, which had a unique expression pattern, was clustered with AtSPX4 and OsSPX4. Under −Pi treatment, the expression level of DoSPX4 reached a peak on 5 d in roots, while showing a downward trend in the aboveground parts. DoSPX4 was located on the cell membrane. Overexpression DoSPX4 promoted Pi content in the stem and the expression level of NtPHT1/2 in Nicotiana tabacum. The results of Yeast two-hybrid showed that DoSPX4 could interact with Phosphate High-Affinity Response factor (DoPHR2). These results highlight the role of DoSPX4 in response to low phosphorus, which provides a theoretical basis for further study on the response mechanism of −Pi in D. officinale.

Introduction

Phosphorus (Pi) plays an essential role in plant growth (Chen and Liao, 2017). The low content of available Pi in the soil is difficult to meet the Pi requirements accompany with plant growth. Plants increase effective Pi utilization by sensing and absorbing Pi in the soil, then adapt to low Pi environment in molecular and physiological levels.

Dendrobium officinale is a perennial herb which has a variety of pharmacological effects. It can grow on tree trunks, rocky cliffs, or fern surfaces in natural conditions. It is subjected to various environmental stresses in the harsh growth environment, among which low Pi stress is the main factor affecting its growth and development (Liu N. et al., 2018). The previous research found that D. officinale could respond to −Pi and accumulate effective active components in low phosphorus. The expression of the key genes in the secondary metabolism were significantly correlated with Pi concentration, while the early response genes to −Pi in D. officinale have not been reported (Liu L. et al., 2021).

Proteins, containing the SPX domain, have been identified as early response factors which participate in Pi signal transduction in plants (Zhou et al., 2021). The N-terminal of plant SPX protein contains a highly conserved SPX domain, named by the first letter of SYG1, PHO81, and XPR1 genes (Secco et al., 2012). At present, SPX has been classified into four subfamilies by the C-terminal domains of the protein. The four subfamilies are SPX (containing only one SPX domain), SPX-EXS (containing SPX and one EXS domain), SPX-MFS (containing SPX and MFS domain), and SPX-RING (containing ring-type star finger domain) (Wang et al., 2004; Chen et al., 2009; Chiou and Lin, 2011; Lin et al., 2013; Su et al., 2015; Wang et al., 2015; Liu J. et al., 2016; Zhang et al., 2016; Yue et al., 2017; Yang et al., 2018). Among them, the SPX subfamily plays an important role in the early stage of Pi signal recognition.

Generally, SPX can respond to Pi signals and then interact with MYB to change the transcriptional activation of downstream PSI genes. At present, Phosphorus-related MYB includes PHR (MYB-CC) and several R2R3-MYB. In Arabidopsis, AtSPX1/2/3/4 proteins are upstream regulators of AtPHR1 (Duan et al., 2008). AtSPX1 and AtSPX2 had functional redundancy. Both of them could regulate AtPHR1, and the extent of this interaction was affected by AtSPX3 (Puga et al., 2014). In stem, AtSPX4 was a repressor of AtPHR1 (Osorio et al., 2019). In Oryza sativa, there were 6 SPXs (OsSPX1–OsSPX6). Under Pi deficiency, the expression of OsSPX4 was downregulated, while the other five OsSPX genes showed an upward trend (Secco et al., 2012; Lv et al., 2014). OsSPX1/2/4 could interact with OsPHR2 and affect the regulation of OsPHR2 on downstream PSI genes (Shi et al., 2014; Wang et al., 2014). OsSPX3, OsSPX5, and OsSPX6 were homologous genes, which were involved in functional redundancy in response to phosphorus. OsSPX3 and OsSPX5 could form homodimers and participate in complex regulation in O. sativa (Shi et al., 2014). OsSPX4 was rapidly degraded by the proteasome pathway under low phosphorus, which had unique subcellular localization (Lv et al., 2014). These studies showed that SPX played an important role in phosphate response in plants, and SPX4 (OsSPX4 in O. sativa and AtSPX4 in A. thaliana) may have a different regulatory pattern from other SPXs.

Among them, gene prediction based on similarity comparison through published genome sequence has become the main method to screen key genes and analyze molecular mechanisms (Bhatt et al., 2021). It has become the main method to screen key genes and analyze molecular mechanism that gene prediction based on similarity comparison. The previous studies showed that low phosphorus could promote the accumulation of effective active substances in D. officinale (Liu L. et al., 2021). However, the molecular mechanism of D. officinale response to −Pi has not been reported. In this study, the bioinformatics analysis of the early Pi response factor DoSPX was carried out. The expression pattern of DoSPX in D. officinale was analyzed by qRT-PCR. DoSPX4 had a unique expression pattern, which was subcellular localization was further investigated. The interaction between DoSPX4 and DoPHR2 was studied by Yeast two-hybrid. The function of DoSPX4 to plant low phosphorus was verified by heterologous overexpression of DoSPX4 in N. tabacum.

Materials and Methods

Treatment and Preservation of Plant Materials

The D. officinale tissue culture seedlings used in the experiment were from the Anhui Provincial Engineering Technology Research Center for Development and Utilization of Regional Characteristic Plants, School of Life Sciences, Anhui Agricultural University. The seedlings of D. officinale with uniform size, shape, and color were cultured in the plant tissue culture room under a constant temperature of 25°C at 8L:16D photoperiod. The tissue culture seedlings were cultured on Murashige and Skoog medium (MS) for 8 months, then put into MS medium with different Pi concentrations. The 5 levels of KH₂PO₄ are used to set different Pi concentrations on MS medium (2.5, 1.25, 0.625, 0.0625, and 0 mM), and K⁺ in different Pi concentration media were supplemented with different concentrations of KCl. Samples were taken on 0, 1, 5, 10, and 40 days after treatment and put into a 10-ml centrifuge tube and immediately froze in liquid nitrogen and store it in the refrigerator at −80°C for standby. The three biological replicates were set for each treatment.

Identification of SPX Gene Family in Dendrobium officinale

By reference SPX protein sequences in Oryza sativa (Supplementary Table 1) and Arabidopsis thaliana (Supplementary Table 1), SPX proteins in D. officinale genome (Supplementary Table 1) sequence were selected with a threshold of E-value < 1E–5. Then the obtained sequences were submitted to CD-HIT¹ (Fu et al., 2012) to remove the redundant sequence. Finally, the candidate sequences are submitted to SMART² (Letunic et al., 2021) and PFAM³ (Mistry et al., 2021) to identify the conserved motifs. The basic information of protein sequence was obtained online by the ExPasy website⁴ (Gasteiger et al., 2003).

Construction of DoSPXs Phylogenetic Tree

The 7 SPX sequences in D. officinale were compared with the SPX protein sequences of 27 A. thaliana, 12 O. sativa and 12 Phalaenopsis equestris by ClustalW (Thompson et al., 1994). A phylogenetic tree was constructed using the MEGA7.0 NJ method with the bootstrap = 1,500 and beautified with ITOLS online website⁵ (Letunic and Bork, 2021). The names of the gene name and accession numbers of SPX can be found in Supplementary Table 2.

Cis-Acting Element Analysis

The TBTOOLS (Chen et al., 2020) was used to extract the promoter region of the genome sequence by using the genome annotation file of D. officinale. The 1,500 bp upstream sequences of SPX CDS was submitted to PLANTCARE⁶ (Lescot et al., 2002) and NEWPLACE⁷ (Higo et al., 1999) online websites to analyze the possible cis-acting elements of the promoter. Use TBTOOLS for drawing.

Subcellular Localization

In order to understand the subcellular localization of DoSPX4, the recombinant plasmid pCAMBIA1305-DoSPX1-GFP was constructed by amplifying the fragment of DoSPX4 by sense and antisense primers (Supplementary Table 3). The recombinant plasmid was introduced into Agrobacterium tumefaciens EHA105. EHA105 Infect tobacco leaves and DoSPX4 is transiently expressed in Nicotiana benthamiana leaf epidermal cells. A confocal laser microscope was used to observe the GFP fluorescence signal.

Yeast Two-Hybrid

In order to verify the interaction of DoSPX4 with DoPHR2, the AD-DoSPX4 recombinant vector was constructed by amplifying DoSPX4 open reading frame into AD, and the DoPHR2-BD recombinant vector was constructed by amplifying the DoPHR2 fast-play reading frame into BD, the primers were shown in Supplementary Table 3. The constructed AD-DoSPX4 and DoPHR2-BD were used to transform AH109 jointly, and the successfully transformed clones were screened on SD-WL, and the clones were screened on SD-HAWL. The X-α-Gal is used to identify positive interactions (SD-WL indicates the SD medium without Leu and Trp, and SD-HAWL means the SD medium without Ade, His, Leu, and Trp).

Nicotiana tabacum Transformation

In order to understand the function of DoSPX4, the recombinant plasmid pCAMBIA1305-DoSPX4 was constructed by amplifying the fragment DoSPX4 by sense and antisense primer (Supplementary Table 3), pCAMBIA1305-DoSPX4 transformed into A. tumefaciens GV3101. The GV3101 was transformed into N. tabacum leaf discs via an A. tumefaciens-mediated leaf disc procedure (Topping, 1988). Regenerated plants are obtained by inducing callus, budding, rooting, and transplanting. The positive strain is screened and selected by using 50 mg/L Hygromycin B and 200 mg/L antibacterial Cefotaxime. The transgenic N. tabacum were cultured in MS medium with 1.25 mM Pi concentration (HP) and 0.0625 mM Pi concentration (LP) for 7 days.

Determination of Available Pi Content

The content of available Pi in plants was determined by the ammonium molybdate method (Nanamori et al., 2004). To determine the Pi concentration of the transgenic N. tabacum, the transgenic N. tabacum was ground with liquid nitrogen and 10% (w/v) perchloric acid (PCA). The supernatant was centrifuged after 10 times dilution with 5% (w/v) PCA. The working solution [sulfuric acid-ammonium molybdate (solution A) and ascorbic acid solution (solution B) were mixed in proportion (6:1)] extracts the available Pi from the supernatant. The absorbance was measured at 820 nm by a UV spectrophotometer.

Quantitative Real-Time PCR Analysis

The RNA was extracted from the Liquid nitrogen quick-frozen plant tissues using a Plant Total RNA Isolation Kit (Sangon Biotech, Shanghai, China). A One Step RT-qPCR Kit (BBI Life Science, Shanghai, China) was used to obtain cDNA. 2× TaqMan Fast qPCR Master Mix (BBI Life Science, China) was used to execute qRT-PCR. Reaction conditions were performed according to Liu’s method (Liu L. et al., 2021). The qRT-PCR primers were designed using NCBI PRIMER-BLAST⁸ (Supplementary Table 3). Each experiment was set up with three biological replicates, and the results were calculated using 2^–ΔΔCT method.

The Temporal Expression Patterns Analysis of DoSPXs

The transcriptome data of 8 D. officinale tissues (root, stem, leaf, flower buds, column, lip, and sepal) were downloaded from the NCBI SRA database (PRJNA348403). Trimmomatic (Bolger et al., 2014) is used to filter and trim data. A retrieval file of D. officinale genome was established by HISAT2 (Pertea et al., 2016), and high-quality reads were compared to D. officinale genome. Samtools (Li et al., 2009) was used for sorting and format conversion to obtain BAN format files. Finally, StringTie (Pertea et al., 2016) was assembled for sequence, and transcript abundance was estimated. TBOOLS (Chen et al., 2020) is used for the visualization of results.

Results

Identification of SPX Family Genes in Dendrobium officinale

By blasting the published genome sequences of D. officinale using the conserved SPX sequences reported in O. sativa and A. thaliana, the possible DoSPX sequences were obtained, and the redundancy of the sequences with high similarity was removed, then verified these sequences with SMART and PFAM, the result was shown in Table 1. Seven possible DoSPX sequences were obtained, all of which contain SPX conserved domains. Combining with the naming method of A. thaliana, two of them contain EXS domains named DoSPX-EXS1 and DoSPX-EXS2. One sequence contains the MFS sequence, named DoSPX-MFS. A sequence containing the RING field, called DoSPX-RING. The three sequences containing only the SPX domain were named DoSPX1, DoSPX3, and DoSPX4. The amino acid sequence analysis showed that the size of all proteins was 2.75–10.12 kDa and the isoelectric point was 5.08–9.22. The average hydrophilic coefficient (GRAVY < 0) shows that the other six are hydrophilic proteins except for DoSPX-MFS.

TABLE 1

Table 1. Analysis of amino acid sequence encoded by DoSPXs.

System Evolution Analysis of DoSPXs

Through CLUSTERW alignment, the phylogenetic trees of D. officinale, P. equestris, O. sativa, and A. thaliana were established by the NJ method of MEGA7.0. The result was shown in Figure 1. It was found that all sequences were divided into four subclasses. The sequences of each subclass of SPX in D. officinale were well classified from those in O. sativa and A. thaliana. DoSPX1 is clustered with AtSPX1, 2, and OsSPX1; DoSPX3 were clustered with OsSPX3, 5, 6, and AtSPX3; while DoSPX4 is clustered with AtSPX4, OsSPX4. DoSPX-EXS1, DoSPX-EXS2, DoSPX-MFS, and DoSPX-RING are well clustered with the three subfamilies reported in O. sativa and A. thaliana.

FIGURE 1

Figure 1. Phylogenetic analysis of SPX proteins in Dendrobium officinale, Phalaenopsis equestris, Oryza sativa, and Arabidopsis thaliana. The neighbor-joining (NJ) tree was created with 9 D. officinale, 12 P. equestris, 12 O. sativa, and 27 A. thaliana SPX protein sequences.

Analysis of DoSPXs Cis-Acting Elements

By analyzing the upstream promoter sequence of DoSPXs, many cis-acting elements necessary for plant growth, development, and stress response were excavated. The result was shown in Figure 2. In these elements, MSA like, CAT-box and BOX-4 related to plant growth and development; ERE and TGACG-motif were involved in response to hormones; TC-rich repeat were involved in response to abiotic stress. These results suggested that DoSPXs may participate in the growth, development, and stress response of D. officinale.

FIGURE 2

Figure 2. Analysis of the cis-acting elements in DoSPXs of D. officinale. (A) The number in each cell represents the number of cis-acting elements in different SPX genes. (B) Each bar diagram represents a different type of cis-acting element. (C) The pie chart represents the proportion of promoters in other groups.

Expression Pattern Analysis of DoSPXs

In order to understand the expression patterns of the DoSPXs in D. officinale, the expression level of the DoSPXs in different tissues was analyzed. The results were presented in heat map form in Figure 3. DoSPX1 and DoSPX4 showed similar expression patterns in various tissues and had high expression levels in D. officinale. DoSPX3 had high expression level in sepals, showed that DoSPX3 played an important role in the sepals. DoSPX-EXS1 had high expression level in the root tips and stem. DoSPX-EXS1, DoSPX-RING, and DoSPX-MFS had low expression levels in all eight tissues.

FIGURE 3

Figure 3. Expression profiles of DoSPX genes in 8 tissues of D. officinale. Blue to red indicates low to high expression levels of the gene.

In order to understand the response of DoSPXs to −Pi stress, D. officinale was taken at 1, 5, 10, and 40 days after the −Pi treatment and determined the expression level of DoSPXs. The results were shown in Figure 4. Under the −P, the transcript abundance of most PSI genes increased. However, each gene showed its unique expression pattern at 0 day of the −P treatment. In D. officinale buds, the expression levels of DoSPX-EXS1, DoSPX-MFS, and DoSPX-RING reached the highest at 1 day after the −P treatment, DoSPX1 and DoSPX3 reached the highest at 5 day after the −P treatment; DoSPX-EXS2 peaked at day 10. In roots, DoSPX1-DoSPX4, DoSPX-EXS1, DoSPX-EXS2, and DoSPX-MFS reached the highest expression level at day 5 after the −P treatment; their expression level increased with the increase of −Pi treatment time. These results suggested that DoSPXs mostly reached a high expression level in the early stage of −P stress, while DoSPX4 showed different changes from other genes in the buds under the −P stress. The expression of DoSPX4 reached its highest level at 0 day of −P treatment, and the expression down-regulated with −Pi treatment time.

FIGURE 4

Figure 4. (A) Temporal and spatial expression patterns of PSI gene in shoot and roots under −P stress in D. officinale. The actin was used as an internal reference gene to regulate gene expression (B) SPX genes expression in shoot and root of D. officinale. Treated with −Pi for 5 day. Green, yellow, and red indicate low, medium, and high expression.

Subcellular Localization of DoSPX4

The CDS region without the stop codon of the DoSPX4 gene was cloned into the pCAMBIA1305.1-GFP vector, and the subcellular localization of DoSPX4 in epidermal cells of N. tabacum was observed by a laser confocal microscope. The results showed that DoSPX4-GFP recombinant protein had a fluorescence signal on the cell membrane, while GFP was distributed in the whole cell (Figure 5).

FIGURE 5

Figure 5. Subcellular localization of DoSPX4 protein in Nicotiana tabacum epidermal cells. The results were observed by confocal laser microscopy. The pictures show a green fluorescent field, a bright field and an overlay of two fields (merge). The numerical reading of the red ruler is 20 μm.

DoSPX4 Interact With DoPHR2 by Y2H

To identify the interaction between DoSPX4 and DoPHR2, the CDS of DoSPX4 was cloned into pGADT7, and the segment (C192aa-225aa) of DoPHR2 without transcriptional activation domain was cloned into the pGBKT7 vector. AD-DoSPX4 and BD-DoPHR2^{192aa–225aa} were transformed into yeast strain AH109. The yeast strain could grow normally on SD/-Trp-Leu-His-Ade medium, indicating that DoSPX4 was tender enough to interact with DoPHR2^{192aa–225aa} (Figure 6).

FIGURE 6

Figure 6. The DoSPX4 interacts with DoPHR2. The pGADT7 and pGBKT7 were used as the negative control. SD-WL indicates the SD medium without Leu and Trp, and SD-HAWL means the SD medium without Ade, His, Leu, and Trp.

Overexpression DoSPX4 in Nicotiana tabacum

To analyze the function of DoSPX4 in plants, we obtained transgenic N. tabacum overexpression DoSPX4. As shown in Figure 7, through −Pi treatment, the length of leaves and root in OE-DoSPX4 N. tabacum plants decreased significantly, and the root–shoot ratio was 1.97 times than that of the empty vector (EV) group.

FIGURE 7

Figure 7. Transgene analysis. (A) Phenotypic difference of OE-DoSPX4 transgenic N. tabacum. (B) Identification of positive transgenic N. tabacum lines by PCR (M represent DL2000 DNA marker, a represent EMPTY VECTOR, b represent OE-DoSPX4). (C) The leaf number of OE-DoSPX4 transgenic N. tabacum. (D) The root length of OE-DoSPX4 transgenic N. tabacum. (E) The root–shoot ratio of OE-DoSPX4 transgenic N. tabacum. Each sample included at least three replicates, and the values are the means ± SDs. *Indicates that the difference is significant, **Indicates that the difference is very significant.

To understand the function of DoSPXs on Pi absorption and utilization, the available Pi contents of transgenic N. tabacum under + P and −P culture were measured (Figure 7). Under the −P stress, P content in the aboveground part of OE-DoSPX4 N. tabacum was significantly higher than that in the EV group. These results indicated that the DoSPX4 overexpression promoted the Pi accumulation in the shoot.

The expression levels of Pi response genes (NtPHRs and NtPTs) in OE-DoSPX4 N. tabacum were also detected (Figure 8). Under + P, the expression levels of NtPHR1 and NtPHR2 were significantly lower in OE-DoSPX4 N. tabacum than those in the control group. Under the −P condition, the expression levels of NtPHR1 and NtPHR2 were significantly up-regulated except the NtPHR2 in the aboveground of OE-DoSPX4 N. tabacum. NtPT1 and NtPT2 were basically induced in OE-DoSPX4 N. tabacum, especially under the −Pi stress. These results suggested that DoSPX4 may play a negative regulatory role in the expression of NtPHR1 and NtPHR2, and then affect the process of Pi transport from N. tabacum roots to shoot.

FIGURE 8

Figure 8. Determination of indices in transgenic N. tabacum. (A) The available Pi concentration in shoot in OE-DoSPX4 N. tabacum. (B) The available Pi concentration in root in OE-DoSPX4 N. tabacum. (C) The expression analysis of NtPHR1/2 and NtPT1/2 in shoot in OE-DoSPX4 N. tabacum. (D) The expression analysis of NtPHR1/2 and NtPT1/2 in root in OE-DoSPX4 N. tabacum. Each sample included at least three replicates, and the values are the means ± SDs. *Indicates that the difference is significant, **Indicates that the difference is very significant.

Discussion

The organic Pi that plants can absorb in the environment cannot satisfy the need of plants (Grennan, 2008). Medicinal plants have also evolved complex mechanisms to adapt to the −Pi responses (Liu L. et al., 2016, 2018; Wang et al., 2020). The proteins, containing the SPX domain, participate in the molecular regulatory network of plant response to Pi stress (Liu N. et al., 2018). Through the genome analysis of D. officinale, we obtained seven DoSPX proteins. DoSPX proteins have strong homology in P. equestris and D. officinale, suggesting that the regulatory network of plants responding to −Pi stress may be conserved. Through phylogenetic analysis and conservative motif analysis, we found that seven DoSPXs belong to 4 subfamilies (SPX, SPX-MFS, SPX-EXS, and SPX-RING) and have homology with O. sativa and A. thaliana.

The cis-acting elements of DoSPXs promoter region include MSA-like elements involved in cell cycle regulation and CAT-box elements involved in meristem development, which indicates that DoSPXs may be involved in the growth of D. officinale. In addition, the promoter region contains ERF, TGACG-motif, TC-rich, and ARE elements, which can respond to ethylene, plant hormones, plant defense and stress (Feng et al., 2020; Huo et al., 2021; Zhang et al., 2022). These results indicated that DoSPXs may involve in abiotic stress such as invasion and drought.

Tissue special expression showed that DoSPXs had different expression patterns, which indicates that DoSPXs may play different functions. The expression level of DoSPX1 and DoSPX3 was induced in the roots and shoot of D. officinale under −P and reached the peak at day 5, which indicated that DoSPX1 and DoSPX3 may participate in the early response of D. officinale to −P, this was similar to the expression pattern of AtSPX1/2/3 (Duan et al., 2008; Puga et al., 2014) and OsSPX1/2/3/5/6 (Wang et al., 2009; Shi et al., 2014).

Under the −P, DoSPX4 had a different expression pattern compared to DoSPX1 and DoSPX3. According to homology comparison analysis, DoSPX4 had high homology with OsSPX4 (Lv et al., 2014) and AtSPX4 (Duan et al., 2008). In A. thaliana, the localization of AtSPX4 is different from AtSPX1 and AtSPX3, which shows the uniqueness of the function of AtSPX4 in A. thaliana Pi response. The subcellular localization analysis indicates that DoSPX4 is located on the membrane, which is consistent with AtSPX4 and OsSPX4. These results imply that DoSPX4 may have similar functions to AtSPX4 and OsSPX4, but this still needs a lot of experimental to prove.

It has been reported that SPX can interact with MYB-CC transcription factor PHR and then affect the downstream PSI genes (Lv et al., 2014). InsPs can promote the interaction between SPX and the MYB-CC domain of PHR (Duan et al., 2008). The C-terminal of DoPHR2 contains the MYB-CC domain. At Yeast two-hybrid test showed that DoSPX4 interacted with the C-terminal of DoPHR2. The results indicated that DoSPX4 worked by DoPHR2 in −P. However, the recognition of SPX protein conserved domain by InsPs and the regulation of DoPHR2 at the protein level need an in-depth study.

The DoSPX4 was overexpressed in N. tabacum, which can observe the changes in the root system, Pi content, and Pi transporter expression levels. In OE-DoSPX4 N. tabacum, the root–shoot ratio increased, which was conducive to the absorption of Pi from the environment. the qRT-PCR analysis found that the expression of NtPHR1/2 and NtPT1/2 in OE-DoSPX4 transgenic N. tabacum shoot and root increased. The effective Pi content decreased in the root, while the effective Pi content increased in the aboveground part. It is speculated that DoSPX4 is involved in the induction of NtPT1/2 by NtPHR1/2. NtPT1/2 is a high-affinity phosphate transporter responsible for the long-distance transport of Pi in plants. The high expression of NtPT1/2 promotes the transport of Pi from root to aboveground part in vivo, which increases the Pi content of aboveground parts. The absorption and utilization efficiency of Pi in N. tabacum was improved (Figure 9).

FIGURE 9

Figure 9. Possible DoSPX4 impact mechanism. DoSPX4 promoted the expression of NtPHR1/2 in roots, induced the expression of NtPT1/2 and high-affinity phosphate transporter in roots and shoot, and promoted the transportation of Pi from roots to stems in transgenic N. tabacum in low phosphorus.

Conclusion

In this study, seven SPX family proteins were identified from D. officinale. The qRT-PCR analysis showed the SPX family had different expression patterns. The DoSPX4 was located in the cell membrane and had the ability to interact with DoPHR2. The heterologous expression of N. tabacum showed that DoSPX4 could activate NtPHR1/2, increase the expression of NtPT1/2 in roots and stems, and promote the transport of Pi from roots to shoot. These results provide an experimental basis for further study on the adaptation mechanism of D. officinale to low phosphorus.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Author Contributions

HF organized and designed the experiment. LL and HX conducted the experiment and completed the manuscript writing. JS and HS analyzed the data and put forward valuable suggestions to XS and LT in the process of manuscript modification. All authors read and approved the manuscript.

Funding

This work was supported by the Anhui University Natural Science Foundation (KJ2019A0181 and K1942003), the Hefei Natural Science Foundation (2022036), and the Anhui Natural Science Foundation (1908085MC59).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

Thanks to the authors for their contributions to the experiment, and thanks to the foundation for supporting the project.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.943788/full#supplementary-material

Footnotes

1. ^ http://weizhong-lab.ucsd.edu/cdhit_suite/cgi-bin/index.cgi
2. ^ http://smart.embl-heidelberg.de/
3. ^ http://pfam.xfam.org/
4. ^ http://web.expasy.org/protparam/
5. ^ https://itol.embl.de/
6. ^ http://bioinformatics.psb.ugent.be/webtools/plantcare/html/
7. ^ https://www.dna.affrc.go.jp/PLACE/?action=newplace
8. ^ https://www.ncbi.nlm.nih.gov/tools/primer-blast/

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