Label‑free quantitative proteomic analysis
After protein extraction, protease solution, LC-MS/MS and label-free data analysis, a total of 4329 protein groups (Additional file 2) and 23,598 peptides (Additional file 3) were identified from six DXWR root samples (RLP and RCK with three biological repetitions, respectively). Among these proteins, we designated 3589 proteins that were detected in at least two replicates as identified proteins (Figure 1a-c). In addition, clustering analysis of proteins identified in different samples showed that the similarities between the three biological repetitions of the same treatment were high, and between the different treatments were very low (Figure 1d). Based on this, it was indicated that changes in expression of these target proteins could represent a significant effect of biological treatment on samples. Using these data, we selected proteins with ≥ 1.5-fold changes as an additional standard (refer to [19]) and the volcano pot was drawn by using two factors of protein expression fold changes and p value obtained from t test to show the significant differential protein accumulation between RLP and RCK (Figure 1e). The results showed that 60 protein groups were up-regulated (Table 1) and 15 down-regulated (Table 2). Those results indicated that P deficiency treatment could affect the accumulation of some gene expression products in DXWR.
Functional classification by gene ontology (GO)
To gain insight into the functional roles of the proteins significantly different between the RCK and RLP samples, Gene Ontology (GO) annotation and enrichment was conducted and the results were listed in Additional file 4, schematically represented in three ontologies as molecular function, cellular component, and biological process, as in Figure 2a. Enrichment of biological process involved in metabolic process and cellular process were significantly observed. The most significantly enriched molecular function were catalytic activity and binding. A significant enrichment of cellular compartments involved in cell part, cell, membrane, membrane part and organelle part were identified. From the above description, we can give a conjecture that low-P deficiency could affect cell proliferation, enzyme synthesis as well as the ability of cell or membrane to bind to certain stimulus signals.
Kyoto encyclopedia of genes and genomes (KEGG) pathway mapping
KEGG Pathways analysis were performed on the 75 significantly different expression proteins (SDEPs, 60 up-regulated and 15 down-regulated) identified in this study (Additional file 5). These proteins were involved in 31 metabolic pathways. Four proteins were enriched to the spliceosome pathway, which was one of the top 20 KEGG pathways predicted to be affected by low-P deficiency (Figure 2b), including up-regulated U6 snRNA-associated Sm-like protein 8 (LSm8, LOC_Os05g51650), U1 small nuclear ribonucleoprotein A (U1A, LOC_Os05g06280) and down-regulated splicing factor of arginine/serine-rich (SR, LOC_Os01g06290), pre-mRNA-splicing factor SPF27 (BCAS2, LOC_Os01g16010). Furthermore, two proteins enriched in the pathway of branched-chain amino acids (BCAAs, include valine, leucine and isoleucine) biosynthesis, including up-regulated branched-chain-amino-acid aminotransferase (BCAT, LOC_Os03g01600) and acetolactate synthase small subunit (ALS, LOC_Os11g14950). Two proteins enriched in the pathway of glyoxylate and dicarboxylate metabolism, containing down-regulated ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL, encoded by leucoplast), as well as up-regulated phosphoglycolate phosphatase (PGP, LOC_Os09g08660). In addition, two proteins enriched in amino sugar and nucleotide sugar metabolism, inclusive of up-regulated chitinase (LOC_Os01g49320) and glycosyl hydrolase (LOC_Os01g47070). Two proteins enriched in glutathione pathway, containing up-regulated GST (LOC_Os10g38740 and LOC_Os10g38360). These results indicated that genes involved in these pathways might respond to low-P stress in DXWR.
Protein-protein interactions (PPI) between the identified proteins
Protein is an important component of biological organisms, which do not perform biological function independently, but through the interaction of proteins to regulate physiological and biochemical processes. Therefore, we performed a PPI analysis of the proteins identified by label-free quantitative analysis. As shown (Figure 2c), there was a strong interaction only between a few proteins after screening.
A2X5V0 (Uniprot ID, LOC_Os02g33850, up-regulated) with the function of elongation factor Tu family protein (EF-TU) has 5 mutual proteins, including Q0D840 (LOC_Os07g08840, up-regulated) with function as Thioredoxin, A2YMF8 (LOC_Os07g36490, up-regulated) with the function of RNA recognition, Q5TKF9 (LOC_Os05g51650, up-regulated) with the function of U6 snRNA-associated Sm-like protein LSm8 which involved in RNA splicing, B8AHU1 (LOC_Os02g05880, down-regulated) with function of RNA polymerase which participate in the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates, and B8AC69 (LOC_Os01g16010, down-regulated) with the function of BACS2 protein which is the core component of Prp19 related complex along with involved in important life activities such as splicing of precursor RNA.
Additionally, B8AC69 and A2YMF8 both have 4 mutual proteins involved in splicing of precursor RNA and translation beginnings. What’s more, Q0DKM4 (LOC_Os05g06280, up-regulated) and A0A0P0XSK6 (LOC_Os10g07229, up-regulated) have 3 mutual proteins and encode U1 small nuclear ribonucleoprotein A and dehydrogenase, respectively. Q6EP66 (LOC_Os09g08660, up-regulated) and A6MZ96 (LOC_Os01g06290, down-regulated) have 2 mutual proteins and encode Phosphoglycolate phosphatase and splicing factor, respectively. These results suggested that low-P stress induced reduction of transcription-related genes, and the increase of most RNA splicing related genes along with intensification of the gene expression associated with the elongation during translation.
Comparison of abundance expression patterns between proteins and transcripts verified by qRT-PCR
In present study, among 75 SDEPs, there were 24 proteins with fold changes larger than 1.5 both in proteomics and transcriptome level that were associated with P deficiency treatment in DXWR (Table 3). In addition, we also verified its expression at the transcriptome level by qRT-PCR, which showed consistency in transcriptome data and qRT-PCR results, shown in Figure 3. Among these proteins, 21 up-regulated, 2 down-regulated at both transcriptome and proteome level, and only one had the opposite abundance trend (up-regulated in proteome level but down-regulated in transcriptome level). Gene expression abundance was increased at both two levels including OsPT2 (LOC_Os03g05640), OsPAP10c (LOC_Os12g44020), OsPAP10a (LOC_Os01g56880), OsPHF1 (LOC_Os07g09000), as well as a gene encoding glycerophosphoryl diester phosphodiesterase family protein (GDPD, LOC_Os03g40670), three genes encoding glycosyl hydrolase (LOC_Os07g23850,LOC_Os05g15770 and LOC_Os01g47070) and one gene encoding glucan endo-1,3-beta-glucosidase precursor (LOC_Os07g35560). Furthermore, a gene (LOC_Os10g35500) encoding epoxide hydrolase increased by low-P stress at proteomic level, but the corresponding transcription levels decreased, possibly by increasing the stability of the corresponding mRNA and reducing the number of transcriptions to reduce energy consumption.
Conjoint analysis of proteomic and QTLs related to P deficiency tolerance
As shown in table 4, there are two P-deficiency tolerance related QTLs have been identified in DXWR [17] and 12 QTLs existing in different positions on the chromosome related to P-deficiency stress have been found in Oryza sativa based on the Gramene QTL database. Among genes corresponding to 75 SDEPs identified by the proteome in this study, we located 9 genes among these QTL intervals, as shown in Table 5. In the midst of them, two genes (LOC_Os12g44020, OsPAP10c and LOC_Os04g41970, OsGLU3) have been characterized in previous studies [20, 21], and two of the other seven uncharacterized genes (LOC_Os12g09620 and LOC_Os03g40670) have been detected at both transcriptome and proteome levels. Furthermore, the functional expression characteristics of the remaining five genes (LOC_Os01g57450, LOC_Os03g29240, LOC_Os03g13540, LOC_Os03g29190 and LOC_Os06g07600) have not been reported yet.
The expression pattern in DXWR of key genes responded to low P in cultivated rice detected by qRT-PCR
Based on previous studies on P response mechanism in cultivated rice, the homologous genes of PHR1 (OsPHR2 and OsPHR1), OsPHO2, OsPHO1 as well as its NAT genes play an important role in the low-P response process. Therefore, we examined the expression levels of OsPHR2, OsPHO2 as well as three members of OsPHO1 (OsPHO1;1, OsPHO1;2 and OsPHO1;3) and three NATs correspondently, as shown in Figure 4, and found that OsPHR2 was down-regulated after low-P treatment in DXWR roots, which was contrary to the results of previous studies on cultivated rice [7, 22]. We suspect that maybe OsPHR1 plays a major role during the P starvation signaling pathway in DXWR. For validation, we examined the expression of OsPHR1 in DXWR. But the result was consistent with OsPHR2 and was also down-regulated. Such results may indicate that the transcription levels of OsPHR1 and OsPHR2 are inhibited by low-P signals in DXWR. In addition, OsPHO2 was down-regulated in both DXWR and NP. Among OsPHO1;1, OsPHO1;2 and OsPHO1;3, only OsPHO1;3 expression was up-regulated and the other two were down-regulated in DXWR, but three genes all up-regulated in Nipponbare (NP). In the corresponding NAT, only OsPHO1;2 NAT expression change trend is consistent with OsPHO1;2 and the other two NATs change trend is opposite to OsPHO1;1 and OsPHO1;3 in DXWR, but all three NATs up-regulated in NP. These results indicate that there may be a difference in the mechanism of resistance to low-P in DXWR compared to cultivated rice.