Physiological responses of stylo to Pi starvation
To assess the dynamic alternations of stylo under P-sufficient (+Pi) and P-deficient (-Pi) conditions, a time-course of hydroponic experiment was performed. Results showed that the decreased in the shoot dry weight was observed after 7 d of -Pi treatment, and the differences between -Pi and +Pi became increasing significantly as the treatment time increased (Fig. 1). Although no difference in root dry weight was found under two P treatments for 7 d. Root dry weight was increased by 40.3 and 31.2% after 10 and 15 d of -Pi treatment compared to that in +Pi treatment, respectively (Fig. 1a, b; Additional file 1: Fig. S1). Consistently, the total root length, root surface area and root volume were increased after 10 and 15 d of -Pi treatment (Fig. 1c, d, e). The maximum increased ratios of total root length and root surface area were 44.6 and 56.1% at 15 d of P treatments, respectively (Additional file 1: Fig. S2a, b).
The ratio of root/shoot was significantly increased under Pi starvation. Furthermore, the maximum ratio of root/shoot was reached at 15 d of -Pi treatment, which was 2.6-fold higher than that of +Pi treatment (Additional file 1: Fig. S3). After 15 d of -Pi treatment, although total P content was declined, the activity of root acid phosphatase (APase) and phenylalanine ammonia (PAL) were increased by 209.0% and 100.5%, respectively (Additional file 1: Fig. S4a, b, c). Similarly, root total phenol, flavonoid, and total antioxidant capacity (T-AOC) were also increased by -Pi treatment compared to those of +Pi treatment (Additional file 1: Fig. S4d, e, f). Furthermore, it was found that the internal malate concentration of stylo roots was decreased by 69.5%, but the external malate exudation rate of stylo roots was increased by 44.3% under -Pi treatment (Additional file 1: Fig. S4g, h). These results suggest that a series of morphological and physiological changes occur in stylo in response to Pi starvation.
Characterization of expansin gene family in stylo roots
Expansin protein family, localized to the cell wall, is implicated in regulating plant root growth . In this study, a total of 16 expansin genes were identified in stylo (Additional file 2: Table S1). Phylogenetic analysis showed that 16 stylo expansin members were divided into four subfamilies, including 9 SgEXPA, 1 SgEXLA, 3 SgEXPB and 3 SgEXLB (Additional file 1: Fig. S5). The expression patterns of these genes were further analyzed in roots of stylo with 15 d of P treatments. Results showed that 4 SgEXPAs (SgEXPA1, 2, 4 and 8), SgEXPB2 and SgEXLB1 were up-regulated, whereas SgEXLB3 was down-regulated by Pi starvation. The expressions of the remaining stylo expansin genes were not affected by low P treatment (Fig. 2). These results suggest that some of the stylo expansin genes participate in modifying root growth under low P condition.
Overview of metabolome in stylo roots response to Pi starvation
To evaluate metabolome response to Pi starvation, an LC-MS/MS analysis was performed on stylo roots under two P treatments. A total of 708 metabolites were identified in stylo roots at two P levels (Additional file 3: Table S2). Principal component analysis (PCA) showed that principal component one (PC1) nicely defined the difference between +Pi (triangles) and -Pi (circles) stylo materials, which represented about 80.15% of the variation (Additional file 1: Fig. S6a). The metabolites with the ratio of -Pi/+Pi more than 2 or less than 0.5 and the variable importance in project (VIP) more than 1.0 were considered as differentially accumulated metabolites (DAMs). A total of 256 DAMs were identified in stylo roots at two P treatments, including 136 low P up-regulated metabolites and 120 low P down-regulated metabolites (Fig. 3a), which can be clustered into the up-regulation cluster (color in red) and down-regulation cluster (color in blue), respectively (Additional file 1: Fig. S6b). The root samples were also clustered into +Pi and -Pi treatment branches (Additional file 1: Fig. S6b). All of the identified 256 DAMs were classified into 14 categories, including flavonoids, phenylpropanoids, phenylamides and its derivatives, amino acids and cholines, the numbers of the up-regulated these DAMs were higher than those of the down-regulated DAMs (Fig. 3b). However, the numbers of up-regulated amino acid derivatives and sugars were less than those of the down-regulated (Fig. 3b).
Changes in sugars, cholines, nucleotide and its derivatives in stylo roots response to Pi starvation
Based on the existence of phosphate group in metabolites, sugars, cholines, nucleotide and its derivates can be classified into two groups, namely P-containing or non-P-containing metabolites. For the 13 sugars-related DAMs, the levels of 6 P-containing sugars were declined by-Pi treatment. Among them, 2-deoxyribose 1-phosphate, ribulose-5-phosphate, mannose-6-phosphate and fructose-1-phosphate were decreased by more than 10-fold under -Pi treatment compared to those under +Pi treatment. However, the levels of 3 non-P-containing sugars were significantly increased, including glucose, inositol and gluconic acid (Fig. 4). Furthermore, the levels of 4 non-P-containing cholines were increased, but the levels of P-containing cholines, such as glycerol-3-phosphocholine (GPC) and phosphocholine (PCho), were decreased by 58.43- and 20.96-fold under -Pi treatment compared to those under +Pi treatment, respectively.
In addition, 28 DAMs were identified as nucleotide and its derivates. Among them, the levels of 10 out of 14 non-P-containing nucleotides were increased, while the levels of 11 out of 14 P-containing nucleotides were decreased in stylo roots exposed to low P stress. Nicotinic acid mononucleotide, cytidylic acid, uridine 5’-diphospho-D-glucose, uridine 5’-monophosphate, nicotinic acid adenine dinucleotide and adenosine monophosphate were declined by more than 10-fold in P-deficient stylo roots (Fig. 4).
To detect the response of hydrolases participating in P-containing metabolites catabolism to low P stress, the expressions of 5 stylo purple acid phosphatase (PAP) and 3 ribonuclease (RNS) genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Result showed that the expressions of 4 out of 5 SgPAPs were up-regulated by-Pi treatment, especially for SgPAP10/12/23, and the expressions of 3 out of 4 SgRNSs were enhanced under low Pi stress.
Alterations of amino acid and its derivatives in stylo roots response to P deficiency
Among the identified amino acids, 9 out of 22 were considered as DAMs (Additional file 4: Table S3), 7 out of 9 amino acids-related DAMs were significantly induced under -Pi treatment, whereas glutamate (Glu) and cystine (Cys) were decreased by Pi starvation in stylo roots. Citrulline was the amino acid with the highest level (more than 5-fold) (Fig. 5). In addition, 81 amino acid derivatives were also identified in this study. Among them, 29 amino acid derivatives were DAMs, including 9 up-regulated and 20 down-regulated amino acid derivatives (Additional file 4: Table S3; Fig. 5).
Analysis of flavonoids in stylo roots response to P deficiency
A total of 54 DAMs belonged to flavonoids, including 23 flavonoes, 16 flavonols, 6 flavanones, 4 isoflavanes, 2 flavanols, 2 anthocyains and 1 chalcone. The accumulation of a larger number of flavonoids was increased in stylo roots under low P stress (Additional file 1: Fig. S8).
For the differentially accumulated flavones, the accumulation of 19 out of 23 flavones was significantly increased, including 4 glycoside derivatives of apigenin and 4 glycoside derivatives of chrysoeriol, while the concentrations of the remaining flavones were declined by -Pi treatment (Fig. 6; Fig. 7).
For the differentially accumulated flavonols, the levels of 9 out of 16 flavonols was increased, whereas the levels of the other 7 flavonols were decreased by -Pi stress. Although the levels of kaempferol and quercetin were increased under Pi starvation, their glycoside derivatives displayed different responses to low P stress. Three glycoside derivatives of kaempferol were up-regulated, whereas 5 glycoside derivatives of quercetin were down-regulated by low P stress (Fig. 6; Fig. 7).
For the differentially accumulated flavanones, the abundances of the most flavanones were increased in stylo roots under low P stress, except hesperetin O-hexoside. The liquiritigenin was found to be the strongest up-regulated by 4.97-fold under P deficiency (Fig. 6; Fig. 7). Similarly, the concentrations of 4 isoflavones were increased under -Pi stress. The concentrations of three isoflavones, including rotenone, daidzein and daidzein 7-O-glucoside, were increased by more than 5-fold in low P-deficient roots (Fig. 6).
Subsequently, the expressions of 2-hydroxyisoflavanone dehydratase (SgHID) and uridine diphosphate glycosyltransferase (SgUGT) involved in daidzein and daidzein 7-O-glucoside synthesis, respectively, were further analyzed. qRT-PCR analysis showed that the expressions of 3 out of 5 SgHIDs and 2 out of 3 SgUGTs were up-regulated in stylo roots by low P stress (Fig. 8). In addition, the expressions of flavonoid 3’-hydroxylase (SgF3’H-1), flavonol synthase (SgFLS-1) and flavanone 3-hydroxylase (SgF3H-1) associated with flavonoids synthesis were also detected (Fig. 7). Results showed that the expressions of SgF3’H-1, SgFLS-1 and SgF3H-1 were up-regulated in stylo roots under P deficiency (Fig. 8).
Analysis of phenylpropanoids, phenylamides and its derivatives in stylo roots response to P deficiency
In addition to flavonoids, other differentially accumulated phenylpropanoids were also identified in this study, including 7 phenolic acids and 8 non-phenolic acid phenylpropanoids. For phenolic acids, the concentrations of 5 DAMs were significantly increased, especially for, caftaric acid, which was increased by more than 10-fold under -Pi treatment. For non-phenolic acid phenylpropanoids, the concentrations of 5 DAMs were enhanced, whereas 3 DAMs were decreased in roots exposed to low P treatment. Among the increased DAMs, the relative level of sesamin was increased by more than 10-fold in P-deficient stylo roots.
In addition, fifteen differentially accumulated phenylamides and its derivatives were identified, including 13 up-regulated and 2 down-regulated DAMs (Fig. 9). The relative levels of 3 phenylamides (N-benzoyl-tryptamine, N-feruloyl-cadaverine and N-feruloyl-putrescine) were increased by more than 5-fold in roots under low P stress. As shown in Fig. 10, numerous metabolites relative to phenylpropanoids metabolic pathway were up-regulated by P deficiency. Interestingly, the concentrations of all of the ρ-coumaric acid and four corresponding derivatives were increased in stylo roots in response to Pi starvation.