Physiological responses of stylo under Pi starvation
To assess the dynamic alternations of stylo under P-sufficient (+Pi) and P-deficient (-Pi) conditions, a time-course of hydroponic cultivation experiment was performed. Results showed that the inhibition on the shoot dry weight was observed after 7 d of -Pi treatments, and the difference between -Pi and +Pi became increasing significantly as the treatment time increased (Fig. 1). In contrast, no difference in root dry weight was found between -Pi and +Pi treatments for 7 d. However, root dry weight was increased by 40.3 and 31.2% after 10 and 15 d of -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 starvation (Fig. 1c, d, e). The maximum ratio of total root length and root surface area under -Pi versus +Pi treatments was observed at 15 d of treatments. The ratio of total root length and root surface was increased by 44.6 and 56.1%, respectively (Additional file 1: Fig. S2).
The ratio of root/shoot was also influenced by Pi availability, as reflected by a significant increase in ratio of root/shoot 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). Additionally, after 15 d of -Pi treatment, total P content was declined, whereas the activity of root acid phosphatase (APase) and phenylalanine ammonia (PAL) were increased by 209.0% and 100.5%, respectively. Root total phenol, flavonoid, total antioxidant capacity (T-AOC) were also increased by -Pi treatment compared to 15 d of +Pi treatment (Additional file 1: Fig. S4). These results suggest that a series of morphological and physiological changes are occurred in stylo under Pi starvation.
Characterization of expansin gene family in stylo roots
Expansin protein family, located in the cell wall, is associated with regulating plant root morphology and architecture . A total of 16 stylo 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 out of 9 SgEXPAs, 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 Pi availability (Fig. 2). These results suggest that stylo expansin genes participate in modifying root morphology under low P condition.
Overview of metabolome in stylo roots response to Pi starvation
To evaluate metabolic responses to Pi starvation, an LC-MS/MS analysis was performed on stylo roots at two P treatments for 15 d. A total of 708 metabolites were identified under 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) plant material, which represented about 80.15% of the variation. The intragroups of three biological repeats of +Pi and -Pi treatments were similar to each other on the PC1, suggesting a good reproducibility of the data (Fig. 3a). The metabolites with the ratio of -Pi/+Pi ≥ 2 or ≤ 0.5 and the variable importance in project (VIP) ≥1 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 down-regulated metabolites (Fig. 3b). In the heatmap, the DAMs were clustered in two branches: the down-regulation cluster (color in blue) and up-regulation cluster (color in red). The root samples were also clustered into +Pi and -Pi treatment branches (Additional file 1: Fig. S6). All of the identified 256 DAMs were classified into 14 categories, including flavonoids, phenylpropanoids, phenylamides and its derivatives, amino acids and cholines, the up-regulated numbers of which were higher than that of the down-regulated. However, the numbers of up-regulated amino acid derivatives and sugars were less than that of the down-regulated (Fig. 3c).
Changes of 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 were classified into P-containing and non-P-containing two groups. For the 13 DAMs belonged to sugars, the relative 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 more than 10-fold in the -Pi treatment compared to those of in the +Pi treatment. In contrast, the relative levels of 3 non-P-containing sugars were significantly increased, including glucose, inositol and gluconic acid (Fig. 4). The relative levels of 4 non-P-containing cholines were increased. The levels of P-containing cholines, such as glycerol-3-phosphocholine (GPC) and phosphocholine (PCho), were decreased by 58.43- and 20.96-fold in the -Pi treatment compared to those of in the -Pi treatment, respectively.
In addition, 28 DAMs were identified as nucleotide and its derivates. Among them, 10 out of 14 non-P-containing nucleotides were increased, while 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 more than 10-fold.
To detect the response of hydrolases participating in P-containing metabolites catabolism to low Pi 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. 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), and 7 out of 9 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 induction (more than 5-fold) (Fig. 5). In addition, 81 amino acid derivatives were also identified in metabolome. 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 (Fig. 6a).
For the differentially accumulated flavones, the relative levels of 19 out of 23 flavones were significantly increased, including 4 glycoside derivatives of apigenin and 4 glycoside derivatives of chrysoeriol, while the remaining flavones were declined by -Pi treatment (Fig. 6b; Fig. 7).
For the differentially accumulated flavonols, the relative levels of 9 out of 16 flavonols were increased, whereas the levels of 7 flavonols were decreased under -Pi stress. Although the relative levels of kaempferol and quercetin were increased after 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 (Fig. 6b; Fig. 7).
For the differentially accumulated flavanones, the abundance of most flavanones was increased in stylo roots under low P stress, except hesperetin O-hexoside. The flavanone with the strongest induction was liquiritigenin, which was up to 4.97-fold (Fig. 6b; Fig. 7). Similarly, the concentrations of 4 isoflavones were increased under -Pi stress. Three of them (rotenone, daidzein and daidzein 7-O-glucoside) were increased more than 5-fold (Fig. 6b).
Subsequently, the expressions of stylo 2-hydroxyisoflavanone dehydratase (SgHID) and uridine diphosphate glycosyltransferase (SgUGT) involved in daidzein and daidzein 7-O-glucoside synthesis were further analyzed. Results showed that 3 out of 5 SgHIDs and 2 out of 3 SgUGTs were significantly up regulated by low P stress (Fig. 8). In addition, the expression of stylo flavonoid 3’-hydroxylase (SgF3’H-1), flavonol synthase (SgFLS-1) and flavanone 3-hydroxylase (SgF3H-1) encoding enzymes involved in flavonoids synthesis were detected (Fig. 7). Results showed that the expression of SgF3’H-1, SgFLS-1 and SgF3H-1 were up-regulated under P deficient condition (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 metabolome, including 7 phenolic acids and 8 non-phenolic acid phenylpropanoids. For phenolic acids, the concentrations of 5 DAMs were significantly increased, especial for, caftaric acid, which was increased more than 10-fold under -Pi treatment. For non-phenolic acid phenylpropanoids, the concentrations of 5 DAMs were enhanced, whereas 3 DAMs were decreased. Among the increased DAMs, the relative level of sesamin was increased more than 10-fold.
Fifteen differentially accumulated phenylamides and its derivatives were identified in the metabolome, 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 more than 5-fold. As shown in Fig. 10, numerous metabolites in phenylpropanoids metabolic pathway were up-regulated by P deficiency. Interestingly, all ρ-coumaric acid and four corresponding derivatives were increased in stylo roots in response to Pi starvation. These results indicate that the induction of phenylpropanoids and phenylamides may be the adaptive mechanism of stylo in response to low P stress.