Dynamic changes of stylo biomass and root growth in response to Pi-starvation.
In order to detect the dynamic alternations of stylo growth under +Pi (Pi-sufficiency) and -Pi (Pi-deficiency) treatments, dry weight, root total length, surface area and volume were measured at 0, 7, 10, 15, 20 day time points. Results showed that Pi-limitation stress significantly affected stylo growth (Fig. 1). Under low Pi availability condition, stylo shoot dry weight was significantly inhibited that grew more evident, reflected by 20.3% ~ 65.9% decreases after 7 ~ 20 days of -Pi treatment, being compared with stylo plants grown in +Pi condition (Fig. 1b). Nevertheless, root growth exhibited a significant increase, reflected by 40.3% and 31.2% increases in dry weight after 10 and 15 days of Pi-starvation, respectively (Fig. 1a, b; Additional file 2: Fig. S2). Consistent with this result, low P level led to significantly increases of total root length, root surface area and root volume at 10, 15 day, but not at other time points (Fig. 1c, d, e). Furthermore, the increased ratio of total root length and root surface area were the highest after 15 days low P treatment (Additional file 1: Fig. S1). Taking together, the stylo seedlings were more intense responses to Pi-deficient stress at 15 day than 10 day, as reflected by more decreases in shoot dry weight, more increases in total root length and root surface area (Fig. 1; Additional file 1: Fig. S1; Additional file 2: Fig. S2).
Changes of physiological and biochemical indicators under Pi-deprivation of stylo roots
Along with more altering in root morphology after 15 days low P treatment, the alternations of physiology and biochemistry were analyzed. Results showed that total P concentration of stylo was significantly decreased under low Pi availability stress at 15 day (Fig. 2a), on the contrary, the markedly increase of root APase activity was observed (Fig. 2b). In addition, we performed the antioxidant capacity measurement, it revealed that root antioxidant capacity was elevated significantly under low P level at 15 day, the increases were reflected by higher T-AOC and PAL activity, increased accumulations of secondary metabolites total phenol and flavonoid (Fig 1c, d, e, f).
Overview of metabolome analysis at two P levels in stylo roots
An LC-MS/MS analysis was performed on the stylo roots after exposed in low P condition for 15 days to evaluate stylo metabolite responses. Results displayed that a total of 708 metabolites were identified between two P levels (Additional file 4: Table S2). To simplify complex data and identify patterns in all metabolites, a principal component analysis (PCA) was performed with 708 metabolites (Fig. 3a). PCA indicated that principal component one (PC1) nicely defined the difference between +Pi (triangles) and -Pi (circles) plant material, and represented about 80.15% of the variation, while the intragroup three repeat samples of +Pi and -Pi treatments were similar to each other on the PC1 (Fig. 3a). According to the criteria of differentially accumulated metabolites (DAMs), namely, -Pi/+Pi ≥ 2 or ≤ 0.5, and VIP ≥1, it was found that 452 metabolites were unchanged, 256 DAMs significantly responded to Pi-limitation, including 136 up-regulated and 120 down-regulated metabolites (Fig. 3b). Clustered heatmaps provided an overview of the normalized values (Z score) of these 256 DAMs (Fig. 3c). In the heatmap, metabolites were obviously clustered in two branches of down cluster and up cluster, the root samples were also clustered into +Pi and -Pi treatment branches, the high variation suggested that the obvious alteration of DAMs responded to Pi-starvation.
A reproducible metabolic response to Pi-inadequacy in stylo roots
All identified 256 DAMs were classified into 14 categories, including phenylpropanoids (27.7%), amino acid (13.7%), nucleotide (10.2%), organic acid (7.8%), sugars (5.1%), and the other 9 groups with a proportion of 35.5% (Fig. 4; Additional file 5: Table S3). Thereinto, most of the phenylpropanoids (71.8%) showed a significant increase, and 76.9% sugars were inhibited in responding to Pi-limitation. In addition, a little larger proportion of amino acids (57.14%) and organic acids (57.14%) were down-regulated and up-regulated, respectively. These above results indicated that the metabolic responses of stylo roots to Pi-limitation stress were mainly focusing on the phenylpropanoid metabolism and following the primary metabolism including sugar, organic acid, nucleotide, and amino acid.
Declined accumulation of sugar abundance responding to Pi starvation in stylo roots
Totally, 13 DAGs associated with sugar metabolism were detectable in stylo roots (Fig. 4, 5; Additional file 6: Table S4). It was found that only three sugar metabolites were increased accumulation in low P stylo roots, containing glucose, inositol, and D-gluconate. Whereas, 10 sugars were decreased, six of which were phosphorylated sugars and reduced extremely low, especially 2-deoxyribose-1P (-41.47 fold), ribulose-5P (-30.75 fold), D-mannose-6P (-26.64 fold), and fructose-1P (-16.58 fold, Fig. 5; Additional file 6: Table S4). Glucose and D-gluconate were up-stream substrate of glycolysis metabolism, the increased accumulation of which might closely be associated with the reduced of down-stream phosphorylated sugars (Fig. 6). In brief, the accumulation of glucose was remarkably enhanced and some of the phosphorylated sugars sharply dwindled in Pi-deficient stylo roots.
Changes of organic acid and its derivatives to adapt to low P supply in stylo roots
Total 20 DAGs of organic acid and its derivatives were identified in stylo roots with 10 up-regulated and 10 down-regulated in response to Pi-limitation (Fig. 4, 5; Additional file 6: Table S4). It was found that Pi-starvation led to the metabolite increases by 2.07-fold (4-guanidinobutyric acid) to 8.97-fold (2-phosphoglycerate). By contrast, down-regulated organic acids showed a decrease between 2.13-fold (kynurenic acid) and 56.83-fold (argininosuccinate, Fig. 5; Additional file 6: Table S4). 2-phosphoglycerate the strongest accumulated was a component of the glycerophospholipids of biomembranes. Following salicylic acid (4.45-fold) and anthranilate O-Hex-O-Hex (4.82-fold) as aromatic organic acids were the next highest accumulated in low P stylo roots. As for the weakened organic acids, which were blocked the source of synthesis or secreted out of the roots, argininosuccinate decreased immensely (56.83-fold). 2-isopropylmalate, α-aminoadipate as well as malate were also weakened 9.23, 7.76 and 3.17-fold in turn (Fig. 5, 6; Additional file 6: Table S4).
Alternation of amino acid and its derivatives in response to P deficiency in stylo roots
Altogether, the concentrations of 15 amino acids increased and 20 amino acids reduced in the stylo roots during Pi-limitation (Fig. 4, 5; Additional file 6: Table S4). The positive response amino acids (AAs) included 33.33% (5/15) proteinogenic amino acids, namely L-tryptophan, L-isoleucine, L-leucine, L-asparagine and L-methionine. The concentration of L-citrulline, a precursor of arginine, displayed the highest accumulation (5.13-fold) in roots (Fig. 5; Additional file 6: Table S4). By contrast, the negative response AAs and derivatives had only one proteinogenic amino acid (L-glutamic acid) decreased by 2.49-fold. Moreover, the top four AAs decreasing immensely were N-acetylphenylalanine, D-pipecolinic acid, L-pyroglutamic acid, and saccharopine, and which were weakened by 8.30, 5.09, 4.95 as well as 4.83-fold, respectively (Fig. 5, 6; Additional file 6: Table S4).
Characteristics of nucleotide and its derivatives response to P deficiency in stylo roots
Total 26 DAEs of nucleotide and its derivates were detected in stylo roots responding to Pi-inadequacy (Fig. 4, 5; Additional file 6: Table S4). Among the 13 up-regulated metabolites, 10 metabolites (76.92%, 10/13) were identified as base or nucleosides without phosphonic groups. Thereinto, the levels of dIMP (2’-Deoxyinosine-5’-monophosphate) and 2’-deoxyadenosine were the stronger elevated by 258.02 and 8.97-fold, respectively. In contrast, 69.23% (9/13) of the declined 13 compounds pertained to nucleotides or its derivates with phosphonic groups. A lot of reduced compounds displayed the higher diminishing ratio, and the top three decreased compounds were CMP (cytidine-5’-monophosphate), UDP-glucose (uridine 5’-diphospho-D-glucose) as well as UMP (uridine 5’-monnophosphate), reduced by 50.55, 19.02, 14.93-fold in turn. In the schematic diagram of the metabolic biosynthesis pathway (Fig. 6), some up-regulated nucleic acid metabolites without phosphate were the precursors or substances to biosynthesize the corresponding of downstream down-regulated nucleosides metabolites with phosphate, for instance, uridine (2.92-fold) to UMP (-14.93-fold) and UDP-glucose (-19.02-fold), 2’-dexoyadenosine (8.97-fold) to ADP (adenosine 5’-diphosphate, -3.00-fold), Inosine (2.19-fold) to IMP (-8.07-fold).
Changes of metabolite abundance in the phenylpropane metabolic pathway
Altogether, 71 secondary DMEs related to phenylpropane metabolic pathway were obtained in stylo roots under Pi-deficiency stress, and 72.60% of those measured metabolites accumulated (Fig. 7; Additional file 7: Table S5). The 71 secondary DAMs were divided into six subgroups, namely flavone (28.17%, 20/71), phenylpropanoid (26.76%,19/71), flavonoid (16.90%, 12/71), flavonol (16.90%, 12/71), isoflavone (7.04%, 5/71), proanthocyanidin and anthocyanin with the least ratio 4.23% (3/71). For every subgroup, the up-regulated metabolites were more than down-regulated metabolites, and even there were none of down-regulated metabolites in subgroup isoflavone, proanthocyanidin, anthocyanin (Fig. 7, 8; Additional file 7: Table S5).
Responses of flavone and flavonol metabolites to Pi-starvation in stylo roots
Total 20 flavones significantly varying between -Pi and +Pi treatments were identified in the roots of stylo (Fig. 7, 8; Additional file 7: Table S5). Thereinto, the concentrations of 17 metabolites were markedly higher in Pi-deficiency. Nonetheless, there were only three flavones decreased, namely quercetin 3-O-galactoside, baicalein, as well as trifolin. Of course, the levels of 12 flavonol metabolites were significantly different under -Pi or +Pi treatments (Fig. 7, 8; Additional file 7: Table S5), but the accumulations of which varied slightly from -3.94-fold (quercetin 3-O-rutinoside) to 3.54-fold (quercetin O-hexosyl-O-malonylhexoside). Taking together, the two subgroup 24 increased accumulation metabolites, 14 compounds (58.33%, 14/24) were stored as glycosylation derivative (Fig. 8; Additional file 7: Table S5).
The biosynthesis metabolic pathway of flavone and flavonol was clearly presented (Fig. 9). Most majority of glycosylation derivatives were significantly accumulated, in all of the precursors of which, only kaempferol (2.46-fold) and quercetin (2.89-fold) were affected significantly, and downstream of kaempferol and quercetin had more than three glycosylation derivatives significantly changed, such as quercetin 3-O-galactoside, quercetin 3-O-rutinoside, and quercetin O-acetylhexoside, being the downstream glycosylation derivatives of quercetin, were markedly diminished under Pi-limitation, which might make some contributions to the accumulation of kaempferol and quercetin.
To assess the expressions of synthetases for metabolites kaempferol and quercetin at transcriptional level, the transcriptional responses to Pi-starvation of SgF3H, SgFLS and SgF3’H genes in stylo roots were detected (Fig. 9, 10). It was found that the genes of Sg F3H-1 (4.24-fold), SgFLS-1 (11.09-fold) and SgF3’H-1 (9.23-fold) were significantly up-regulated expression under Pi-limitation treatment in stylo roots, compared with Pi-sufficient condition (Fig. 10).
Analysis of phenylpropanoid, flavonoid and isoflavone metabolites in Pi-starvation stylo roots
Based on the concentration ratio of Pi-limitation stress relative to Pi-sufficient control, 17 phenylpropanoid DAMs were identified in stylo roots, with more than a half compounds (63.16%) increased (Fig. 8; Additional file 7: Table S5). The 17 metabolites were increased by 2.03-fold (4-methylumbelliferyl phenylphosphonate) ~ 12.68-fold (caftaric acid), reduced by 2.03-fold (chrysin) ~ 6.14-fold (1-O-b-D-glucopyranosyl sinapate) in low P level stylo root tissues.
Among flavonoid metabolites, there were 8 metabolites annotated and displayed in flavonoid biosynthesis pathway, the most of flavonoid levels were enhanced with dihydromyricetin concentration increased the highest 6.11-fold (Fig. 8, 9; Additional file 7: Table S5). 5 flavonoid metabolites were identified decreasing in stylo roots under Pi-limitation stress, the highest reducing metabolite was hyperoside by 69.97-fold, which might contribute to the accumulation of flavonoids, especially dihydromyricetin. Accordingly, the genes related biosynthesis of dihydromyricetin (SgF3’H-1, SgF3H-1), were discovered significantly up-regulated expression through qRT-PCR analysis (Fig. 9, 10).
In isoflavonoid biosynthesis pathway, 5 metabolites were measured accumulation in stylo roots under Pi-deficiency condition (Fig. 8, 9; Additional file 7: Table S5). The most changing three metabolites were daidzein (5.97-fold), daidzein 7-O-glucoside (one of the glycoside derivatives of daidzein, 6.93-fold), rotenone (one of the downstream derivatives of daidzein, 9.39-fold). Above all, daidzein was identified as playing a more important role in this metabolic pathway. At the same time, the two genes related to daidzein and rotenone metabolism were analyzed at transcriptional level, three SgHID genes were revealed markedly up-regulated expression by 2.12-fold (SgHID-1), 16.55-fold (SgHID-2), as well as 2.69-fold (SgHID-3), two SgUGT genes were detected significantly up-regulated expression by 6.13-fold (SgUGT-1), 8.13-fold (SgUGT2), that seemed to be strongly associated with the significant increases of daidzein and rotenone in stylo roots at low P levels (Fig. 9, 10).