Metabolic alterations provide insights into Stylosanthes roots responding to phosphorus defciency

Jiajia Luo Hainan University https://orcid.org/0000-0002-8488-1911 Yunxi Liu Hainan University Huikai Zhang Hainan University Jinpeng Wang Hainan University Zhijian Chen Hainan University Lijuan Luo Hainan University Guodao Liu Hainan University Pandao Liu (  [email protected] )

Research article

Keywords: Metabolome, Stylosanthes, Phosphorus defciency, Root, favonoid

Posted Date: August 19th, 2019

DOI: https://doi.org/10.21203/rs.2.13121/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Version of Record: A version of this preprint was published on February 22nd, 2020. See the published version at https://doi.org/10.1186/s12870-020-2283-z.

Page 1/35 Abstract

Background Low phosphorus (P) availability is a major constraint on the growth of plants, especially in acid soils. Stylosanthes (stylo) is a pioneer tropical legume with several adaptive strategies to cope with P defciency, but its metabolic reprogramming during P limitation remain poorly understood. Results To shed light on the acclimation of stylo roots to low P stress, morphophysiological and metabolic response were investigated in this study. After 15 days of low P treatment, shoot dry weight and total P concentration were markedly hampered, whereas root growth, root APase activity, root antioxidant activity were markedly enhanced. Corresponding investigation of metabolic profling showed that a total of 256 metabolites with differential accumulation in response to P defciency were identifed in stylo roots, mainly including sugars, organic acids, amino acids, nucleotides, phenylpropanoids and their derivatives. P defciency leads to signifcant reduction in the levels of phosphorylated sugars and nucleotides, indicates that the known strategies of P scavenging from P-containing metabolites are observed. The metabolisms of organic acids and amino acids were also remodeled by P limitation, which suggests that P-defcient stylo roots redistribute their carbohydrates in responding P deprivation, by releasing organic acids into the rhizosphere to mobilize phosphate from P complexes and employing amino acids as alternative carbon resource for energy metabolism. In phenylpropanoid metabolism pathway, the increased accumulation favonoids, as well as up-regulated expression of involved genes SgF3H, SgF3’H, SgFLS, might contribute to enhance the antioxidant activity of stylo roots, especially kaempferol, quercetin, dihydromyricetin. The enhanced accumulation isofavonoids with differential expression of related genes (SgHID and SgUGT) supported the opinion of isofavonoids secreted to function with rhizosphere microbes in responding to P defciency condition, especially daidzein and rotenone. Conclusions This study provides valuable insights generated from stylo roots into the various adaptation responses to Pi-starvation, identifed candidate genes and metabolites will make some contributions to detect potential target region for future developing Pi-efciency breeding research.

Background

Phosphorus (P) is an essential macronutrient element for plants that participates in structural components, substance and energy metabolisms, signal transduction cascades. Soluble inorganic phosphate (Pi) is the major form of P that plants can be directly uptake from soil [1]. Agriculture faces the challenge of low Pi availability, especially in acid soils, which limits the growth and yield of crops, due to the complexes that free Pi forms with soil inorganic minerals and organic compounds [2]. Farmers have maximized crop yields by applicating large amounts of phosphate fertilizer, but the minable global P resources are non-renewable and fnite, geographically unevenly distributed, and agricultural Pi runoff is a primary factor of the lake and marine estuary eutrophication, toxic cyanobacteria blooms [3]. Therefore, a more comprehensive understanding the mechanisms of plant adaptation to low P stress is imperative for the sustainable development of agriculture, as well as the reduction of environmental damage caused by Pi fertilizer application [4, 5].

Page 2/35 Plant roots are the vital organ, absorbing nutrients from soil, it is also sensitive to soil environment. In long term adaptation procession, plant roots have evolved a multifaceted set of strategies within species dependent limits to adapt P limitation stress to thrive in nature, such as remodeling root morphology and architecture [6], secreting organic anions and favonoids [7, 8], scavenging and recycling internal and extracellular P [9, 10]. In the aspect of P capture, plant roots seem to change root architectural and structural traits, the architectural traits associated with P exploration include shallower root growth angle, greater dispersion of lateral roots, more basal root whorls, the structural traits associated with P acquisition include enhancing the growth of lateral roots, producing longer and denser hairs, more adventitious roots [11]. In addition, the secretion of organic acids and phosphatases by plant roots also make great contributions to P acquisition [12,13].

The molecular responses of gene, protein, metabolite to Pi-limitation are three more vital factors. But transcript abundances of various genes are not always indicative of protein accumulation and provide no information about either the subcellular location of gene products or posttranslational modifcations [13]. Furthermore, recent a lot of studies are emphasizing the importance of protein expression and activity in response to Pi-limitation stress [14-17]. However, there is limited information on the mechanism of metabolite alternation on the adaptation of Pi-limitation stress by plants. In detecting of primary metabolites, metabolic profling studies under P-limitation are available for plant species, including barley (Hordeum vulgare), Arabidopsis thaliana, and maize (Zea mays), and it is found that disaccharides, trisaccharides, amino acids, and organic acids are highly accumulated, P-containing metabolites are decreased [18-20], which indicates four points associated with P acquisition and utilization: (1) the accumulated saccharides are supposed to act as Pi-starvation signal to induce morphological, biochemical, as well as gene expression changes. (2) An increase in amino acids might be the result of protein degradation and a suppressed protein biosynthesis during Pi-limitation stress. (3) The enhanced organic acids might be secreted into the rhizosphere to liberate Pi from P complexes. (4) Under Pi- limitation conditions, plant internal P-containing metabolites are usable resources of P, which will be scavenged and recycled Pi to mitigate Pi-starvation stress [6]. As for secondary metabolites, favonoids, functioning in growth, development, reproduction, played diverse roles in stress responses in plants [21, 22]. Researches have reported that the concentrations of phenylpropanoid, favonoid, favonoid glycoside, glucosinolate metabolites increased during Pi-starvation in Arabidopsis and white lupin (Lupinus albus) roots [19, 6]. when exposed to Pi-defciency, it was found that plant roots had an increased reactive oxygen species (ROS) production, and the increased secondary metabolite favonoids with antioxidant activity might make contributions to scavenge ROS and protect the root from oxidative stress [23, 24]. When favonoids are released into the rhizosphere by roots, which not only prevent microbial degradation of the exuded organic acids but also directly mobilize P in soil [8, 25]. Furthermore, researches had illustrated that several genes regulating the synthesis of favonoids played pivotal roles in plant acclimatization to environmental stresses. Such as favanone 3-hydroxylase (F3H) and favonoid 3’- hydroxylase (F3’H) are considered to respond to ultraviolet-b (UV-B) radiation, drought, salt, chilling stress in plants [22, 26-29], 2-hydroxyisofavanone dehydratase (HID) is found to be involved in the interactions with pathogens [30, 31], uridine diphosphate glycosyltransferase (UGT) play crucial roles in the rhizobia-

Page 3/35 mediated nodulation in leguminous plants [32], favonol synthase (FLS) is proposed to up regulated by exogenous abscisic acid (ABA), salicylic acid (SA) and sodium chloride (NaCl) in tartary buckwheat (Fagopyrum tataricum) [33]. However, few researches have been reported the gene resources involved in favonoid metabolic pathway responding to low P stress, especially the down-stream genes directly regulating the synthesis of someone specifc favonoid compound.

Stylo (Stylosanthes spp.) is a critical legume forage native to the tropical and subtropical areas [34]. Given its acid soil-adapted nature, it provides an ideal candidate to investigate the molecular and biochemical mechanisms of response to Pi-limitation stress in plants [35]. A set of previous researches were mainly focused on the underlining mechanisms of stylo adaptation to low-P stress, for instance, three stylo pureple acid phosphatases (SgPAPs), SgPAP7, SgPAP10, SgPAP26 have been characterized to be participated in extracellular deoxy-ribouncleotide triphosphate (dNTP) utilization [36]. Besides, another PAP from stylo, SgPAP23 function in exogenous phytate-P utilization [37]. In physiological response aspects, earlier investigations uncovered that the increase of secreted citrate and malic acid were observed from stylo roots at low P levels [35, 38]. However, a metabolomics profling in P-defcient stylo roots, covering a wide range of metabolites was missing. In the current study, we performed a comprehensive and detailed overview of the metabolome changes associated with the adaptation of Pi- limitation environment in stylo roots by untargeted LC-MS/MS method, tightly linking our data with physiological measurements and qRT-PCR analyses. Thus, our investigation rendered insights into metabolic changes of stylo roots under Pi-defciency condition, and that might interpret a fraction of plant adaptation Pi-limitation stress, which laid the theoretical foundation for further understanding the metabolic mechanism of plant responding to Pi-deprivation and made contributions to future breeding towards the species with adapting to Pi-defcit ability improvement.

Results

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-sufciency) and -Pi (Pi- defciency) 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 signifcantly affected stylo growth (Fig. 1). Under low Pi availability condition, stylo shoot dry weight was signifcantly inhibited that grew more evident, refected 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 signifcant increase, refected by 40.3% and 31.2% increases in dry weight after 10 and 15 days of Pi-starvation, respectively (Fig. 1a, b; Additional fle 2: Fig. S2). Consistent with this result, low P level led to signifcantly 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 fle 1: Fig. S1). Taking together, the stylo seedlings were more intense responses to Pi-defcient stress at 15 day than 10 day, as refected by more decreases in shoot dry weight,

Page 4/35 more increases in total root length and root surface area (Fig. 1; Additional fle 1: Fig. S1; Additional fle 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 signifcantly 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 signifcantly under low P level at 15 day, the increases were refected by higher T-AOC and PAL activity, increased accumulations of secondary metabolites total phenol and favonoid (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 identifed between two P levels (Additional fle 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 defned 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 signifcantly 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 identifed 256 DAMs were classifed 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 fle 5: Table S3). Thereinto, most of the phenylpropanoids (71.8%) showed a signifcant 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

Page 5/35 Totally, 13 DAGs associated with sugar metabolism were detectable in stylo roots (Fig. 4, 5; Additional fle 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 fle 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-defcient 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 identifed in stylo roots with 10 up-regulated and 10 down-regulated in response to Pi-limitation (Fig. 4, 5; Additional fle 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 fle 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 fle 6: Table S4).

Alternation of amino acid and its derivatives in response to P defciency 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 fle 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 fle 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 fle 6: Table S4).

Characteristics of nucleotide and its derivatives response to P defciency in stylo roots

Total 26 DAEs of nucleotide and its derivates were detected in stylo roots responding to Pi-inadequacy (Fig. 4, 5; Additional fle 6: Table S4). Among the 13 up-regulated metabolites, 10 metabolites (76.92%, 10/13) were identifed 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

Page 6/35 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-defciency stress, and 72.60% of those measured metabolites accumulated (Fig. 7; Additional fle 7: Table S5). The 71 secondary DAMs were divided into six subgroups, namely favone (28.17%, 20/71), phenylpropanoid (26.76%,19/71), favonoid (16.90%, 12/71), favonol (16.90%, 12/71), isofavone (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 isofavone, proanthocyanidin, anthocyanin (Fig. 7, 8; Additional fle 7: Table S5).

Responses of favone and favonol metabolites to Pi-starvation in stylo roots

Total 20 favones signifcantly varying between -Pi and +Pi treatments were identifed in the roots of stylo (Fig. 7, 8; Additional fle 7: Table S5). Thereinto, the concentrations of 17 metabolites were markedly higher in Pi-defciency. Nonetheless, there were only three favones decreased, namely quercetin 3-O- galactoside, baicalein, as well as trifolin. Of course, the levels of 12 favonol metabolites were signifcantly different under -Pi or +Pi treatments (Fig. 7, 8; Additional fle 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 fle 7: Table S5).

The biosynthesis metabolic pathway of favone and favonol was clearly presented (Fig. 9). Most majority of glycosylation derivatives were signifcantly accumulated, in all of the precursors of which, only kaempferol (2.46-fold) and quercetin (2.89-fold) were affected signifcantly, and downstream of kaempferol and quercetin had more than three glycosylation derivatives signifcantly 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.

Page 7/35 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 signifcantly up-regulated expression under Pi-limitation treatment in stylo roots, compared with Pi-sufcient condition (Fig. 10).

Analysis of phenylpropanoid, favonoid and isofavone metabolites in Pi-starvation stylo roots

Based on the concentration ratio of Pi-limitation stress relative to Pi-sufcient control, 17 phenylpropanoid DAMs were identifed in stylo roots, with more than a half compounds (63.16%) increased (Fig. 8; Additional fle 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 favonoid metabolites, there were 8 metabolites annotated and displayed in favonoid biosynthesis pathway, the most of favonoid levels were enhanced with dihydromyricetin concentration increased the highest 6.11-fold (Fig. 8, 9; Additional fle 7: Table S5). 5 favonoid metabolites were identifed 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 favonoids, especially dihydromyricetin. Accordingly, the genes related biosynthesis of dihydromyricetin (SgF3’H-1, SgF3H-1), were discovered signifcantly up-regulated expression through qRT-PCR analysis (Fig. 9, 10).

In isofavonoid biosynthesis pathway, 5 metabolites were measured accumulation in stylo roots under Pi- defciency condition (Fig. 8, 9; Additional fle 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 identifed 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 signifcantly up-regulated expression by 6.13-fold (SgUGT- 1), 8.13-fold (SgUGT2), that seemed to be strongly associated with the signifcant increases of daidzein and rotenone in stylo roots at low P levels (Fig. 9, 10).

Discussion

Low Pi availability in soil impedes the growth of plants, meanwhile, applying numerous P fertilizers is not an ideal solution with low utilization efciency and being bad for environment [39-41]. In worldwide, sustainable agriculture development, based on deeply understanding the mechanisms by which plants responding to low P soil conditions, is badly needed [42]. Stylosanthes is considered as one of the most important tropical and subtropical non-grain legume forage, can thrive in acid soil with Pi-limitation [43]. And so far, there is not global profling the mechanisms responding to Pi-defciency in stylo with omics technology. In this study, the responses of stylo roots to Pi-starvation were detected by metabolome Page 8/35 analyses combining with physiological and biochemical determination, which resulted in more information on adaptive strategies to limited Pi availability condition in stylo for further investigation.

Response of stylo root phenotype, physiology and biochemistry on P defciency

Plant roots directly touch with soil environment, when the growth soil condition alters, roots are the frst one of sensors to be affected. In previous researches, the response of root morphology on low P level had been widely reported in recent years [6, 44, 45]. When plants suffered from low Pi availability stress, in order to capture more P nutrient, the carbohydrates were altered allocation to root biomass, which affected root morphology, refected by enhancing the growth of taproots, lateral roots, root hairs [11, 46], and even producing cluster roots in Proteaceae plants [47]. Consistent with that, the root dry weight, total root length, root surface area and volume of TF0291 stylo were also signifcantly increased after 10, 15 days of low P stress, and the roots were augmented more at the time point of 15 day than 10 day (Fig. 1). Therefore, the roots of stylo seedling after 15 days Pi-defciency stress were selected for further detection. In our study, total P concentration and root APase activity of stylo roots were markedly changed at 15 day of low P level treatment. It indicated that the internal P resource of stylo roots was affected by the external environment of P supply. Subsequently, the physiological and biochemical responses were analyzed, results showed that low P supply led to increase of T-AOC and PAL activity, contents of total phenol and favonoid (Fig. 2), these secondary metabolites mediated by PAL enzyme might make contributions to the increased antioxidant capacity to protect stylo roots from the oxidative damage induced by low P stress. P defciency induced the overproduction of ROS, led to oxidative damage, which had been reported in roots of Brassica napus [48], Oryza sativa [49], Phaseolus vulgaris [50]. However, the changes of stylo root phenotype and APase activity roughly refected the response of P capture, similarity, a few sporadic antioxidative indicators only suggested that metabolites in phenylpropane metabolic pathway might participate in resisting oxidative damage under low level condition. Therefore, high throughput metabolomic technology was applied to detail the possible P absorption and utilization, low P protection strategy in stylo roots.

Decrease of phosphorylated sugars and nucleotides to optimize internal Pi utilization

Plants alter the internal phosphorylated metabolites for scavenging and recycling Pi to adapt to Pi- deprivation, that is well documented [10, 13, 51]. Phospholipids, phosphorylated sugars, and nucleic acids are the typically organic P compounds containing usable Pi resource for plant cells. In our study, sugars declined dramatically in responses to Pi-defciency, especially 6 phosphorylated sugars (Fig. 5, 6; Additional fle 6: Table S4), at the same time, some phosphorylated sugars and the corresponding degradation residues were the opposite response to Pi-limitation. Such as, D-gluconate-6P was reduced by 2.92-fold, but D-gluconate was increased by 2.06-fold (Fig. 5). Furthermore, there were only three enhanced accumulation sugars without P, and the level of D(+)-Glucose (Glc) was the most increased by 10.09-fold. In previous study, Glc has been proposed to act as transcription signal call for gene involved in cell division and respiration in Arabidopsis roots under P-starvation [52]. Based on the results, the conclusion was strongly supported that phosphorylated sugars containing phosphoesters might be

Page 9/35 decomposed to salvage Pi for essential cellular functions in response to Pi-starvation [53, 54]. In addition, when plants subjected to P-starvation, plant glycolysis metabolism possess the bypass reaction to maintain normal function, such as relying on inorganic pyrophosphate (PPi) dependent phosphorylation function instead of Pi-dependent phosphorylation step, the phosphorylated sugars generating in PPi- dependent glycolysis metabolized faster than Pi-dependent glycolysis under P-limitation, maybe the other possible reason explaining the sharply dwindled levels of phosphorylated sugars in stylo roots [6, 55].

The nucleotides containing phosphate groups are the largest organic P pool in plant cells, especially RNA, representing 47% of the organic P [41]. RNA pool of plants constantly adjusts in accordance with the growing conditions, for example, as the leaves of Eucalyptus globulus development, RNA levels increase to accommodate the rapid synthesis of proteins, at full expansion, RNA levels decrease by up to 75% [56]. Under Pi-limitation conditions, the degradation of RNA is well documented. The nucleic acid concentration in plant cells declined sharply under low P stress, being a direct consequence of inhibiting synthesis or promoting degradation of phosphorylated nucleic acid to redistribution P source, which was reported in white lupin [57], soybean (Glycine max) [58] and Arabidopsis [54]. In the present study, 10 metabolites classifed as nucleosides, bases and derivatives without phosphate exhibiting elevated levels were observed in stylo roots under Pi-limitation stress, and in stark contrast with that, 9 metabolites identifed as nucleotides or its derivates possessing phosphate groups had levels dropped substantially (Fig. 5, 6; Additional fle 6: Table S4). Thereinto, the increased phosphorylated metabolites without phosphate groups were the direct degradation products of phosphorylated metabolites, such as uridine (2.92-fold) for UMP (-14.93-fold) and UDP-D-glucose (-19.02-fold), 2’-dexoyadenosine (8.97-fold) for ADP (-3.00-fold), Inosine (2.19-fold) for IMP (-8.07-fold). Those results suggested that remobilization of P in nucleotides was another attempt to maintain cellular Pi homeostasis presented in stylo roots during P- defciency.

Remodeling of organic acids and amino acids metabolism to adjust P capture and self-metabolism

Organic acids are released into the rhizosphere to mobilizes Pi bound in metal complexes, enhancing Pi availability to adapt to Pi-defcient conditions [59, 60]. The quantity and type of organic acids by roots highly depended on the plant species, genotypes and growth conditions and so on. According to published literature, under low P stress, the organic acids released into rhizosphere was mainly citrate and malate by white lupin cluster roots [2], piscidic acid by pigeonpea (Cajanus cajan) roots [61], malate and oxalate by soybean roots [62], citrate by Stylosanthes roots [38]. In the study, malate participating in the tricarboxylic acid (TCA) cycle was observed markedly reduced accumulation in stylo roots, as well as its derivative 2-Isopropylmalate. Argininosuccinate, the derivative of succinate in TCA cycle containing three carboxyls, was the most decreased organic acid for -56.83-fold. Legumes are efcient secretors of organic acids, secretion could be a signifcant contributor to the reduced levels of organic acids in stylo roots [60]. 2-phosphoglycerate and salicylic acid all containing one carboxyl were the top two organic acids enhanced accumulation in stylo roots (Fig. 5, 6; Additional fle 6: Table S4). Consistent with our results, salicylic acid was also found increased in Arabidopsis roots [19]. Even though Li et al. [38] reported a bulk of citrate secreted by stylo roots, but there were less pronounced changes of citrate in our

Page 10/35 TF0291 stylo roots, the difference might be due to the various genotype of stylo material used in two research work. Above all, the changes of organic acid accumulation hinted that organic acid secretion might also be a strategy for TF0291 stylo to adapt to Pi-limitation stress.

A substantial accumulation of free AAs was observed in the roots of severely P defcient plants, including tobacco (Nicotiana tabacum) [63], barley [18], rice (Oryza sativa) [64], Arabidopsis [19], tea (Camellia sinensis) [65]. Furthermore, under P-impoverished condition, the genes involved in protein degradation were up-regulated expression at transcriptional level, and the genes related to protein synthesis were down-regulated expression in both Arabidopsis and common bean species [66, 67]. Therefore, the increased concentrations of AAs are likely associated with enhanced degradation of protein and inhibition of protein synthesis. In our investigation, 15 AAs and derivatives were elevated by 2.20-fold (L- Methionine) to 5.13-fold (L-citrulline) in stylo roots under Pi-starvation, including 5 proteinogenic AAs, namely L-tryptophan, L-isoleucine, L-leucine, L-asparagine and L-methionine (Fig. 4, 5; Additional fle 6: Table S4). By contrast, 20 AAs were observed suppressed accumulation in stylo roots. This might result from some AAs being utilized as an alternative carbon resource for energy production in P-defcient plants [18]. It was typical that proteinogenic amino acid, L-glutamic acid was decreased (Fig. 5,6). Similarly, the reduction of glutamic acid level was also observed in barley and soybean plants caused by P-defcient [18, 58]. Glutamic acid is catalyzed deamination by glutamate dehydrogenase to produce a- ketoglutarate, which participates in the TCA cycle for energy production under Pi-starvation situation [18], it was line with that the increased activities of glutamate dehydrogenase in Arabidopsis root and the enhanced protein abundance of glutamate dehydrogenase in maize root were observed under P-defcient stress [54, 68]. These data indicate that amino acid metabolism is dramatically remodeled by degrading or inhibiting the synthesis of protein, and the free AAs might be employed as alternative carbon resource for energy metabolism to maintain the function of plant cells in stylo roots under low P supply condition.

Accumulation of phenylpropanoid metabolites to enhance low P adaptation

Phenylpropanoid biosynthesis linked to the shikimate pathway is a vital secondary metabolic pathway, which contains numerous branch metabolic pathways, engendering diverse aromatic secondary metabolites, collectively termed (poly) phenolics, critically important for plant growth and environmental adaptation [69]. Phenylpropanoid metabolites were widely documented participating in diverse biological activities in P-defcient plants [8, 25]. Thereinto, favonoids were reported acting as antioxidants to prevent injury caused by free radicals through scavenging of ROS, activating of antioxidant enzymes, inhibiting oxidases, increasing in antioxidant properties of low molecular antioxidants [70-72]. Previous studies .- demonstrated that P defciency induced the accumulation of ROS in plant roots, including H2O2, O2 , which caused oxidative stress to plants [48, 49]. In our study, physiological analysis showed that the T- AOC activity, the contents of total phenols and favonoids in stylo roots were increased under Pi- impoverished condition (Fig. 2). In addition, metabolic profling showed that 71 phenylpropanoid metabolites were affected by Pi-starvation in stylo roots, including 51 increased accumulation and most of the glycosylated metabolites, which was divided into down-stream four branch metabolic pathways (Fig. 7, 8, 9). Thereinto, favone and favonol biosynthesis pathway possessed 32 metabolite members

Page 11/35 with 22 metabolites being modifed by glycosylating (Fig. 8; Additional fle 7: Table S5). Kaempferol and quercetin were enhanced signifcantly, and downstream of which had more than three glycosylation derivatives signifcant alternation (Fig. 9). Consistently, three genes SgF3H, SgFLS as well as SgF3’H involved in kaempferol and quercetin synthesis were found to up-regulated expression by Pi-starvation (Fig. 10). In the favonoid synthesis metabolic pathway, the concentration of dihydromyricetin was most highly increased by 6.11-fold. Furthermore, the transcriptional levels of SgF3’H and SgF3H-1 related to dihydromyricetin metabolism were confrmed highly enhanced expression (Fig. 10). Previously experiments had demonstrated that kaempferol, quercetin, myricetin had the antioxidant properties, the most effective radical scavengers were quercetin and myricetin [73, 74]. When the favonoids were modifed by glycosylation, the antioxidant activity would be reduced [75-77], so numerous glycosylation favonoids increased accumulation in stylo roots might act as stable storage forms for the corresponding aglycons. The results herein suggest that favone and favonol, favonoid metabolism pathways are dramatically modulated by Pi-starvation, which might associate with enhancing the antioxidant activity in stylo roots, especially kaempferol, quercetin, dihydromyricetin. In isofavonoid biosynthesis pathway, rotenone, daidzein 7-O-glucoside and daidzein were the top three metabolites enhanced accumulation by Pi-limitation (Fig. 8, 9). Simultaneously, the regulating genes of which were found up-regulated expression at transcriptional level, such as SgHID, SgUGT (Fig. 10). Consistent with our results, Malusà et al. [78] reported that the content of daidzein considered as a signal molecule in the chemical trafcking soil microorganisms was higher in low P bean roots, and which was considered playing a role in plant-soil- microbe interactions and rhizosphere modifcation [79]. Moreover, daidzein has been studied extensively for anti-cancer activity by inhibiting the growth of cancer cell [80, 81]. In agriculture, rotenone was used as insecticide, the mechanism was that rotenone being the inhibitor of mitochondrial complex I, induced apoptosis through enhancing mitochondrial ROS production [82, 83]. Plus, literatures reported that phenolic compounds, mainly isofavonoids, were excreted by P-impoverished white lupin roots to repel microbial degradation of the P-solubilizing agents and mobilize inorganic P [8, 25]. Those evidences strongly indicate that stylo roots adjust isofavonoid metabolism to adapt to Pi-starvation, and isofavonoids might play a role for functioning with rhizosphere microbes, daidzein and rotenone are worth more noting.

Conclusions

In summary, the physiological, biochemical, metabonomic, qRT-PCR studies have elucidated the mechanisms underlying stylo root adaptations to Pi-defciency. Stylo plants might enhance the growth of root and root APase activity to acquire more Pi, increase the antioxidative activity to protect from low P damage. In global metabolomics analysis, phosphorylated sugars and nucleosides might be salvaged Pi to maintain cellular Pi homeostasis. In low P supply stylo roots, organic acids might be released into the rhizosphere to liberate Pi from metal complexes and AAs might be employed as an alternative carbon resource for energy metabolism, the increased favonoids and related genes might contribute to enhance the antioxidant activity, isofavonoids with differential expression of related genes hint which functioning with rhizosphere microbes in vitro. Taken together, this study provides a more comprehensive theoretical

Page 12/35 basis for understanding the diverse responses to Pi-starvation and the identifed key genes will make contributions to genetic engineering for promoting the ability of adaptation P-insufcient condition in plants.

Methods

Plant growth and treatment

In the current study, stylo genotype ‘TF0291’ (Stylosanthes guianensis) was used in this study, which was originally introduced to Chinese Academy of Tropical Agriculture Sciences (CATAS) from International Center for Tropical Agriculture (CIAT) in Colombia. The seeds of stylo TF0291 were provided by the Tropical Pasture Research Center, Institute of Tropical Crop Genetic Resources, CATAS, Hainan Province, China.

Plant growth conditions were following the experiment described by Liu et al. [36] with some appropriate modifcations. In brief, stylo seeds were pre-germinated at 28°C for 3 days on wet flter paper before planting. The uniform germinating seeds were then selected and planted on supporting medium, pre- cultured in blue plastic pots (25L in volume) flled with modifd Magnavaca’s nutrient solution according -1 -1 -1 to Famoso et al. [84], containing KCl (1 mmol L ), CaCl2 (1 mmol L ), Fe-EDTA (77 μmol L ), MgSO4 (200 -1 -1 -1 -1 μmol L ), Mg(NO3)2 (500 μmol L ), NH4NO3 (1.5 mmol L ), MnCl2·4H2O (11.8 μmol L ), ZnSO·7H2O -1 -1 -1 -1 (3.06 μmol L ), CuSO4·5H2O (0.8 μmol L ), H3BO3 (33 μmol L ), Na2MoO·4H2O (1.07 μmol L ), -1 -1 MgCl2·6H2O (155 μmol L ), KH2PO4 (250 μmol L ). The plants were grown in a greenhouse at Hainan University, China (E121°54′, N30°52′) under a natural day-night cycle and environment. After 7 days, the seedlings were transplanted and exposed to modifed Magnavaca’s nutrient solution supplied with 250

(+Pi) or 0 (-Pi) μmol/L KH2PO4. The nutrient solution was replaced every fve days. To measure dry weight, total P content, total root length, root surface area, and root volume, triplicate or quadruplicate biological of root and shoot materials were collected under both +Pi and -Pi conditions after 0, 7, 10, 15, 20 days of treatments. Roots exposing in P treatments after 15 days were separated, cleaned quickly, frozen in liquid nitrogen and then stored in -80°C ultra-freezer until further physiological measuring, metabolite analysis and RNA isolation.

Assay of root parameters, total P concentration, and other biochemical parameters

The dry biomass of shoot and root samples were weighted after overnight at 65°C until constant weight. For measuring root parameters (root length, surface area, volume), stylo roots spread gently without overlap were scanned at 400 dpi by a scanner (Epson, Japan). Scanned images were used for documenting root parameters by using WinRHIZO program (2009, Regent, Canada). Total P content was assessed in the samples treated by Pi defciency for 15 days, using the phosphorus-molybdate blue color reaction method according to the description of Murphy and Riley [85]. A standard curve generated with

KH2PO4 was used for determining the total P. In addition, the APase activity of stylo roots after 15 days P defciency stress was measured according to previous published methods by Liu et al. [36], APase can

Page 13/35 catalyze ρ-nitrophenyl phosphate to produce blue ρ-nitrophenol, the APase activity was evaluated by detecting the produce rate of ρ-nitrophenol, which had the strongest absorbance at 405 nm.

For evaluating other physiological and biochemical parameters of stylo roots after -Pi stress for 15 days, commercial chemical assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used. Total antioxidant capacity (T-AOC) activity was detected by colorimetry kit (Code: A015) according to the manufacturer’s guide and Qiao et al. [86]. Briefy, T-AOC level was determined by ferric reduced / antioxidant power reaction system, Fe3+ - tripyridine triacridine (TPTZ) is reduced to F2+ - TPTZ by reductive substance, F2+ - TPTZ can be detected by measuring absorbance at 520 nm. The produce rate of F2+ - TPTZ was used to evaluate the T-AOC level. Phenylalanine ammonia lyase (PAL) catalyzed phenylalanine deamination to produce cinnamic acid, to assay the PAL activity, the cinnamic acid produce rate was tested by spectrophotometry, the procedure followed the manufacturer’s guide of kit (Code: A137-1-1). To calculate the T-AOC level and PAL activity, the protein content was determined based on the Coomassie Brilliant Blue method [87]. Finally, under alkaline condition, phenolic substances reduced molybdic acid for blue compounds, the concentration of blue compounds was assessed to calculate total phenols content by kit (Code: A143-1-1). In alkaline nitrite solution, favonoids and aluminum ions reacted to form red complex, and the concentration of red complex was measured to analyze favonoids content by kit (A142-1-1).

Metabolite analysis

The metabolomic approach was conducted at MetWare Biotechnology Limited Company (http://www.metware.cn/, Wuhan, China). All six root samples (three biological repetitions for +Pi and -Pi groups) harvested after 15 days of +Pi and -Pi treatment were used for metabolic analysis. Metabolites were evaluated through untargeted LC-MS/MS technology. Extraction and metabolite analysis were performed as reported by Chen et al. [88]. Briefy, root materials were crushed into fne powder using grinding mill (MM 400, Retsch), 100 mg powder of samples were used for metabolites extraction with 1.0 ml 70% aqueous methanol overnight at 4°C in refrigerator. Then supernatant components were collected and fltered with a 0.22μm micropore flter (SCAA-104, ANPEL, Shanghai, China) after centrifuged for 10 min at 10,000 g before LC-MS/MS analysis.

A 2 μl liquid of each sample was injected into an Ultra Performance Liquid Chromatography (UPLC, Shim- pack UFLC SHIMADZU CBM30A) equipped with a tandem mass spectrometry (MS/MS, Applied Biosystems 6500 Q TRAP). In UPLC, samples were separated with a reverse-phase Waters Acquity UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100mm). The mobile phase contained eluent A (0.04% acetic acid in aqueous solution) and B (0.04% acetic acid in acetonitrile solution) The gradient of separation program was set at 95:5 (A:B, v/v) at 0 min, 5:95 (A:B, v/v) at 11.0 and 12.0 min, 95:5 (A:B, v/v) at 12.1 and 15.0 min. The fow rate and column temperature were maintained at 0.4 ml/min and 40°C, separately. In MS/MS, electrospray ionization (ESI) source operation parameters were set as follows: positive ion mode was used in the instrument; ion source was turbo spray; source temperature was set to 550°C; ion spray voltage was adjusted to 5.5 kV; the ion source gas I, gas II, curtain gas were set at 55, 60,

Page 14/35 25 pounds per square inch, respectively. For triple quadrupole (QQQ) scans, each ion pair was scanned according to the optimized declustering potential and collision energy as multiple reaction monitoring (MRM) experiments.

The qualitative and quantitative analysis of metabolites were conducted according to the previous report [89]. The quantitation of metabolites was performed by matching the secondary spectral information with self-built metware database (MWDB) and public database of metabolite information. The quantitation of metabolites was analyzed through MRM of QQQ MS/MS. In addition, differentially accumulated metabolites (DAMs) were identifed by orthogonal projection to latent structure-discriminant analysis (OPLS-DA), according to the criteria of fold change (-Pi/+Pi) ≥ 2 or ≤ 0.5, and the variable importance in project (VIP) being ≥ 1 [90].

Gene expression pattern analysis by qRT-PCR

Total ribonucleic acids (RNAs) were extracted using the RNA-solve reagent (Omega Biotech, USA) following the manufacturer’s guide. First complementary deoxyribonucleic acid (cDNA) strand synthesis of deoxyribonuclease (DNase) I-treated total RNA (2 μg) was performed by M-MLV reverse transcriptase (Promega, Madison, WI, USA). The quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using SYBR Premix Ex Taq II (Takara, Japan) on the Rotor-Gene 3000 qRT-PCR system (Corbett Research, Australia) with the following protocol: 95 °C for 30 s, 45 cycles at 95 °C for 10 s, followed by 58 °C for 30s, and 72 °C for 30 s. The Stylosanthes elongation factor 1-alpha (SgEF-1α, accession number: JX164254) gene was used as an inner control to normalize gene expression for stylo roots. To reveal possible key secondary metabolites response to Pi-starvation, according to the ribonucleic acid sequencing (RNA-Seq) data (unpublished), the following genes were examined the transcript relative abundance: SgF3’H, SgFLS, SgF3H, SgHID, SgUGT. The primer pair of genes for qRT- PCR were listed in Table S1, and the nucleic acid sequence of gene was deposited into National Center of Biotechnology Information (NCBI) database, the accession numbers of which were also displayed in Additional fle 3: Table S1, and the nucleic acid sequence of genes were submitted to the NCBI database, the accession numbers were also displayed in Additional fle 3: Table S1. Four biological replicates were used in this study, and the relative expression levels were calculated according to Liu et al. [37].

Statistical analysis and visualization

Dry weight, total phosphorus concentration, root parameters, physiological indicators and relative expression of genes were entered into Excel 2016 (Microsoft Corporation, Redmond, WA, USA), one -way anysis of variance (ANOVA) with least signifcant difference (LSD) Duncan and Student’s t-test were performed at 95% confdence interval (CI) and 0.05 level of signifcance in SPSS (Version 19.0, IBM, Chicago, IL, USA) software. Principal component analysis (PCA), volcano diagram, clustered heatmap and circular fgures were carried out by using the ‘prcomp’ function of ‘stats’ package, ‘ggplot’ function of ‘ggplots’ package, ‘heatmap’ function of ‘stats’ package, ‘chordDiagram’ function of ‘circlize’ package in program R ( v 3.5.1), respectively. Before analysis, the raw data was Z score-transformed for normalization in heatmap. The metabolic category fgure was created with Microsoft Excel 2016. Page 15/35 Metabolic pathway maps were constructed according to pathway analysis in the KEGG metabolic database (http://www.kegg.jp/). For the metabolic change fold, untransformed mean values (n = 3) were employed to calculate -Pi/+Pi ratios.

List Of Abbreviations

AAs: Amino acids; ADP: Adenosine 5’-diphosphate; ADP-glucose: adenosine 5’-diphosphate-glucose; ANOVA: Anysis of variance; APase: Acid phosphatase; CATAS: Chinese academy of tropical agriculture sciences; cDNA: complementary deoxyribonucleic acid; CI: Confdence interval; CIAT: International center for tropical agriculture; CMP: Cytidine-5’-monophosphate; dIMP: 2’-Deoxyinosine-5’-monophosphate; DMAs: Differentially accumulated metabolites; DNA: Deoxyribonucleic acid; DNase: Deoxyribonuclease; dNTP: Deoxy-ribouncleotide triphosphate; ESI: Electrospray ionization; F3’H: Flavonoid 3’-hydroxylase; F3H: Flavanone 3-hydroxylase; FLS: favonol synthase; ABA: abscisic acid; Glc: D(+)-Glucose; HID: 2- hydroxyisofavanone dehydratase; ID: Identifcation; LC-MS/MS: Liquid chromatography-mass spectrometry; LSD: Least signifcant difference; MRM: Multiple reaction monitoring; MWDB: Metware database; NCBI: National Center of Biotechnology Information; OPLS-DA: Orthogonal projection to latent structure-discriminant analysis; P: Phosphate; PAL: Phenylalanine ammonia lyase; PC1: Principal component one; PCA: Principal component analysis; PHO2: Ubiquitin-like modifer E3 ligase; PHR1: Phosphate starvation response 1; Pi: Inorganic phosphate; PPi: Inorganic pyrophosphate; QQQ: Triple quadrupole; qRT-PCR: Quantitative real-time polymerase chain reaction; RNA: Ribonucleic acid; ROS: Reactive oxygen species; SA: salicylic acid; NaCl: sodium chloride; SgEF-1α: Elongation factor 1-alpha; SgPAP: Stylosanthes pureple acid phosphatase; SPX: SYG1/PHO81/XPR1; Stylo: Stylosanthes; T-AOC: Total antioxidant capacity; TCA: Tricarboxylic acid; TPTZ: Tripyridine triacridine; UDP-glucose: Uridine 5’- diphospho-D-glucose; UGT: uridine diphosphate glycosyltransferase; UMP: Uridine 5’-monnophosphate; UPLC: Ultra performance liquid chromatography; UV-B: Ultraviolet-b; VIP: Variable importance in project; VPE: Vacuolar Pi efux transporter.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The datasets are included in this article to support the conclusions and the additional fles are also available, the nucleic acid sequence of genes have been submitted to the NCBI database

Page 16/35 (https://www.ncbi.nlm.nih.gov/) via BankIt repository with the sequence identifer displayed in Additional fle 3: Table S1.

Competing of interests

The authors declare no competing interests.

Funding

The research was fnancially supported by the Natural Science Foundation of Hainan Province (318QN265), the National Natural Science Foundation of China (31701492, 31861143013, 31672483), the Modern Agro-industry Technology Research System (CARS-34, CARS-22-Z11), and the Central Public- interest Scientifc Institution Basal Research Fund for CATAS (1630032018004). The authors declare that none of the funding bodies have any role in the design of the study or collection, analysis, interpretation of data and writing the manuscript.

Authors’ contributions

P.L. and L.L conceived and designed the study. J.L., Y.L. and J.W. performed the physiological experiments. J.L. and J.W. carried out the metabolic and qRT-PCR analysis. P.L. and J.W. analyzed the data. J.L. wrote the draft manuscript. P.L., G.L., L.L. and Z.C. provided many critical suggestions to revise the manuscript. All authors read and approved the fnal manuscript.

Acknowledgments

The authors sincerely thank MetWare Biotechnology Limited Company (Wuhan, China) for helping to identify the metabolites.

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Additional Files

Additional fle 1: Fig. S1 Effects of Pi availability on the ratio of root increasing. (a) Total root length. (b)

Root surface area. (c) Root volume. After precultured under +Pi (250 μmol/L K2HPO4) for 7 days,

Seedlings were grown under +Pi or -Pi (0 μmol/L K2HPO4) after 7, 10, 15, 20 days for Pi-starvation treatment. Data are means of six independent replicates with standard error bars. Different lowercase letters indicate signifcant difference among groups (P < 0.05), tested by one-way ANOVA with LSD. Ratio of increased under -Pi (%) = [(-Pi - +Pi)/+Pi] * 100. (DOCX 69.8 kb)

Additional fle 2: Fig. S2 Growth of stylo under +Pi and -Pi conditions for 15 days. Plants were grown in hydroponics for 15 days with 250 μmol/L KH2PO4 (+Pi) or 0 μmol/L KH2PO4 (-Pi). Every bottle contained six plants. (DOCX 318 kb)

Page 23/35 Additional fle 3: Table S1 A list of primers used for qRT-PCR and gene accession numbers in NCBI database. (DOCX 17.3 kb)

Additional fle 4: Table S2 General information of identifed metabolites from metabolomic analysis in stylo roots. (XLSX 71 kb)

Additional fle 5: Table S3 General information about differentially accumulated metabolites (DAMs) from metabolomic analysis in stylo roots. (XLSX 45.6 kb)

Additional fle 6: Table S4 Differentially accumulated metabolites of sugar, organic, amino, nucleotide and its derivatives. (XLSX 18.2 kb)

Additional fle 7: Table S5 Differentially accumulated metabolites associated with phenylpropane metabolic pathway. (XLSX 25.1 kb)

Figures

Page 24/35 Figure 1

Effects of Pi availability on stylo growth at different treatment times. (a) Root phenotype, (b) dry weight of shoot and root, root parameters including (c) total root length, (d) root surface area, (d) root volume of stylo after 0, 7,10,15, 20 days two P levels treatment, +Pi: 250 μmol/L KH2PO4, Pi: no KH2PO4 added. Six biological repeats were contained in each experimental group (n = 6). Error bars indicated standard error. Quartile (box), maximum and minimum (whiskers) and outlying values (circles) were shown. *: signifcantly different between +Pi and Pi treatment in the Student’s t-test (P < 0.05), ns: no signifcant different between +Pi and Pi condition.

Page 25/35 Figure 2

Physiological and biochemical levels in response to Pi-limitation stress in stylo roots. (a) Total P concentration of shoot and root, (b) root APase activity, (c) T-AOC activity, (d) PAL activity, contents of (e) total phenol, and (f) favonoid. Stylo seedlings were precultured in hydroponics for 7 days with 250 μmol/L KH2PO4 and subsequently transferred in +Pi (250 μmol/L KH2PO4) or Pi (no KH2PO4 added) liquid medium for 15 days. Error bars denoted standard errors (SE, n = 3 or 4). Signifcance analysis between +Pi and Pi treatment was performed by Student’s t-test (two-tailed, equal variance): * P ≤ 0.05, ** P ≤ 0.01, the same as below. P: phosphate, APase: acid phosphatase, T-AOC: total antioxidant capacity, PAL: phenylalanine ammonia lyase, DW: dry weight, FW: fresh weight.

Page 26/35 Figure 3

Overview of metabolomics changes of stylo roots under +Pi and Pi conditions. (a) Principal component (PC) scores of metabolic the frst two variances in stylo roots (n = 3). Stylo roots were grown in +Pi (triangles) and Pi (circles) solutions for 15 days. The confdence level in the grey confdence circle is 95%. (b) The volcano diagram of the distribution of metabolites responsive to Pi-limitation in stylo roots. Red circles, green circles and black circles indicated the up regulated, down regulated and the unchanged metabolites, respectively. (c) Clustered heatmap of differentially accumulated metabolites (DAMs) of stylo roots under low P supply. Individual metabolites are represented by rows, and nutritional status are represented by columns. Heatmap visualization of metabolites is based on standardized transformation (Z score) of metabolite concentrations (n = 3).

Page 27/35 Figure 4

Category of DAMs in stylo roots during Pi-limitation stress. Data are shown as the number of metabolites, and only DAMs that are altered signifcantly (VIP ≥ 1.0, Pi/+Pi ≥ 2.0 or ≤ 0.5) are shown. Up-regulated: Pi/+Pi ≥ 2.0; Down-regulated: Pi/+Pi ≤ 0.5. DAMs: differentially accumulated metabolites.

Page 28/35 Figure 5

Repertoire of four categories primary DAMs in stylo roots during Pi-starvation. Data are shown as heatmap for the response ratio of Pi relative to +Pi (n = 3). Response ratios are shown as positive numbers for an increase and negative inverted number for a decrease. Only DAMs are shown in this fgure. In addition, the column of KEGG ID is listed the metabolites identity in KEGG database, and those metabolites must meet requirements of having ID, being annotated into known metabolic pathways in KEGG database and being easy to integrate into the profle of metabolic pathway (Fig. 6) synchronously. "--" in the fgure indicates the ID of metabolites in KEGG database not existing or not being annotated into known metabolic pathways.

Page 29/35 Figure 6

Core metabolism overview of the four groups primary DAMs in stylo roots during Pi-limitation stress. Only metabolites being noted the KEGG ID in Fig. 5 were shown. The schematic diagram was constructed and modifed based on KEGG pathway database (https:// www. Keg.jp/keg/pathway.html). The response ratio for each metabolite was given for Pi/+Pi, and the decreased response ratio number was inverted for a negative value.

Page 30/35 Figure 7

Changes in metabolite abundances related to phenylpropane metabolism in stylo roots under difference P condition. The fgure showed the changes of six subcategories metabolites, the digital scale represented the numbers of corresponding metabolites, the up-regulated or down-regulated metabolites were screened according to the criterion of fold change ≥ 2.0 or ≤ 0.5, and VIP ≥1.0 (n = 3).

Page 31/35 Figure 8

Alterations in secondary metabolite levels due to Pi-limitation in stylo roots. Fold changes of metabolites (Pi/+Pi) were displayed as numbers, and the ratio of Pi/+Pi less than 0.5 was inverted as negative a number (n = 3). The higher metabolite abundances in stylo roots were given by positive ratios (red or purple), the lower abundances metabolites were presented by negative ratios (green or blue). The metabolite ID of KEGG database was listed in the last column, asterisks indicated that the metabolites were glucose derivatives, which could be found in metabolic pathways of KEGG database. Finally, these known source metabolites were extracted and matched into the metabolic pathway (Fig. 9).

Page 32/35 Figure 9

Changes of phenylpropane pathway in stylo roots under Pi-defciency stress. The fgure showed metabolic heatmaps of ffty metabolites, mean values of Pi/+Pi ratio were presented under the corresponding metabolites (n = 3), on the false color scale red and purple indicated increase, blue and green indicated decrease in metabolites. The solid arrow showed direct and dotted arrow represented speculated steps in the pathway. In addition, the more vital genes were marked on the metabolic procedure. The pathways were designed based on KEGG pathways (http://www.kegg.jp/kegg/pathway.html) and researches of Zhang et al. [24], Kc et al. [65].

Page 33/35 Figure 10

Expression profling of candidate genes associated with phenylpropane metabolism in stylo roots under Pi-limitation stress. Twelve genes were selected and analyzed under both +Pi and Pi treatments for 15 days. Transcript expression levels were normalized using the internal control SgEF1α, and relative expression values were calculated based on means of four biological replicates (n = 4), the SE was marked by error bars. Transcripts with statistically signifcant changes in expression were tested by

Page 34/35 Student’s t-test (two-tailed, equal variance): * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and ns represented insignifcant (P ≥ 0.05), the same as below.

Supplementary Files

This is a list of supplementary fles associated with this preprint. Click to download.

supplement1.xlsx supplement2.xlsx supplement3.pdf supplement4.xlsx supplement5.pdf supplement6.xlsx supplement6.pdf

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