Phosphate Starvation Responsive Protein Phosphatase GmHAD1-2 Regulates Flavonol Biosynthesis And Lateral Root Development In Soybean

Phosphorus(P) is a major constituent of biomolecules in plant cells,and thus is considered as an essential macronutrient for plant growth.Low phosphate(Pi) availability in soils is a major constraint for plant growth.Although complex responses of plants to Pi starvation are well documented,few studies were performed to integrate both global transcriptome and metabolome analyses,and thus shed light on molecular mechanisms underlying metabolite alterations in plants responsive to P deficiency.In this study,it is the first time that global metabolites and transcripts were investigated in soybean(Glycine max)roots subjected to Pi starvation through targeted liquid chromatography mass spectrometry(LC-MS/MS)and RNA-sequencing analyses.Finally,functions of a Pi starvation up-regulated gene,haloacid dehalogenase 1-2(GmHAD1-2)was characterized to be involved in regulating soybean root growth by altering flavonol biosynthesis and auxin accumulation via enzymatic analyses,interacting protein identification and other related molecular techniques.The main results are shown as follows:(1)The RNA-seq technique was used to analyze global changes of gene expression profiles in soybean roots responsive to P deficiency.It was observed that P deficiency led to increases of soybean root growth at the initial P treatments,but decreases after the long-term P treatments.RNA-seq sequencing was conducted in soybean roots treated with two P levels for 12 days.A total of 1,644 differentially expressed genes(DEGs)were identified in soybean roots,including 1,199 up-regulated genes and 445 down-regulated genes by P deficiency.Among them,39 DEGs might be involved in the synthesis of flavonoids and their related metabolites in soybean roots.(2)The LC-MS/MS technique was used to analyze global changes of metabolic profiles in soybean roots responsive to P deficiency.Totally,155 metabolites differentially accumulated in soybean roots at two P levels,including 73 metabolites with increased accumulations and 82 metabolites with reduced accumulations by P deficiency.Among them,43 metabolites with a phosphate group were significantly reduced under low P conditions.Moreover,integration of metabolome and transcriptome analyses revealed that transcripts of a set of DEGs mainly contributed to alterations of metabolic processes related to flavonoids in soybean roots by Pi starvation,including,chalcone synthase(CHS),chalcone reductase(CHR),chalcone isomerase(CHI),flavanoid 3'-hydroxylase(F3'H).(3)A Pi starvation up-regulated gene,GmHAD1-2 was selected for further studies.The enzymatic analysis of GmHAD1-2 showed that its optimal substrates were phosphoethanolamine and phosphorylated serine.The maximum reaction rate(Vmax)was 48.318±0.788 ?mol min-1 mg-1 and the Michaelis constant(Km)was 0.415±0.0138 m M.Expression patterns of GmHAD1-2 were analyzed through GUS staining of PGmHAD1-2:GUS transgenic soybean plants.It was found that GmHAD1-2 was mainly expressed in leaves and roots of soybean.Moreover,the expression levels of GmHAD1-2 was significantly increased in roots,especially in primary root tips,lateral root primordia and tips after 12 d of P deficiency.(4)Functions of GmHAD1-2 were further examined in soybean transgenic lines with GmHAD1-2 overexpression and suppression.It was found that GmHAD1-2 suppression significantly inhibited soybean growth and P content,especially for lateral root growth(e.g.,lateral root length and the largest root width).Furthermore,it was observed that suppressing GmHAD1-2 significantly decreased GUS staining in the lateral root tips of the transgenic hairy roots harboring the auxin responsive DR5:GUS via the soybean hypocotyl injection transformation system,suggesting reduced auxin concentrations in lateral root tips.Subsequently,LC-MS/MS was used to analyze changes of global metabolic profiles in soybean roots between wild type and the transgenic line with GmHAD1-2 suppression.The results showed that suppressing GmHAD1-2 led to differential accumulations of 59 metabolites,including 8 flavonols.The results strongly suggested that GmHAD1-2 might affect soybean lateral root growth by regulating flavonol synthesis and auxin accumulation.(5)The yeast two-hybrid system was conducted to identify the target proteins interacted with GmHAD1-2.A total of eighteen proteins were found to be interacted with GmHAD1-2,including one chalcone reductase(i.e.,Gm CHR1).The interaction between GmHAD1-2 and Gm CHR1 was further verified by yeast two-hybrid,bimolecular fluorescence complementation(Bi FC)and pull down analyses.Furthermore expression patterns of Gm CHR1 were analyzed through q RT-PCR.It was found that Gm CHR1 was mainly expressed in leaves,roots,flowers and pods.Furthermore,expression levels of Gm CHR1 were significantly increased in soybean roots responsive to P deficiency.Meanwhile,Gm CHR1 was found to be localized in the plasma membrane,nucleus and cytoplasm through transient expression analysis in tobacco leaves.Moreover,suppressing Gm CHR1 significantly decreased biomass,total root length and flavonol accumulations in transgenic hair roots,suggesting that Gm CHR1 might affect soybean root growth by regulating flavonol metabolism.In summary,integrated analyses of metabolome and transcriptome revealed complex molecular mechanisms underlying soybean root adaptation to P deficiency by RNA-seq and LC-MS/MS.Subsequently,functional characterization of a Pi starvation responsive gene,GmHAD1-2 suggested that GmHAD1-2 could regulate flavonol biosynthesis and auxin accumulations,and thus influence soybean lateral root growth.Results from the study could provide theoretical basis for further understandings of mechanisms underlying soybean root morphology and architecture with high P efficiency,enrich the Pi signaling network,supply candidate genes and transgenic resources for development of soybean cultivars with high phosphorus efficiency.