Strigolactone-mediated Mechanism Of Phyllostachys Edulis Root Response To Phosphate Stress

Moso bamboo(Phyllostachys edulis)is the most valuable bamboo species in China and widely distributed in Southern China where the soil is generally phosphate(Pi)deficient,resulting in low shoot product.While excessive application of phosphorus fertilizer led to high Pi stress in the bamboo forest,resulting in the degradation of bamboo forest.A novel plant hormone,strigolactone(SL),plays a key role in root growth and development under Pi stress.However,its regulation mechanism of root development has not been systematically reported,and SL-mediated mechanism in response to Pi stress is still unclear.This study analyzed the molecular mechanism of SL-mediated root response to Pi stress in moso bamboo by integrating physiological,biochemical,microstructural and molecular results.Main results are as follows:1.The aboveground biomass reached the highest under 100 μM Pi for 4 weeks.Compared with 100 μM Pi,1 μM Pi treatment significantly increased primary root length and promoted root hair growth,but decreased lateral root density.While 1000 μM Pi treatment significantly decreased primary root length and inhibited root hair growth,but increased lateral root density.After 4 weeks of treatment at 1,100,1000 μM as low Pi,sufficient Pi and high Pi levels,there were significant difference in primary root length between low Pi and high Pi at day 4,and significant differences among three Pi levels at day14.There were significant differences in lateral root density between low Pi and high Pi at day 8,and significant difference among three Pi levels at day 14.Under low Pi,root elongation is significantly promoted by prolongation of root apical meristem,and root branching is significantly reduced by reduction of lateral root primordium(LRP)number.Under high Pi,root elongation is inhibited by the shortening of root apical meristem,while root branching is promoted by increased LRP number.2.Analysis of differentially expressed genes(DEGs)among different Pi treatments in the same period shows that DEGs in LRP zone are significantly enriched in the pathway of SL synthesis and cell response to phosphorus starvation.DEGs in primary root tips are enriched in cellular response to phosphorus starvation and divalent inorganic anion homeostasis.Analysis of DEGs among different periods at the same Pi level indicates that DEGs in LRP zone are significantly enriched in plant hormone signal transduction,phenylpropane synthesis and photosynthesis,and DEGs in primary root tips are significantly enriched in other polysaccharide degradation pathways,plant hormone signal transduction and photosynthesis.Combined with Weighted correlation network analysis,SL pathway is the main pathway under Pi stress,and Pi-starvation response genes and SL synthesis and signaling genes have highly similar expression patterns.These results indicates that there is a strong interaction between the genes responding to Pi starvation and the genes related to SL synthesis and signal transduction in bamboo roots.3.Twenty-three genes related to phosphorus starvation,strigolactone,auxin,gibberellin,ethylene,abscisic acid and root growth were screened out from the genes significantly enriched in LRP zone and primary root tip region.Analysis of q RT-PCR were consistent with the expression trend.In LRP zone,SPX1,SPX6(phosphorus starvation responsive genes),Os I＿08463(phosphorus transport gene),ABCB1,NCS1(auxin transport-related genes)and MAX1,D27,CCD7,D10 a,D10b,D3 a,D3b(SL biosynthesis and signal genes)were upregulated gradually under low Pi,and reached the highest expression level at336h;D14a/b(SL receptor),while ERD15(negatively regulates ABA response),and ESR1(related to cell proliferation)were significantly down-regulated after 96 h of low Pi.There was no significant difference of DEGs between high/low Pi and sufficient/low Pi.In primary root tip region,CML16(related to root growth)and ERF4(ethylene-responsive gene)were significantly down-regulated at 24 h,and CCD7 and D10 b were significantly up-regulated at 336 h under sufficient and low Pi.SAUR11(auxin-responsive gene),GID1(GA receptor),and ERF5(ethylene-responsive gene)were significantly down-regulated at 48 h,ERF071(ethylene-responsive gene)was significantly up-regulated at 96 h,and ASPGB1(related to root growth)was significantly down-regulated gradually under high Pi.4.During 14 days,GR24 induced significant phosphorus starvation response,including gradual decrease of primary root length and lateral root density with the increase of GR24 concentration.1μM GR24 decreased primary root length and lateral root density significantly through delaying root growth and reducing LRP formation,and lateral root density reached the same level as that under low P.With the increase of TIS108 concentration,primary root length decreased and lateral root density increased gradually.1μM TIS108 significantly reduced primary root length by decreasing root apical meristem length,but increased the lateral root density,which was similar to that under high Pi.TIS108 promoted more pericycle cells to form LRP and promoted root growth process.These results indicate that SL participates in the growth and development of bamboo root and plays an important role in primary root elongation and lateral root formation.5.Two kinds of SL in the root exudates of moso bamboo were significantly increased under low Pi.GR24 significantly increased and TIS108 decreased the root-shoot ratio under low Pi,but GR24 and TIS108 had no significant effect on root-shoot ratio under sufficient or high Pi,suggesting that SL affects the distribution of dry matter to tissues depending on Pi concentration.TIS108 reduced PAE significantly only under low Pi,indicating that SL can promote PAE of moso bamboo under low Pi.Application of TIS108 under low Pi resulted in deformation and swelling of primary root tip cells,indicating that SL plays an important role in maintaining cell morphology,especially under low Pi.Primary root length and root apical meristem length were significantly reduced by GR24 under both low and sufficient Pi,but not under high Pi.TIS108 significantly reduced primary root length and root apical meristem length,with the largest reduction under low Pi,indicating that SL affects root elongation depending on Pi concentration.TIS108 increased lateral root density significantly by promoting more pericycle cells to form LRP under low and sufficient Pi,but not under high Pi,suggesting that SL inhibits lateral root formation depending on Pi concentration.6.In primary root tip region,GR24 inhibited and TIS108 increased the expression of CML16 and ERF4 within 24h under low Pi;GR24 inhibited the expression of ERF071 and ERF5,and promoted the expression of ASPGB1 and SL-biosynthesis genes under high Pi.TIS108 inhibited the expression of ASPGB1 throughout the experiment and increased the expression of ERF5 at 48 h,but had no effect on ERF071.Therefore,SL may affect root elongation by regulating the expression of CML16,ERF4,SPX1,SPX6 and Os I＿08463 under low Pi,while the absence of SL under high Pi may release the inhibition of ERF071 and ERF5,as well as the activation of ASPGB1,thus affecting root elongation.In LRP zone,GR24 promoted the expression of Os I＿08463 and ABCB1,and slightly decreased the expression of ESR1 in 96 h under low Pi.GR24 significantly increased the expression of all the Pi starvation responsive genes and SL genes except CCD7 at 96 h or 192 h under high Pi,and inhibited the expression of ABCB1 and ESR1 throughout the experiment.Under low Pi,TIS108 inhibited the expression of NCS1 at 24 h and SPX1,SPX6,SL genes at 96h;promoted the expression of ESR1 at 96 h,and ERD15 and ABCB1 at 192 h.Under high Pi,TIS108 significantly promoted the expression of ABCB1,NCS1,ESR1 and ERD15 at 192 h.Therefore,SL may reduce lateral root formation by promoting Os I＿08463,SPX1,SPX6,ABCB1,NCS1 and inhibiting the expression of ESR1 and ERD15 under low Pi,while the absence of SL under high Pi may release the activation of Os I＿08463,SPX1,SPX6,ABCB1 and NCS1,as well as the inhibition of ESR1 and ERD15,thus promoting lateral root formation.In summary,this study analyzed the morphological changes and physiological characteristics of bamboo roots under different Pi levels,resolved the microstructure characteristics of bamboo roots under Pi-deficiency and Pi-excess condition,and clarified the key pathway of bamboo primary root tip region and lateral root primordium zone in response to Pi stress.The key candidate genes were screened from biological processes such as SL-biosynthesis and-signaling and cell response to phosphorus starvation.SL content increases significantly and is involved with the regulation of primary root elongation and lateral root formation under low Pi.In this process,SL can also regulate the homeostasis and signaling pathways of other plant hormones such as auxin,ethylene and abscisic acid.