Study On The Mechanisms Of Low Phosphorus Stress And OsGRF1 Gene On The Regulation Of Rice Morphological And Physiological Traits

GRF1 belongs to a family of plant specific growth regulators and plays an important role in plant growth and development regulation and stress resistance.The previous research results of our research group showed that low phosphorus stress led to morphological remodeling of rice,including significant reduction of tiller number and significant lengthening of roots.At the same time,low phosphorus stress significantly induced the expression of OsGRF1 gene(Log2FC=2.149,P-value=0.000215).Therefore,in this study,the OsGRF1 gene overexpression line OE and the OsGRF1 gene knockout line KO were constructed using low phosphorus-sensitive rice variety Tongjing 981 as materials.The above rice materials were cultivated under normal phosphorus and low phosphorus conditions.Through morphological measurement,physiological and biochemical measurement,hormone content determination,expression profile sequencing,qRT-PCR detection and yeast one hybrid point-to-point verification,this study clarified the gene expression and hormone regulation mechanisms of low phosphorus stress and OsGRF1 gene on rice morphology and physiology and pinpointed the corresponding gene expression regulation pathways.At the same time,the study elucidated the possible role of OsGRF1 gene in the regulation of rice morphological and physiological traits by low phosphorus stress.The main research results are as follows:1.After identification,both the OsGRF1 overexpression line OE and the OsGRF1 gene knockout line KO were transgenic positive seedlings,and the OsGRF1 gene expression level of the OsGRF1 overexpression line was significantly up-regulated,the OsGRF1 knockout line lost 33 bases(corresponded to 11 amino acids)at OsGRF1 target site,causing OsGRF1 gene function inactivation.Therefore,both the OsGRFl overexpression line OE and the OsGRF1 gene knockout line KO could be used as OsGRF1 gen mutation materials for subsequent research.2.Low phosphorus stress and OsGRF1 overexpression induced the gene expression of NSP2,D27,D17,D10 and MAX1,then promoted the synthesis and signal transduction of strigolactone,led to an increase of the content of strigolactone and inhibited rice tillering;At the same time,low phosphorus stress and OsGRF1 overexpression inhibited the gene expression of LOGLs and NAC2,which promoted tillering and resulted in a significant decrease of the tiller number of rice.On the contrary,OsGRF1 knockout inhibited the gene expression of NSP2,D27,D17,D10 and MAX1,leading to a decrease of the content of strigolactone(the above results were consistent with the measurement of strigolactone content);Simultaneously,OsGRF1 knockout induced the expression of genes which could promote tillering,such as LOGL9,NAC2,MOC1,FON1,RAX2,HD3A,GLN1-2,BZR1 and WOX11,and led to a significant increase of rice tillering.In addition,low phosphorus stress and OsGRF1 overexpression led to a significant decrease of cytokinin content,while OsGRF1 knockout led to a significant increase of cytokinin content.This was also one of the reasons why low phosphorus stress and OsGRF1 overexpression led to a decrease of rice tillers and OsGRF1 knockout led to an increase of rice tillers.The results of the research also indicated that the expression induction of OsGRF1 under low phosphorus stress was correlated with the reduction of rice tiller number caused by low phosphorus.3.Low phosphorus stress induced the expression of positively regulating root growth and development genes PLDZETA1,NPC4,PELPK1,NAC92,MYBS1,MYB30,PSK4,PSK5,P23-1,EL5,SCL3,CEPR1,and inhibited the expression of negatively regulating root growth and development genes CYP20-1,CLE45,BAM3,MAPKK18,GTL1,by which resulted insignificant lengthening of rice roots.Similarly,OsGRF1 overexpression induced the expression of positively regulating root growth and development genes NPC4,PELPK1,NAC92,EL5,SCL3,CEPR2,GRP5,ERF071,and inhibited the expression of negatively regulating root growth and development gene CYP20-1,CLE45,BAM3,MAPKK18,GTL1,IAA20,by which resulted in significant lengthening of overexpression line roots.On the contrary,OsGRF1 knockout induced the expression of negatively regulating root growth and development genes NA C48 and PERK8,and inhibited the expression of positively regulating root growth and development genes LHW,BHLH74,GALT3,BRXL1,RPD1,RAPTOR2,SAB,TORNADO2,by which led to a significant shortening of knockout line roots.Moreover,the expression induction of OsGRF1 under low phosphorus stress was correlated with the lengthening of rice roots caused by low phosphorus.4.Low phosphorus stress induced the expression of negatively regulating stem and leaf development genes GH3.2,GH3.6,GH3.8,ERF113,CYP734A4,CKX5,and inhibited the expression of positively regulating stem and leaf development genes AIPT,BTF3 and D2,by which led to shorter plant height and smaller leaves of rice under low phosphorus.In the same way,OsGRF1 knockout induced the expression of negatively regulating stem and leaf development genes GH3.2,GH3.8,CYP734A,HOX1,GLN1-2,NAC48,and inhibited the expression of positively regulating stem and leaf development genes D2,HPGT3,ARGOS,GA20OX2,D18,BHLH74,ERECTA,TCP 15,KAO,by which led to significant plant height decrease and leaf reduction of knockout lines.On the contrary,OsGRF1 overexpression induced the expression of positively regulating stem and leaf development genes D2,MADS55,FER,CTF7,OSH15,WAK1,WAK2,GSTU17,SKP1A,and inhibited the expression of negatively regulating stem and leaf development genes GH3.8,IAA20,HY5,HOX1,E2FC,leading to an increase of plant height and the enlargement of leaves.In addition,changes in the content of gibberellin,brassinolide,and abscisic acid were also involved in the regulation of low phosphorus stress and OsGRF1 gene on rice plant height and leaf size.Moreover,the expression induction of OsGRF1 under low phosphorus stress may have a certain alleviating effect on the reduction of plant height and leaf size caused by low phosphorus.5.Due to low phosphorus stress and OsGRF1 overexpression inhibited rice tillering,leading to a decrease in the number of rice panicles;On the contrary,due to OsGRF1 knockout promoted rice tillering,which leds to a significant increase in the number of panicles in the knockout line.Low phosphorus stress induced the gene expression of TGW3 and inhibited the gene expression of BG1,leading to decrease of thousand grain weight.OsGRF1 knockout of induced the gene expression of CKX2 and CKX8,and inhibited the gene expression of D.2 and VIL2,causing a decrease of the grain number per panicle and thousand grain weight.On the contrary,OsGRF1 overexpression induced the gene expression of MADS55,D2,ERECTA and VIL2,resulting in the increase of the grain number per panicle and thousand grain weight.This also indicated that the expression induction of OsGRF1 gene under low phosphorus stress was correlated with the decrease in rice panicle number caused by low phosphorus,but there was no significant correlation with the decrease of the grain number per panicle and thousand grain weight caused by low phosphorus.Moreover,the expression induction of OsGRF1 under low phosphorus stress may have a certain alleviating effect on the reduction of thousand grain weight and the grain number per panicle of rice caused by low phosphorus.6.Low phosphorus stress induced the expression of phosphorus transporter genes PHTs,PHO1,PHO3 and G3Pp1 to promote phosphorus absorption and transportation in rice under low phosphorus conditions,and induced the expression of acid phosphatase genes PAPs,IPAPs and PPAs to promote the activation of organic phosphorus in vivo and in vitro and increase the available inorganic phosphorus content,and induced the expression of genes PHR3,SPXs,PAH1,PAH2,DGD2,PLDZETA1,SQD2,NPC1,NPC4,LCAT3,PLA2-Ⅱ,RNLE,NPP to activate the phosphorus starvation signal pathway and phosphorus starvation rescue system,causing membrane lipid remodeling and phosphorus homeostasis maintaining in rice.OsGRF1 overexpression inhibited the gene expression of PHT2-1,which might be related to a significant decrease of total phosphorus content in the aboveground part of overexpressed line.OsGRF1 knockout of induced the gene expression of PHT1-1,PHT1-8 and PHT1-13,and inhibited the gene expression of SABRE,resulting in a significant increase of total phosphorus content in the aboveground part of knockout line.7.Both low phosphorus stress and OsGRF1overexpression significantly reduced the root activity of rice.OsGRF1 knockout resulted in a significant increase of the root activity of rice.Therefore,there was a certain correlation between the expression induction of OsGRF1 gene under low phosphorus stress and the decrease of rice root activity caused by low phosphorus.8.Low phosphorus stress induced the expression of chlorophyll degradation related genes SGR,NAC029,RCCR,CLH2,and inhibited the expression of chlorophyll synthesis related genes CHLG and PTC52,resulting in a significant decrease of chlorophyll content in rice leaves.Similarly,OsGRF1 knockout induced the expression of chlorophyll degradation related genes NAC029 and RCCR,and inhibited the expression of chlorophyll synthesis related genes CHLG,PTC52,PORA,PORB,leading to a significant decrease of chlorophyll content in knockout line.OsGRF1 overexpression induced the expression of chlorophyll synthesis related genes PTC52,leading to a significant increase of chlorophyll content in overexpressed line.Moreover,the expression induction of OsGRF1 gene under low phosphorus stress may have a certain alleviating effect on the decrease in chlorophyll content in rice leaves caused by low phosphorus.9.Low phosphorus stress induced the gene expression of SPC4,PODs,SOD2,MKK5,CATA,CATC,OsGRF1 overexpression also induced the gene expression of PODs,CATB,PNC1,which resulted in the increase of antioxidant enzyme activity in plant under low phosphorus stress and OsGRF1 overexpressed line.Contrarily,OsGRF1 knockout inhibited the gene expression of PODs,PRXIIE-2,FSOD1,FSOD2,leading to a decrease of antioxidant enzyme activity in knockout line Therefore,the expression induction of OsGRF1 gene under low phosphorus stress was correlated with the increase of antioxidant enzyme activity of rice caused by low phosphorus.10.The results of yeast one hybrid point-to-point verification revealed that the upstream promoter region of D17 gene which catalyzed the synthesis of strigolactone,could interact with OsGRF1.