The Mechanism Of OsVPE3-mediated Vessel Differentiation In Root Tip Of Rice Seedlings Under Drought Stress

Rice(Oryza sativa L.)is one of the most principal crops in China.Drought has become the limiting factor in rice production,seriously affecting the reduction of rice yield.In the growth cycle of rice,the seedling stage is the key stage that affects the growth and development of rice.It depends on the root tip of primary root to absorb water and nutrients from the soil to maintain the growth and development of the whole plant.However,drought stress accelerated the differentiation of vessels in the primary root tip of rice seedlings.It affects the length of root tip correspondingly,which affected root growth and plant growth.Therefore,in order to provide theoretical basis for formulating rice drought prevention measures in agricultural production,the differentiation mechanism of root tip ducts of rice seedlings under drought stress was explored.The differentiation of vessels requires two key steps: the generation of programmed cell death(PCD)in vessel molecules and the generation of lignification in the cell wall.It has been confirmed that vacuole processing enzyme(VPE)activate other proteases to produce protein cascade reactions and trigger conduit molecules to induce PCD.Moreover,lignification is associated with lignin synthesis in vessel cell walls.In addition,phenylalanine ammonia lyase(PAL)and cinnamyl alcohol dehydrogenase(CAD)play an important role in lignin synthesis pathway.In this study,different concentrations of polyethylene glycol-6000(PEG)were used to simulate the drought environment.Rice seedlings were treated for 2 d,4 d and 6 d,and then rehydrated for 2 d after 4 d of drought,when the length of primary roots in the rice seedlings was 2 cm.In this experiment,lignin content and cytoplasmic membrane permeability of primary root tips in the rice seedlings were detected.In addition,the lignin staining of primary root tips were observed.Morover,the position of vessels differentiation and seedling growth were examined.To identify the key genes of OsVPE,OsPAL and OsCAD,their transcription levels were determined.Next,the transcription levels and enzyme activity levels of key genes were screened out.Finally,the mechanism of OsVPE3-mediated transcription levels of OsPAL2 and OsCAD1 in regulating vessels differentiation of rice seedlings under drought stress were explored by adding the VPE specific inhibitor Ac-YVAD-CMK.The results showed that:(1)With the increase of PEG,the lignin content in the primary root tip of rice seedlings gradually increased,and the lignin staining deepened.In other words,the degree of lignification is proportional to the degree of drought stress.(2)With the increase of PEG,the position of vessels differentiation moved forward.At the same time,the length of root tip was shortened,which affected the root growth and plant growth.Rehydration after drought can alleviate the forward movement of vessels differentiation position.At the same time,the length of elongation zone and root tip increased correspondingly.Finally,the growth of roots and plants were restored.Among them,20% PEG could inhibit the growth,root and root tip length of rice seedlings,but it still retained some recovery ability after rehydration.(3)With the increase of PEG,plasma membrane permeability of root tip cells of rice seedlings increased,while cell activity decreased.Consequently,the number of cell death increased.In comparison,the order of activity recovery ability in the root tip cells of the rice seedlings was 10% PEG,15% PEG,20% PEG and 25% PEG,respectively.(4)OsVPE3,OsPAL2 and OsCAD1 play an important role in the root tip growth of rice seedlings.(5)In the early stage of drought treatment,with the increase of stress degree and stress time,the gene transcription levels of OsVPE3,OsPAL2 and OsCAD1 and the activity levels of VPE,PAL and CAD showed a "gradual increase" trend.After 4 days of drought and rehydration culture after drought,the transcription levels and activity levels showed a change trend of "rising first and then flattening".(6)20% PEG and VPE specific inhibitor AC-YVAD-CMK were selected for further experiments.It was found that the activity of OsVPE3 in the root tip of rice seedlings was down-regulated either by Ac-YVAD-CMK treatment alone or by 20% PEG and Ac-YVAD-CMK co-treatment.(7)VPE specific inhibitor Ac-YVAD-CMK can inhibit the gene transcription level of OsPAL2,OsCAD1 and the activity level of PAL,CAD in the root tip of rice seedlings.At the same time,it can delay the process of vessel differentiation position in the primary root tip of rice seedlings under drought stress.In conclusion,OsVPE3 increased the degree of lignification and promoted the differentiation of vessels,leading to the shortening of elongation zone and root tip length,then,influenced the root growth and plant growth of rice seedlings by up-regulating the transcription levels of OsPAL2 and OsCAD1 and the activity levels of PAL and CAD in the root tip of rice seedlings under drought stress,.