The Molecular Mechanisms Of The Root Hair Elongation Regulation Under Low Phosphorus Stress Of Sorting Nexin Protein SNX1 Via PIN2

Phosphate,as an important nutrient element necessary for plant growth and development,is a component of many biological macromolecules and important organelles.Plants have acquired a series of complex mechanisms to cope with low phosphorus stress in the long-term evolution process,among which the changing root system(such as shortening of main root,increasing of lateral root and root hairs)is an important way for plants to adapt to low phosphate stress.The expression of PLD?2 is obviously induced and PLD?2-derived Phosphatidic acid(PA),an important cell signaling molecule,can affect plant growth and development by regulating the location and activity of downstream proteins.As one of the most important plant hormones,auxin plays an important role in root morphogenesis.The content and distribution of auxin in root directly affect root development under low phosphate stress.Cell sorting nexin 1(SNX1)is involved in plant stress response and morphogenesis through PIN2 which can mediate the polar auxin transport.How PA and SNX1 regulate the development of plant root hairs under low phosphate stress has not been studied yet.Further studies on this fine regulatory molecular mechanism can provide new ideas and approaches for crop breeding for low phosphate tolerance.In this study,we found that wheat SNX1 gene is significantly induced by low phosphate stress.Functional analysis showed that Ta SNX1 and At SNX1 are highly conservative.In this study,we systematically analyzed the regulation of root hair development in Arabidopsis thaliana under low phosphate conditions,revealed the molecular mechanism of SNX1 and PLD derivative PA regulating root hair development through PIN2,and constructed the wheat lines with over-expression of At PLD?2.The main results are as follows:1.The protein sequence homology of wheat SNX1(Ta SNX1)and Arabidopsis thaliana SNX1(At SNX1)is very high.The subcellular localization of them are completely overlap.The binding strength of Ta SNX1 to PA is like that of At SNX1.Like At SNX1,Ta SNX1 can form homodimers by interacting with each other.The protein which the heterologous expression of Ta SNX1 in Arabidopsis thaliana is accumulation under low phosphate stress.Our results show that Ta SNX1 and At SNX1 are functionally conservative.2.The extension of root hairs of pin2 mutant was inhibited under low phosphate stress,while another phenotype of root remains unchanged.The root structure of over-expression or mutant of At SNX1 is not changed when compared with Col under low phosphate stress,which may be due to functional redundancy of At SNX gene family.When the gene function of At SNX family is inhibited by Wortmannin treatment,root hair elongation is inhibited under low phosphate stress.The over-expression and mutant of At PLD?2 showed that the root systems are consistent with that of Col under low phosphate stress.Under low phosphate stress,when At PLD?2 enzyme activity is specific inhibited by PLD2 inhibitors,the extension of root hairs is inhibited and other phenotype of roots remained unchanged.Combined with the above results,At PIN2,At SNX1 and At PLD?2 were involved in the regulation of root hair extension.3.By fat western,At SNX1 can bind to PA.The binding strength of At SNX1 to PA is increased with the increase of PA concentration.By using the Liposome binding,PA liposome also could bind to At SNX1.4.By used western blot,the accumulation of At SNX1 on the plasma membrane is induced by low phosphate stress.When treatment with the PA synthesis inhibitor(1-butanol)or in pld?2 mutant,the accumulation of At SNX1 is not changed under low phosphate stress.Under normal condition,when treatment with the PA or in PLD?2-over background,the accumulation of At SNX1 is increased on the plasma membrane.5.By used cytological observations,under normal condition,the degradation of PIN2-GFP in vacuole is gradually increased,and under low phosphate stress,the degradation of PIN2-GFP in vacuole is reduced.When treatment with the PA synthesis inhibitor(1-butanol)or in pld?2 mutant,the accumulation of PIN2-GFP is increased under low phosphate stress.Under normal condition,when treatment with the PA or in At PLD?2-over background,the accumulation of PIN2-GFP is inhibitor invacuole.Under low phosphate stress,the increase of PA can reduce the degradation of PIN2-GFP in vacuole.6.The degradation of PIN2-GFP regulated by PA under low phosphorus stress is depended on the presence of SNX1.In the snx1 mutant,the degradation of PIN2-GFP in vacuole is no difference when treatment with PA or 1-butanol.7.Under normal condition and low phosphate stress,the endocytosis and exocytosis of SNX1-RFP and SNX2a-GFP is not changed.The endocytosis of PIN2-GFP is inbibited under low phosphate stress,but the exocytosis is the same as that under normal condition.The results showed that the accumulation of PIN2 in plasma membrane is due to the combination of SNX1 and PA under low phosphorus condition,which would inhibit the separation and transportation of PIN2 from plasma membrane to vacuole,and ultimately affect the distribution of auxin and root hair extension.8.In view of the important role of PA,a derivative of PLD 2,as a signal molecule,in plant adaptation to low phosphate tolerance,the wheat strains with over-expression of At PLD?2 are constructed.The 14 different wheat variety positive lines as receptors are obtained,which laid an important foundation for further research and application.The study elucidated the molecular mechanism that the vacuole degradation of PIN2 is regulated by PA and SNXI in plants under low phosphate conditions,and then affect root hair development.Our researches fill in the key link of phospholipid signal to auxin signal in plant response to low phosphate,and can provide new ideas and approaches for crop breeding for low phosphate tolerance.