Proteome And Phosphoprotein Investigation Of Jasmonic Acid In Regulation Potato Tuber Development

Potato(Solanum tuberosum L.)is an important crop.As the primary economic organ of potato,the number and weight of tubers directly affect the final yield of potato.Environmental conditions and hormones regulate tuberization.Jasmonic Acid(JA)is an essential phytohormone that can initiate the defence response of plants and participate in the regulation of plant growth and development.Previous studies found that JA could promote cell expansion in tubers,increase the fresh weight and tuberization rate,and promote the formation and development of tubers.However,the molecular mechanism of JA regulation in tuberization is still unclear.In this experiment,exogenous JA was used to induce tuberization.The tuber phenotype,cellular structure,tissue morphology,content of endogenous hormones,proteome,phosphoproteome,and expression of the vital protein in the regulation of tuberization were detected and analyzed after JA induction.Our results revealed proteins and phosphoproteins regulation of tuberization by JA in vitro.It provides a theoretical basis for further elucidating the role of specific genes in JA regulation in the process of tuberization.The main results of this study are as follows:1.Statistical analysis of tuber morphology indicated that fresh tuber weight and tuberization rate increased under 0.5 and 5 μM JA treatments and decreased under 50μM JA.With 0.5 μM JA treatment,the transverse cell area of the cortex and pith significantly increased,and the cell density decreased.Under 5 μM JA treatment,the proportion and cell area of the perimedulla significantly increased,and the cell density decreased.Under 50 μM JA treatment,the longitudinal region of the cortex increased while the region of the pith decreased.Moreover,the transverse cells density in the perimedulla increased dramatically while the cell area of the pith decreased.These results displayed that JA mainly regulated the development of perimedulla in the process of tuberization.2.The application of exogenous JA may affect the content of endogenous phytohormones because of the putative interactions among them.Data showed that the change of endogenous JA content was in line with exogenous JA concentration.The changes in JA content were positively correlated with the changes of indole-3-acetic acid(IAA)and salicylic acid(SA)but negatively correlated with dihydrozeatin,DHZ,and 1-aminocyclopropane-1-carboxylic acid(ACC).Endogenous hormone analysis showed that exogenous JA could increase the content of endogenous JA in tubers and induce changes in the content of other plant hormones,which could regulate the development and formation of tubers.3.To reveal vital proteins in the regulation of tuberization by JA,the proteomic analysis of potato tuber revealed that a total of 257 differentially expressed proteins(DEPs)were induced by exogenous JA.Especially,5 μM JA had the most significant impacted on DEPs.DEPs were divided into nine categories,and the top three protein groups were metabolism-related proteins(Pm,39%),protein synthesis and metabolismrelated proteins(Pb,19%),and transcription and translation-related proteins(Pt,13%).Three cell pathways were extracted from DEPs using bioinformatics analysis: cell wall synthesis pathway,primary carbon metabolism pathway,and protein synthesis pathway.JA mainly regulated proteins related to cell wall monosaccharide synthesis,cell wall material transportation,and cytoskeletal structure for changes in tuber cell morphology.JA controlled the carbon-flux from dissimilation into assimilation in primary carbon metabolism by inhibiting malate dehydrogenase(MDH)expression in the tricarboxylic acid(TCA)cycle.In the protein synthesis pathway,the ribosomal protein was the hub of JA regulation.JA regulated the synthesis of other proteins by affecting the expression of ribosomal protein,which was the basis for controlling other pathways.In addition,western blot found a positive correlation between the expression of JA receptor COI1 and tuberization,which implied the dependence of tuber development on COI1.4.Phosphoproteomic analysis was applied to investigate the role of protein phosphorylation in tubers.488 phosphorylated proteins were identified in tubers.Among 1028 phosphorylation sites of serine and threonine detected,676 were outside the conserved homology domains.All phosphorylated proteins share 7 conservative phosphorylation motifs([SP],[Rxx SP],[GSP],[SSP],[Rxx S],[SDx E] and [TP]).Bioinformatics analysis shows that phosphorylated proteins were involved in glucose metabolism,m RNA production,and mitogen-activated protein kinase(MitogenActivated Protein Kinases,MAPK)signalling pathways.Compared with the results of proteomic analysis,we found that phosphorylated proteins are mainly involved in two core pathways in the tuberization process: primary carbon metabolism and protein synthesis.5.The differentially expressed phosphorylated proteins(DEPPs)were 258 in potato tuber after JA treatment.Mainly,0.5 μM JA had the most significant effect on DEPPs.The top three categories with the most proteins were Pt(34%),Pm(12%),and signal transduction-related protein Pg(12%).JA significantly affected the phosphorylated level of UDP-xyl synthase(UXS),α-Tubulin,and microtubuleassociated protein(MAP65-1b)to regulate the cell wall monosaccharide metabolism and cytoskeleton stability.DEPPs related to primary carbon metabolism are 14-3-3 and sucrose non-fermenting-1-related protein kinase 2.4(Sn RK2.4),indicating that JA regulated the primary carbon metabolism by phosphorylation of these critical proteins.The phosphorylated proteins involved in the core of the JA regulation were HSP90 and HSP70 that assist COI1 to form the SCF ubiquitin ligase complex.JA regulated the recognition of COI1 by HSP90 and HSP70 through protein phosphorylation combined with the changes of COI1 expression.In summary,JA played a vital role in promoting the tuberization of potato tuber in vitro: Firstly,JA promoted the volume enlargement of the perimedulla cells and regulated the development of the perimedulla.Secondly,cell wall synthesis,primary carbon metabolism,and protein synthesis were the three core pathways of JA regulating tuber development,among which protein synthesis was the basis.Thirdly,JA regulated phosphorylation of structural and functional proteins in three core pathways,of which sucrose non-fermenting-1-related protein kinase 2.4(Sn RK2.4),14-3-3,HSP90,and HSP70 were key phosphorylated proteins.Fourth,the regulation of the tuberization by jasmonic acid depended on its receptor COI1.