Physiological Mechanisms And Phosphoproteomic Analysis Of Potato(Solanum Tuberosum L.) Tuber Development In Vitro Regulated By Cytokinin

Potato（Solanum tuberosum L.）is one of the most important food and vegetable crops worldwide and also one of the important model plants for studying morphogenesis of underground sink organs.Potato tuber formation and development directly affects tuber yield and quality trait formation.Phytohormones play crucial roles in tuber morphogenesis,and Cytokinin（CK）is one of the most important hormones.Studies on CK-regulating tuberization have important theoretical and practical significances for revealing development mechanism of underground storage organs and elucidating tuber yield and quality trait formation.In this study,potato stolons cultured in vitro were exogenously applied with N₆-（delta 2-isopentenyl）-adenine（2ip,an active CK）and Lovastatin（a CK biosynthetic inhibitor）to analyze changes on tuber morphology,anatomical structure,cell ultrastructure and endogenous hormone levels.And on this basis,the changes of tuber proteome and phosphoproteome were further elucidated,and the key differentially abundant proteins and phosphoproteins involved in tuber development were also identified.It could reveal the key pathways and modes of CK-regulating tuberization,which would provide novel insights into the molecular mechanism of potato tuber development.The main research results are as follows:1.Low t Z or 2ip concentration（0.1μM-5μM）promoted tuber development,whereas higher t Z or 2ip concentration（>10μM or 50μM）showed inhibition effects.0.5μM t Z or 0.1μM 2ip showed the most obvious promoting effects.The beginning time of tuberization was obviously advanced,and the tuberization rate,tuber fresh weight and diameter were significantly increased.After 100μM-200μM t Z or 2ip treatment,the tuber length-width ratio was significantly increased,and the tuber shape was changed.Lovastatin inhibited tuber development.The beginning time of tuberization was significantly delayed.The tuberization rate,tuber fresh weight and diameter were significantly decreased.The tuber length-width ratio was significantly increased,and the tuber shape was changed.2.The tuber tissue microstructure was changed under exogenous 2ip and inhibitor Lovastatin treatments.After 0.1μM 2ip treatment,the average cell number of pith and perimedulla in both transverse and longitudinal section were significantly increased,whereas it was significantly decreased in cortex.And,the average cell area of each tuber tissue was significantly increased.It indicated that the cell division and expansion of pith and perimedulla in both transverse and longitudinal direction were enhanced to promote tuber development.After 1μM Lovastatin treatment,the cell number and area of each tuber tissue in transverse section were significantly decreased.The average cell number of pith and perimedulla in longitudinal section was significantly increased,whereas the cell area was decreased.Both the cell number and area of cortex in longitudinal section were significantly decreased.It appeared that CK biosynthesis inhibition prevented the cell division and expansion in transverse of tuber tissue,and the cell division was mainly occurs along longitudinal direction.3.The tuber cell ultrastructure was changed under exogenous 2ip and inhibitor Lovastatin treatments.After 0.1μM 2ip treatment,the tuber cells were densely arranged,and the cytoplasm was thick with abundant organelles.The mitochondria and amyloplast structure were complete,and their number and volume were increased.The endoplasmic reticulums were more and longer attached with a large number of ribosomes.The nucleus has large nucleoli and thick nuclear matrix.It showed that the intracellular metabolic activity is vigorous and provides sufficient energy and material for tuber morphogenesis.After 1μM Lovastatin treatment,some mitochondrias and endoplasmic reticulums were disintegrated,and many multi-vesicular bodies appeared in cells,which is not conducive to tuber morphogenesis.4.The endogenous hormone levels in tubers were changed under exogenous 2ip and inhibitor Lovastatin treatments.After 0.1μM 2ip treatment,the endogenous hormone level changes were conducive to enhance tuber cell division and accelerate tuber growth rate.The ratios of active CKs/（GA₃,ABA or JA）,IAA/ABA and IAA/GA₃ were increased.The total JAs（JA,JA-Ile and cis-OPDA）and ABA content were significantly decreased,whereas the IAA and SA content were increased.After 1μM Lovastatin treatment,the ratios of active CKs/（GA3,IAA,ABA or JA）were decreased.The IAA and SA content were significantly increased,whereas the ACC content was decreased.5.There were 715 differentially abundant proteins involved in CK-regulating tuber development were identified using TMT-labeled quantitative proteomics.Based on the analysis results of hierarchical cluster,functional classification,GO and KEGG enrichment,CK might mainly participate in regulating Ca²⁺homeostasis and signal transduction,regulating ROS-or phosphorylation-mediated signal transduction,maintaining sucrose-starch metabolism balance,promoting storage protein accumulation,and stabilizing cell structure for promoting tuber morphogenesis.6.Phosphorylation-mediated regulation is a key mechanism of CK-regulating tuber development.A total of 358 differential phosphoproteins were identified during CK-regulating tuber development by TMT-labeled quantitative phosphorylation proteomics.Five types of conserved motifs were extracted,including[s P],[Rxxs P],[Rxxs],[s D]and[s Dx E].These phosphorylated motifs were located on Ser residues,indicating that a lot of Ser phosphorylation events were involved in CK-regulating tuber development.The key protein phosphorylation events were mainly involved in signal transduction,sucrose-starch metabolism,cell structure maintenance,redox homeostasis and protein biosynthesis/degradation.First is to regulate phosphorylation status of Brassinolide BRI1 receptor kinase 1（BAK1）,which might interact with BR signal to co-regulate tuber morphogenesis.The second is to maintain sucrose-starch metabolism balance in tubers by regulating phosphorylation status of related-enzymes.The third is to maintain cytoskeleton and membrane stability for tuber morphogenesis by regulating phosphorylation status of tubulin and membrane proteins.Fourth is to regulate phosphorylation status of respiratory burst oxidase C（Rboh C）,then the induced ROS might trigger tuber development-related signal transduction.Fifth,CK biosynthesis inhibition leads to the down-regulated phosphorylation level of ribosomal proteins and the up-regulated phosphorylation level of protein ubiquitination degradation system-related enzymes,which is not conducive to tuber development-related protein biosynthesis and storage protein accumulation.