Proteome Analysis Of Potato (Solanum Tuberosum L.) Tuber Development In Vitro Regulated By Auxin And Cytokinin

Potato（Solanum tuberosum L.）tuber development process has an important effect on the yield and quality of tuber.The continuous and complex process of tuber development is the result of the interaction of external environmental factors and internal regulator factors.Auxin and cytokinin,as the important plant growth regulators,play an vital role in potato tuber development process.In this study,the potato stolon cuttings（cv.Atlantic）were used as experimental materials to explore the effect of different concentrations indole-3-acetic acid（IAA）or 6-Benzylaminopurineon（BAP）on potato tuber morphology,physiology and proteomics.This study revealed the physiology and proteomics mechanism of tuber formation and development regulated by auxin and cytokinins.The main results were as following:1.The morphological changes and the physiological response of potato tuber development regulated by IAA in vitro.With the increase of IAA concentration from0.5 mg/L to 2 mg/L,the percentage of tuberization,tuber fresh weight and tuber diameter were increased and significantly higher than control,and had a maximum value under 2 mg/L IAA treatment.Compared with 2 mg/L IAA treatment,the percentage of tuberization,tuber fresh weight and tuber diameter were decreased with the increase of IAA concentration from 2.5 mg/L to 3.5 mg/L,but still higher than control.Under 4 mg/L IAA treatment,the percentage of tuberization was lower than control,the tuber fresh weight and tuber diameter were higher than control,but the all changes were no significance.With the increase of IAA concentration from 4.5 mg/L to 11 mg/L,the percentage of tuberization,tuber fresh weight and tuber diameter were declined and significantly lower than control.Under 11.5 mg/L IAA treatment,the tuberization was completely inhibited.The tuberization beginning time was delayed gradually with the IAA concentration increasing.The sucrose,fructose and glucose content of tuber were significantly increased with the increase of IAA concentration,and rapidly reached the maximum value under 11.5 mg/L IAA treatment.The content of reducing sugar,soluble total sugar and starch were sharply increased and had a maximum value under 2 mg/L IAA treatment,and then obviously decreased under higher concentration IAA treatment.The hydrogen peroxide（H₂O₂）content and the superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）activities of tuber were significantly increased with the IAA concentration increasing.Ascorbic acid（AsA）content was increased and reached a maximum value under 2 mg/L IAA treatment,whereas sharply decreased with IAA concentration increasing.2.Comparative proteomics analysis of potato tuber development regulated by IAA in vitro.Proteome analysis of tubers induced by different concentrations IAA（0,2,4 and 11.5 mg/L）was performed using the 2DE and MALDI-TOF/TOF MS spectrometry techniques.Fifty proteins with abundance changing more than 2.5-fold were identified successfully.The results showed that storage-related proteins（such as patatin and proteinase inhibitor）,transcription and translation-related proteins,starch synthesis and glucose metabolism-related proteins,redox homeostasis-related proteins and chaperones were up-regulated under 2 mg/L IAA treatment and then down-regulated under higher concentration IAA treatment.The GO analysis showed that 50 differential abundance proteins were mainly enriched in cells,intracellular,intracellular part and cytoplasm.The main molecular functions had the peptidase inhibitor activities,peptidase enzyme regulator activities,endopeptidase inhibitor and endopeptidase regulator activities.The biological processes were mainly involved in the regulation of cellular protein metabolism process,primary metabolism process,organic substance metabolism process and oxidoreduction coenzyme metabolic process.The KEGG analysis showed that 50 differential abundance proteins were mainly involved in biosynthesis of amino acid,glycolysis,carbon metabolism,ascorbate and aldarate metabolism.3.The morphological changes and the physiological response of potato tuber development regulated by BAP in vitro.With the increase of BAP concentration from0.5 mg/L to 3 mg/L,the percentage of tuberization,tuber fresh weight and tuber diameter were increased and significantly higher than control,and had a maximum value under 3 mg/L BAP treatment.Compared with 3 mg/L BAP treatment,the percentage of tuberization,tuber fresh weight and tuber diameter were decreased with the increase of BAP concentration from 3.5 mg/L to 5.5 mg/L,but still higher than control.Under 6 mg/L BAP treatment,the percentage of tuberization and tuber fresh weight were lower than control,the tuber diameter were higher than control,but the all changes were no significance.With the increase of BAP concentration from 6.5mg/L to 12.5 mg/L,the percentage of tuberization,tuber fresh weight,tuber diameter were declined and significantly lower than control.Under 13 mg/L BAP treatment,the tuberization was completely inhibited.With the increase of BAP concentration from 0.5 mg/L to 10 mg/L,the tuberization beginning time was earlier than control,but delayed with the increase of BAP concentration from 10.5 mg/L to 13 mg/L.The sucrose,fructose and glucose content of tuber were sharply increased with the concentration increasing,and rapidly reached the maximum value under 13 mg/L BAP treatment.The reducing sugar,soluble total sugar and starch content were significantly increased and had a maximum value under 3 mg/L BAP treatment,and then obviously decreased under higher concentration BAP treatment.The POD and CAT activities of tuber were significantly increased with the increase of BAP concentration.The H₂O₂ and AsA content and SOD activity were sharply increased and reached a maximum value under 3 mg/L BAP treatment,whereas decreased with BAP concentration increasing.4.Comparative proteomics analysis of potato tuber development regulated by BAP in vitro.Proteome analysis of tubers induced by different concentrations BAP（0,3,6 and 13 mg/L）was performed using the 2DE and MALDI-TOF/TOF MS spectrometry techniques.Fifty-five proteins with abundance changing more than2.5-fold were identified successfully.The results showed that storage-related proteins（such as patatin and proteinase inhibitor）,transcription and translation-related proteins,starch synthesis and glucose metabolism-related proteins and chaperones were up-regulated under 3 mg/L BAP treatment and then down-regulated under higher concentration BAP treatment.However,the redox homeostasis-related proteins were up-regulated under BAP treatment.The GO analysis showed that 55 differential abundance proteins were mainly enriched in cells,cell part,intracellular part and intracellular.The main molecular functions had the oxidoreductase activities,antioxidant activities,enzyme inhibitors activities and peptidase inhibitor activities.The biological processes were mainly involved in the glycolytic process,metabolism process,reactive oxygen species metabolism process and oxidation reduction process.The KEGG analysis showed that 55 differential abundance proteins were mainly involved in ascorbate and aldarate metabolism,biosynthesis of secondary metabolites,carbon metabolism and glycolysis.5.Bioinformatics analysis was used to analyze the key proteins of potato tuber development regulated by IAA or BAP in vitro.Ten key differential abundance proteins were obtained basing on the principal component analysis under IAA or BAP treatments,respectively.UniProt database and Protscale program in the ExPASY web site was used to analyze differential abundance proteins hydrophobicity/hydrophilicity.The result showed that the score of these 20 key differential abundance proteins was higher and the hydrophobic area was wider when the peak value more than 0.Subcellular localization of these 20 key differential abundance proteins were mainly located in the cytoplasm,nucleus,mitochondria,extracellular tissues（including cell walls）and endoplasmic reticulum by using UniProt database and Wolf PSORT software prediction.UniProt database and NetPhos 3.1 Server software was used to predict differential abundance proteins post-translational phosphorylation modification sites.The result showed that these 20 key differential abundance proteins have multiple post-translational phosphorylation modifications of serine and threonine sites.