Influence And Mechanism Of Protease Inhibitors On Enzymatic Browning Of Potatoes

Potatoes are prone to browning during processing,severely affecting exterior quality and nutritive value of the processed-products,which causes great economic losses to potato processing industry.At present,most of the studies mainly focus on the physiological and biochemical analysis,and the exploration of molecular mechanism of enzymatic browning are still limited.RNA-Seq analysis was performed to compare the differences at transcription levels of potatoes with different browning-susceptibilities in previous studies of author's lab.And results showed the transcript levels of protease inhibitors had significant differences between potatoes with less and more browning,indicating that protease inhibitors(PIs)might play important roles in browning-resistance of potatoes.But no further research has been conducted to prove this hypothesis.Therefore,it has great practical and scientific significance for studying new technologies to control the enzymatic browning and for molecular breeding of anti-browning potatoes,to study the effects and molecular mechanism of PIs on potato browning.Nine PI genes were screened through increasing the screening pressure and re-analyzing the transcript differences between potatoes with different browning.Four PI genes including StPI 143,StPI 146,StPI 128 and StPI 9511 were selected via gene ontology and homology analysis.Overexpressed-PI plants were obtained by agrobacterium-mediated potato transformation and transgenic potato tubers were harvested after cultivation.The effects and mechanism of overexpressed-PIs on browning,physiology and biochemistry of potatoes were studied.Besides,the subcellular localizations of cysteine StPI 146 and cysteine StPI 143 were analyzed by transient transformation of tobacco.Furthermore,c DNA library of potato was constructed and screened,a protein was selected and made sure to interact with StPI 146 and StPI 143 respectively through yeast two-hybrid and pull-down test,which explained the molecular mechanism of StPI 146 and StPI 143 on potato browning preliminarily.Major results are as follow:(1)Through the transcript analysis of potatoes with different browning-susceptibilities(Kexin#4 and Kexin#13,and potatoes with and without PCT),37 common differentially expressed genes(DEGs)were obtained,in which,there were 15 common up-regulated genes including 9 PI genes in less-browning potatoes(Kexin#13 and potatoes with postharvest curing treatment,PCT)compared with more-browning potatoes(Kexin#4 and control potatoes without PCT).GO analysis of DEGs showed the enriched terms mainly focused on the activities of catalytic,hydrolase and peptidase and most of the genes in these enriched terms were protease inhibitors.Therefore,presumably PIs might play important roles in enzymatic browning of potatoes through the regulation of catalytic,hydrolase and peptidase activities.(2)Analysis of nucleic and amino acids sequence showed the accordant rate of protease inhibitor genes StPI 146 and StPI 143 is up to 94.92%,with higher similarity.Purified protein of StPI 146 obtained by prokaryotic expression was used to treat papain in vitro and result showed the inhibition rate of 1.0 mg/L and 2.0 mg/L protein-StPI 146 on papain activity were both around 45%,indicating that protein of StPI 146 had activity of cysteine protease inhibitor.Genes of StPI 146 and StPI 143 were cloned and overexpressed in potatoes respectively.After cultivation in green house,transgenic and wild type(WT)potatoes were harvest and used for browning evaluation.Results showed browning degrees of both overexpressed-StPI 146 and-StPI 143 potatoes were significantly lower than that of WT potatoes.Protease activities from three lines of overexpressed-StPI 146(48.54×10³ U/kg,41.95×10³ U/kg and 56.85×10³ U/kg)and from three lines of overexpressed-StPI 143(37.75×10³ U/kg,40.75×10³ U/kg and49.89×10³ U/kg)were all significantly lower than that from WT(75.77×10³ U/kg).Besides,the total free amino acids(FAA)in overexpressed-StPI 146 potatoes(6.45 g/kg,5.80 g/kg and 5.95g/kg)and-StPI 143 potatoes(6.42 g/kg,5.97 g/kg and 5.76 g/kg)were also significantly less than that in WT potato(8.72 g/kg).However,overexpression of StPI 146 and StPI 143 did not lowered PPO activity of potato tubers.(3)Concentrations of free tyrosine or total FAA in the overexpressed-StPI 143 and-StPI146 potato mash were adjusted to the levels in wild type mash by supplementing exogenous FAAs.Without supplementing amino acids,the browning degrees of overexpressed-StPI 146and-StPI 143 potato mash were between 0.5 and 0.6 while the browning degrees increased to around 0.7 after supplementing with free tyrosine only.And the browning degree of overexpressed-StPI 146 and-StPI 143 potato mash rised up to about 0.91 after supplementing the total FAAs,having no significant difference with the degree of WT mash.These results showed that increasing FAAs promoted potato browning.Besides,browning degree of potato mash supplemented with total free amino acids was higher than that of mash supplemented with tyrosine only,resembling the browning degree of wild-type mash.These results indicated that,besides tyrosine,the decrease of total FAA accumulation was an important factor alleviating enzymatic browning of potatoes.(4)Exogenous aspartic protease inhibitor,Pepstatin A,treatment inhibited browning of potato mash and decreased PPO activity of potato mash.Gene of aspartic protease inhibitor,StPI 9511,was cloned and overexpressed in potatoes to obtain the transgenic potato tubers.Overexpressed-StPI 9511 significantly reduced potato browning compared with WT.Compared with WT,protease activities in three lines of overexpressed-StPI 9511 potato tubers decreased by 45%,46%and 34%,the accumulation of FAAs reduced by 18%,33%and 38%,and PPO activity lowered by 27%,32%and 29%respectively.Besides,Overexpressed-StPI 9511increased SOD and CAT activity,and lowered H₂O₂ and O₂?^-contents.Supplementing with exogenous FAAs into overexpressed-StPI 9511 potato mash to levels of wild type mash,enhanced the browning of transgenic potato mash,but the browning of which were still significantly lower than that of wild type mash.These results indicated that overexpressed-StPI9511 could also alleviate the enzymatic browning of potato via inhibiting PPO activity and enhancing antioxidant ability in addition to decreasing FAA accumulation to reduce potato browning.(5)Cysteine protease inhibitor StPI 143 and StPI 146 are both located in nucleus and cytoplasm.A ubiquitin domain-containing protein,DSK2B,was obtained via constructing and screening c DNA library,which probably interact with StPI 143 and StPI 146.And DSK2B was confirmed to interact with StPI 143 and StPI 146 through Pull-down and yeast two-hybrid tests.The results indicated that StPI 143 and StPI 146 might decrease protein degradation and reduce free amino acids accumulation through participating the process of ubiquitination,besides regulating proteolysis via interacting with protease directly.