Biochemical Characterization Of Polyphenol Oxidase And Its Controlling Genes In Common Wheat

Polyphenol Oxidase(PPO) is the major cause of enzymatic discoloration in Asian noodles and other wheat-based end products,Darkening and discoloration affect consumer acceptance of wheat products,especially yellow alkaline and white salted noodles,so it becomes very important to select low PPO activity variety for breeders,however,there are needs for a rapid,accurate assay for determining PPO activity and relationships between controlling genes and activities.24 cultivars are used to explore the possibility of with catechol instead of L-DOPA to detect wheat kernel PPO activity according to AACC 22-85,and it is further validated by 196 cultivars;3 representative cultivars are used to study the characteristics of PPO for enzymology;changes of isoenzymes are analyzed during kernel development and grain germination use 19 and 54 cultivars which PPO activities have more differences;the inhibition and activation of 11 chemical agents under 5 kinds of concentrations on PPO activities are researched by 100 cultivars;43 ESTs and 7 mRNAs are assemblied with DNAMAN software;the allelic variations of PPO are detected use STS markers located on chromosome 2A and 2D in 300 F₄ single lines and 362 cultivars,and the effections of allele including 2AL(low PPO activity on chromosome 2A),2AH(high PPO activity on chromosome 2A),2DL(low PPO activity on chromosome 2D),2DH(high PPO activity on chromosome 2D) and allelic genotypes on PPO activities are studied,so it is for variation of introns either,the introns may influence gene transcription and expression.The results from above experiments showed as follows:1.According to AACC 22-85,with catechol instead of L-DOPA to detect wheat kernel PPO activity is one low-cost and credible mothed.2.The optimum temperature and pH of wheat polyphonel oxidase was 65℃and pH4.0～4.6,respectively.Km=0.19mol/L,Vmax=4.04×10²mol/min3.There were not isoenzymes detected in the wheat dry grains.The number of isozymes increased gradually in wheat grain germinating period and filling stage,but decreased in the developing kernel during from milk stage to mature stage.The PPO activity got increased rapidly after 12 hours in soaking,reached peak value at 24 hours, then it became smooth.4.The activity of wheat grain PPO could be inhibiting by mercaptoethanol,EDTA,saturated NaCl,SDS,sodium bisulfite,sodium diethyldithiocarbamate,glutathione and vitamin c,and activating by CuCl₂.It presents logarithmic relation between wheat PPO activity and concentration of inhibitors and activating agent,the type of additives and genotype of cultivars affected inhibition and activation either. 5.There are 15 PPO genes in wheat at least,and these genes fall into 3 distinct sequence groups.Groupâ… are tissue-specific expression genes,which have relation to the function of tissue development;groupâ…¡are genes of storage in seed,which participate in germination;groupâ…¢are resistant genes,also are defensive genes,it can be induced by outside factors.6.About 50%variation of kernel PPO activities are supplied by allelic variation located on chromosome 2A and 2D,the influence of allelic variation on PPO activity which located on chromosome 2A is 2-3 times than that of chromosome 2D.7.Kernel PPO activities in most of cultivars is low and medium level.The PPO activity of genotype 2AL/2DL,2AL/2DH,2AH/2DL,2AH/2DH get higher one by one for one degree(40-60AU/min·g);and their activity level is low medium,medium,medium high,high medium,respectively;and they make activity decrease 30.1%,decrease 7.8%, increase 8.3%,increase 30.0%,respectively.8.2AL and 2DL gene make kernel PPO activity decrease 25%and 10%, respectively.2AH and 2DH gene make kernel PPO activity increase around 17%.2AL, 2DL,2AH,2DH keep PPO activity at low,medium,medium high,medium high level, respectively.So,it should be attentioned to select 2AL gene for low PPO activity breeding program.9.GenBank accession DQ889708 may be the major gene fragment located on chromosome 2B,which controlling high PPO activity in wheat kernels.