The Role Of Key Enzymes In Chlorophyll Removal In Senescence Of Different Ploidy Wheat Leaves

The physiological activity of flag leaf has important influence on grain development during wheat aging,the most obvious sign of leaf aging is chlorophyll degradation.Two pathways are known to degrade chlorophyll(Chl)in plants,one is the removal of phytol before removing magnesium ions from chlorophyll a by Chlorophyllase(CLH),the other is removing magnesium ions before removing phytol from chlorophylla by Pheophytinase.(PPH),CLH and PPH are key enzymes for the removal of phytol from chlorophyll.But it hasn't been reported which enzyme is playing a key role in the removal of phytol in different ploidy wheat leaves.In this study,eight wheat cultivars with different ploidy of A A,BB,DD,AABB and AABBDD were used as tested materials.The activities of the CLH and PPH enzymes in the flag leaves during the aging process were measured,and the gene expression was analyzed by qPCR,the relative chlorophyll content(SPAD value)and functional leaf area(GLAD)of different wheat varieties at different stages after anthesis as well as the changes in photosynthetic and fluorescent parameters were analyzed,from the point of view of chemistry,the differences in chlorophyll degradation pathways in different ploidy wheat were analyzed.The purpose was to understand the role of CLH and PPH in chlorophyll degradation during senescence in different genotypes of wheat,to clarify the evolutionary law and mechanism of these changes and correlations in different genotypes of wheat,to provide theoretical basis for the breeding of stay green wheat varieties.the results are as follows:1.With the progress of grain filling process after flowering,the SPAD value and GLAD of the flag leaf of eight wheat varieties gradually decreased,however,the differences between wheat varieties are exhibited at each post-flowering stage.Based on the systematic clustering of SPAD values and GLAD,eight wheat varieties can be divided into three types.The first type,whether based on SPAD values or GLAD,had a larger maximum rate(MRS)?shorter aging duration and shorter holding period,so it is non-stay-green type.The third type is stay-green type,It had larger MRS and later time to reach the maximum rate(TMRS),senescence start time(TS)and the end of senescence(To),this type had a long green holding period.The second type is between the first type and the third type,and it is an intermediate type.2.The enzymatic activities of CLH and PPH in the flag leaves of the eight wheat varieties showed one or two peaks after anthesis,but the height values and the occurrence time of the peaks of wheat varieties were different.In addition,basing on the absolute values of the slope of the linear equation of enzyme activity change,the PPH slightly larger than CLH in most wheat varieties.The results indicating that both PPH and CLH are important in the process of chlorophyll degradation,however the role of PPH may be slightly larger.3.There were significant or extremely significant negative correlations between CLH and PPH enzymatic activities,SPAD values,GLAD,photosynthetic parameters(Tr,Pn),and chlorophyll fluorescence parameters(ETR,Fv/Fm,qp,and Y(?)).The systematic clustering results of eight wheat varieties based on SPAD values and GLAD were basically consistent with the systematic clustering results based on changes of PPH and CLH enzyme activities.This indicated that CLH and PPH have important effects on the decline of chlorophyll content and functional green leaf area,and changes of photosynthetic parameters and Chlorophyll Fluorescence parameters during the post-anthesis of flag leaf senescence.4.The expressions of TaCLH and TaPPH were in different times period after anthesis,and the relative expression of TaCLH was lower than that of TaPPH gene in most of the tested wheat.The enzymatic activities of CLH and PPH were significantly correlated with their average relative expression levels,indicating that the regulation of the transcriptional level of related genes playing an important role in the viability of these two enzymes.The relative expression levels of TaCLH and TaPPH genes were also significantly correlated,further demonstrating that these two genes are co-expressed during the senescence of the flag leaves and together playing a role in the degradation of chlorophyll.5.The enzymatic activities of CLH,PPH and the expressions of TaCLH,TaPPH in different ploidy wheat were not significantly different.The chlorophyll-degrading dephytol process did not show significant differences between ploidies in the evolution of diploids to tetraploids and hexaploids wheat.