The Relationship Between Chlorophyll Metabolism And Tocopherol Biosynthesis In Arabidopsis

Tocopherol and tocotrienol,known as vitamin E,are lipidic antioxidants synthesized in photosynthetic photoautotroph,play important roles in the daily diet of both humans and animals.Considering green plants,especially most oilcrops,have been becoming the major resources for natural vitamin E production to meet the growing demand of the public.With the limited study on the regulation of tocopherol synthesis associated with chlorophyll metabolism,the relationship between them is still not clear.Our research interests are mainly focused on the substrate PDP,which is used for tocopherol biosynthesis and can be attributed to the chlorophyll-linked pathway.To reveal the feasibility way to enhance efficient production of tocopherol level,the relationship between the metabolics of chlorophyll breakdown and chlorophyll synthesis with tocopherols biosynthesis will be discussed in this study for further improving the content in Arabidopsis.The major conclusions in this study achieved as following: 1)Chlorophyll synthase is critical for vitaminE synthesis,and altered expression affects tocopherol levels in Arabidopsis leavesIn this study,a heterozygous Arabidopsis T-DNA mutant of chlorophyll synthase(Salk134433),was applied for comparative analysis of tocopherols content between green and non-green leaves to comfirm the relationship between the metabolics of chlorophyll and tocopherols.Analysis of leaves of plants revealed only low tocopherol concentrations in homozygous chlsyn1-1 plants relative to wild-type plants.After crossing CHLSYN-transgenic lines and heterozygous chlsyn1-1 plants.The selection plants homozygous for the chlsyn1-1 allele but segregating by Dsred marker for the transgene provided a useful tool for additional examination of the involvement of chlorophyll synthase in tocopherol synthesis.This is the direct genetic evidence discovered that chlorophyll plays great role in the biosynthesis of tocopherols in green plants.We explore the contributions of chlorophyll synthase to tocopherol synthesis by up-and down-regulation of this gene,Our findings indicate that suppression of CHLSYN expression increases tocopherol concentrations in leaves,whereas enhanced expression of this gene reduces tocopherol concentrations in leaves.This provided additional support for the quantitative importance of the indirect biosynthetic pathway for the tocopherol precursor PDP in Arabidopsis.In the case of green plants,However,inordinate capacity of chlorophyll synthase for PDP re-synthesized to chlorophyll proved to be a bottleneck for achieving maximal tocopherol production,which lead to the low level of tocopherols.2)The relationship between tocopherol biosynthesis and related phytol hydrolase in chlorophyll degradation of Arabidopsis seedsThe free phytol is a crucial intermediate for the production of substrate PDP,which is being incorporated into the biosynthesis of tocopherols,phytol derived from the process of chlorophyll breakdown was supposed to be the major resource for the biosynthesis of tocopherols.The recent discovered Arabidopsis gene PPH was able to use Mg-free chlorophyll as substrate to generate free phytol during leaf senescence.Meanwhile,the other two Arabidopsis genes AtCLH1 and AtCLH2,which has also shown a capable of catalyzing the breakdown step of chlorophyll to generate the free phytol in vitro substrate assay.The total tocopherol concentration were determined in seeds from T-DNA null mutants of their single and generated double or triple mutants.All of the mutants were nearly indistinguishable compared with wild type.Meanwhile,tocopherol level was elevated in the seeds of PPH-overexpressing plants.This result indicated that only a small portion of free phytol derived forom PPH-mediated dephytylation seems able to enter tocopherol biosynthesis,and an unknown hydrolytic enzyme distinct from PPH and CLH seem to be responsible for producing phytol from chlorophyll for tocopherol biosynthesis.In addition,our study was focus on a crucial gene in the regulaion of chlorophyll degradation,NYEs.The result revealed the seed of nye1/nye2 double mutant showed a stay-green phenotype and tocopherol concentration was altered,but NYEs genes play a limited role in regulating tocopherol biosynthesis.Surprisingly,total tocopherol content of mature seeds was reduced by about 50% in NYE1-overexpressing plants.Together,these data suggest that although overexpression of NYE1 or PPH accelerates chlorophyll breakdown in seeds,neither of these activities contributes substantially to tocopherol biosynthesis.The fate of NYE/PPH-derived phytol in respective overexpressor remains unknown,but it seemingly does not contribute to the main pool of PDP used for tocopherol synthesis.We also can conclude that the tocopherol biosynthesis related chlorophyllase in Arabidopsis is still missing.