title: Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis.
abstract: Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis.
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Chunyu
authors.initials: C
authors.lastname: Zhang
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Guodong
authors.initials: G
authors.lastname: Ren
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Delin
authors.initials: D
authors.lastname: Li
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Cahoon
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Yongming
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.).
authors.forename: Bin
authors.initials: B
authors.lastname: Yu
authors.affiliation: National Research Center of Rapeseed Engineering and Technology and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z., E.B.C.);Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture, Wuhan 430070, China (C.Z., W.Z., D.L., Y.Z.); andCenter for Plant Science Innovation and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (C.Z., G.R., R.E.C., M.C., B.Y., E.B.C.) ecahoon2@unl.edu.
authors.forename: Edgar B
authors.identifier: http://orcid.org/0000-0002-7277-1176
authors.initials: EB
authors.lastname: Cahoon
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D019729:Carbon-Oxygen Ligases / Q000235:genetics* / Q000378:metabolism; D002734:Chlorophyll / Q000378:metabolism; D044127:Epigenesis, Genetic; D018506:Gene Expression Regulation, Plant; D009154:Mutation; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D034622:RNA Interference; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D024505:Tocopherols / Q000378:metabolism*; D014810:Vitamin E / Q000096:biosynthesis*
chemical_list: D029681:Arabidopsis Proteins; D014810:Vitamin E; D002734:Chlorophyll; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; C000598254:CHLOROPHYLL SYNTHASE, Arabidopsis; D019729:Carbon-Oxygen Ligases; D024505:Tocopherols