Deficiency in riboflavin biosynthesis affects tetrapyrrole biosynthesis in etiolated Arabidopsis tissue
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Tetrapyrrole biosynthesis is controlled by multiple environmental and endogenous cues. Etiolated T-DNA insertion mutants were screened for red fluorescence as result of elevated levels of protochlorophyllide and four red fluorescent in the dark (rfd) mutants were isolated and identified. rfd3 and rfd4 belong to the group of photomorphogenic cop/det/fus mutants. rfd1 and rfd2 had genetic lesions in RIBA1 and FLU encoding the dual-functional protein GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase and a negative regulator of tetrapyrrole biosynthesis, respectively. RIBA1 catalyses the initial reaction of the metabolic pathway of riboflavin biosynthesis and rfd1 contains reduced contents of riboflavin and the flavo-coenzymes FMN and FAD. Transcriptome analysis of rfd1 revealed up-regulated genes encoding nucleus-localized factors involved in cytokinin signalling and numerous down-regulated LEA genes as well as an auxin-inducible GH3 gene. Alteration of cytokinin metabolism of rfd1was confirmed by elevated contents of active forms of cytokinin and stimulated expression of an ARR6::GUS reporter construct. An etiolated quadruple ckx (cytokinin oxidase) mutant with impaired cytokinin degradation as well as different knockout mutants for the negative AUX/IAA regulators shy2-101 (iaa3), axr2-1 (iaa7) and slr-1 (iaa14) showed also excessive protochlorophyllide accumulation. The transcript levels of CHLH and HEMA1 encoding Mg chelatase and glutamyl-tRNA reductase were increased in rfd1 and the AUX/IAA loss-of-function mutants. It is proposed that reduced riboflavin synthesis impairs the activity of the flavin-containing cytokinin oxidase, increases cytokinin contents and de-represses synthesis of 5-aminolevulinic acid of tetrapyrrole metabolism in darkness. As result of the mutant analyses, the antagonistic cytokinin and auxin signalling is required for a balanced tetrapyrrole biosynthesis in the dark.
KeywordsTetrapyrrole biosynthesis Chlorophyll ALA biosynthesis Protochlorophyllide Riboflavin biosynthesis Phytohormone Cytokinin Auxin T-DNA insertion mutagenesis
GTP cyclohydrolase II
This work was supported by grants from the Collaborative Research Unit SFB 429 to BG. We are grateful to Thomas Altmann, Leibniz-Institute of Plant Genetics and Crop Plant Research, Gatersleben, for providing the T-DNA-mutagenized seed collection. We are thankful to Hidehiro Fukaki, Kobe University, Kobe, Thomas Schmülling, Free University, Berlin, and Klaus Apel, Rutgers University, Ithaca, for the shy2-101 mutant, the quadruple ckx mutant and the flu mutant, respectively. We thank Markus Fischer, Institute of Food Chemistry, University Hamburg, for fruitful discussion on riboflavin biosynthesis.
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