In-planta analyses of the three Arabidopsis isoforms of the bifunctional RIBA with enzymatic domains for GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase

核黄素是黄素辅酶（FMN和FAD）的前体，是所有生物体所必需的。核黄素生物合成的最初两个酶促步骤是由双功能RIBA酶完成的，包括GTP环水解酶II （GCHII）和3,4-二羟基-2-丁酮-4-磷酸合成酶（DHBPS）。在被子植物中，RIBA基因家族由三个成员组成。从植物核黄素生物合成研究开始，我们发现在拟南芥rfd1突变体（T-DNA插入RIBA1）和RIBA1反义系中强烈降低的AtRIBA1表达不能被同时表达的AtRIBA2和AtRIBA3所补充。目前关于植物核黄素代谢的生物化学和调控方面的研究很少。通过提出的工作方案，旨在验证所有三种RIBA亚型在核黄素生物合成中的功能。目的是研究三种两部分野生型和多种工程型RIBA变体在植物中的酶学特性和生理重要性，这些变体将在致死性RIBA突变体中表达。此外，我们还将对RIBA1基因沉默和过表达的转基因拟南芥进行黄素含量和黄辅酶依赖蛋白及其细胞过程的修饰分析。筛选riba相互作用伙伴的目的是解码核黄素生物合成的功能、组织和控制以及与其他亚细胞过程的相互关系。在初步实验中，利用质体提取物实现了RIBA1和RIBA2的磷酸化。接下来，我们计划鉴定RIBA蛋白的磷酸化位点，并揭示RIBA磷酸化在拟南芥植物中的生理后果。

Riboflavin serves as precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The initial two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprising GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). In Angiosperms the RIBA gene family consists of three members. Starting our studies in plant riboflavin biosynthesis, we found that strongly reduced AtRIBA1 expression in an Arabidopsis thaliana rfd1 mutant (T-DNA insertion in RIBA1) and in RIBA1 antisense lines cannot be complemented by the simultaneously expressed AtRIBA2 AtRIBA3. Only little information is available on biochemistry and control of plant riboflavin metabolism. By means of the proposed working program it is aimed to verify the function of all three RIBA isoforms in riboflavin biosynthesis. It is intended to examine in planta the enzymatic properties and the physiological importance of the three bipartite wild-type and multiple engineered RIBA variants, which will be expressed in a lethal riba mutant. In addition, transgenic Arabidopsis plants with silenced and overexpressed RIBA1 genes will be analysed on the modified content of flavins and the flavocoenzyme-dependent proteins and their cellular processes. Screens for RIBA-interaction partners are aimed at to decode the function, organisation and control of riboflavin biosynthesis and the interrelation to other subcellular processes. In preliminary experiments RIBA1 and RIBA2 phosphorylation was achieved with plastid extracts. In continuation it is planned to identify the phosphorylation site of the RIBA proteins and unravel the physiological consequences of RIBA phosphorylation in Arabidopsis plants.