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

对具有 GTP 环化水解酶 II 和 3,4-二羟基-2-丁酮 4-磷酸合酶酶结构域的双功能 RIBA 的三种拟南芥亚型进行植物内分析

基本信息

批准号：
258667322
负责人：
Professor Dr. Bernhard Grimm
金额：
--
依托单位：
Abteilung Molekulare Physiologie höherer Pflanzen
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/258667322?language=en
关键词：
planta analyses three Arabidopsis isoforms

项目摘要

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.

核黄素是黄酮酶（FMN和FAD）的前体，对于所有生物体都是必不可少的。核黄素生物合成的最初两种酶促步骤是在植物中通过包含GTP环丙基酶II（GCHII）和3,4-二羟基-2-二甲烷-4-二苯甲酮-4-磷酸磷酸盐合成酶（DHBP）的双功能核糖酶（DHBP）进行的。在被子植物中，RIBA基因家族由三名成员组成。开始研究植物核黄素生物合成的研究，我们发现在拟南芥thaliana rfd1突变体（riba1中的T-DNA插入）中，强烈降低了Atriba1的表达，而RIBA1反义线不能同时表达Atriba2 Atriba3。只有很少有关于生物化学和控制植物核黄素代谢的信息。通过提出的工作计划，旨在验证核黄素生物合成中所有三种RIBA同工型的功能。它旨在在Planta中检查酶特性以及三个两分型野生型和多种工程RIBA变体的生理重要性，这些变体将以致命的RIBA突变体表示。此外，将分析具有沉默和过表达的RIBA1基因的转基因拟南芥植物，分析黄素的修饰含量和黄素酶依赖性蛋白及其细胞过程。 RIBA互动伙伴的筛选旨在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，用塑料提取物实现了riba1和riba2磷酸化。在继续，计划鉴定RIBA蛋白的磷酸化位点，并揭示拟南芥植物中RIBA磷酸化的生理后果。