拟南芥叶绿体定位的Hsp90调控胚胎发生和叶绿体发育的机制研究

Chloroplast development is closely connected to embryogenesis, although the molecular mechanisms underlying these two biological processes remain largely unknown. The 90 KDa heat shock proteins (Hsp90s) belong to a highly conserved family of molecular chaperones, which play crucial roles in plant growth and development, and involve in plants' responses to various stresses. Our preliminary studies showed that co-suppression plants of Arabidopsis chloroplast-targeted Hsp90 (AtHsp90.5) displayed an albino phenotype, and T-DNA insertional mutants were lethal. These suggested that AtHsp90.5 may be essential for chloroplast development and embryogenesis. In this application, the function of AtHsp90.5 in chloroplast development and embryogenesis will be systematically characterized by investigation of embryo development, chloroplast ultrastructure, accumulation of thylakoid proteins and differential expression profiles of chloroplast proteins in T-DNA insertional mutants, RNAi lines, over-expression lines and co-suppression plants of AtHsp90.5. Besides, AtHsp90.5 interacting proteins will also be identified and the interaction mechanisms will further be characterized by in-vivo and in-vitro biochemical and molecular approaches.

叶绿体发育和胚胎发生是两个紧密相连的过程，迄今对这两个生物学过程的分子调控机制仍缺乏完整的理解。热激蛋白90（heat shock protein 90, Hsp90）是一类高度保守的分子伴侣，在植物生长发育及对逆境的响应中发挥重要作用。我们发现拟南芥叶绿体定位的AtHsp90.5的共抑制植株具有白化苗表型，且其T-DNA插入突变体是纯合致死的，推测AtHsp90.5可能对叶绿体发育和胚胎发生是必需的。本研究将在此基础上，以AtHsp90.5的T-DNA插入突变体、RNAi株系、过表达株系、共抑制植株为材料，研究AtHsp90.5功能受损对胚胎发育、叶绿体超微结构、光系统蛋白的累积及叶绿体蛋白表达谱的影响。此外，鉴定AtHsp90.5的互作蛋白并进一步用体内体外生化和分子生物学试验分析AtHsp90.5与底物蛋白的互作机制，以期揭示AtHsp90.5调控叶绿体发育和胚胎发生的分子机理。

叶绿体发育和胚胎发生是两个紧密相连的过程，迄今对这两个生物学过程的分子调控机制仍缺乏完整的理解。热激蛋白90（heat shock protein 90, Hsp90）是一类高度保守的分子伴侣，在植物生长发育及对逆境的响应中发挥重要作用。拟南芥基因组含有7个Hsp90家族成员，其中叶绿体定位的AtHsp90.5突变会导致叶绿体发育和胚胎发生受到损害。然而，有关AtHsp90.5具体生物学功能及其发挥作用的机制仍未知。本研究中我们发现AtHsp90.5的共抑制植株具有白化苗表型，且共抑制植株中光系统超级复合体的累积减少，编码光系统的基因表达量降低。进一步发现AtHsp90.5的T-DNA插入突变体是胚胎致死的，其胚胎发育停滞在心型期。AtHsp90.5的表达在开花后4天的果荚中发生上调，其蛋白定位在叶绿体。利用透射电镜发现AtHsp90.5共抑制植株及T-DNA插入突变体的叶绿体中几乎观察不到类囊体膜，表明其叶绿体发育受损。利用BiFC证实AtHsp90.5与VIPP1蛋白互作，而AtHsp90.5共抑制植株中VIPP1多聚体与单体的比例显著升高。本研究证实了AtHsp90.5对叶绿体发育和胚胎发生是必需的，并首次在植物中鉴定了AtHsp90.5的互作蛋白VIPP1。通过进一步研究揭示了AtHsp90.5可能是通过介导VIPP1多聚体解聚为单体而参与类囊体膜的形成和/或维持而发挥作用。