Functional Exploration And Overexpression Of Rice Mg-Chelatase D Subunit

Mg chelatase is the first enzyme in chlorophyll synthesizing pathway (Mg2+branch), and plays an essential role in catalyzing the reaction of Mg2+insertion into protoporphyrin IX. In plants, Mg chelatase consists of three different subunits:ChlH, ChlD and ChlI subunits, which form an enzyme complex to accomplish the reaction and may be involved in other metabolic pathways. Lacking ChlH leads to a gun (genomes uncoupled) phenotype, which indicates that ChlH is related to retrograde-signal in plant. ChlH is also involved in sugar catabolism through regulating a sigma factor, sigE. ChlH was demonstrated to be related to ABA signaling by a series of experiments. ChlI is a target protein of thioredoxin-f, which redox regulates ATPase activity of ChlI in chloroplast. In Arabidopsis, ChlD knock out mutant also shows a gun phenotype. ChlD was considered to be the first case combining an AAA (ATPases associated with various cellular activities) module and a vWA (Von Willebrand factor type A) domain in one protein. However, its110amino acid linking region is sufficient for Mg chelatase catalysis in vitro. These results suggest that vWA and AAA domain may be involved in other metabolic pathway besides chlorophyll synthesis.In this study, we divided OsCHLD into three parts:predicted transit peptide (cTP, Met’-Pro80), OsCHLDN (Leu8’-Ala481) and OsCHLDC (Glu482-Ser754). The cTP of ChlD is sufficient for chloroplast targeting. We got the overexpressed transgenic lines of rice, named OsCHLDN-OX and OsCHLDC-OX, respectively. During whole developing stage, the OsCHLDN-OX plants did not demonstrated any significant phenotype compared with wild type plants (WT). The endogenous ChlD content in leaves at seedling stage, two-leaf stage and young panicle stage of OsCHLDN-OX plants was decreased, unchanged and increased, respectively. During seedling and young panicle stage, the leaves and young panicle in OsCHLDC-OX plants showed a yellow phenotype compared with WT. During two-leaf stage, OsCHLDC-OX plants did not showed any significant phenotype and ChlD content in the leaves stayed in the same level compared with WT. However, during whole developing stage, mRNA level of endogenous ChlD is cosuppressed in OsCHLDC-OX plants but not in OsCHLDN-OX plants. OsCHLDN and OsCHLDC in OsCHLDN-OX and OsCHLDC-OX plants is overexpressed at mRNA level compared with WT, but the corresponding protein can’t be detected in the leaves. We propose this unusual expression pattern as a small RNA-based post-transcriptional regulation. This regulation model leads to the disordered status of photosynthesis associated nuclear genes expression and tetrapyrrole biosynthesis pathway. This study not only helps us understanding the pleiotropic function of Mg chelatase, but also provides the basic theory of improving photosynthetic efficiency.