Characterization Of The Function And Activity Regulation Of Magnesium Chelatase In PEA Plants

The first committed and highly regulated step of chlorophyll biosynthesis is the insertion of Mg2+into protoporphyrin IX to form Mg protoporphyrin IX. This step is catalyzed by Mg chelatase composed of BchI/CHLI, BchI/CHLD and BchH/CHLH subunits. In this study, the different domains in pea CHLI (PsCHLI) are functionally characterized. The expressions of CHLI and CHLD were suppressed in pea(Pisum sativum) plants by means of virus-induced gene silencing (VIGS), respectively. These gene silencing (VIGS-CHLI and VIGS-CHLD) plants were utilized to study the biofunction of Mg chelatase systematically. In addition, the mechanism of thioredoxin-dependent redox regulation on the CHLI and the Mg chelatase activity was also explored in this study. The main results are demonstrated as follows:N-terminal domain of PsCHLI (PsCHLI-N, Val63to Ser336) mediates interactions with CHLI and CHLD, while its C-terminal domain (PsCHLI-C, Ser337to Ser422) is not involved in the interactions but it is responsible for the enzyme activity. Thus, only the integrity of N-and C-terminal domain of PsCHLI (PsCHLI-NC, Val63to Ser422) is essential for ATPase and Mg chelatase activity.Both VIGS-CHLI and VIGS-CHLD plants show a yellowish phenotype with90%decreasing in both Mg chelatase activity and chlorophyll content,50%reduction of5-aminolevulinic acid (ALA) synthesizing capacity and undeveloped thylakoid membranes as well as altered chloroplast nucleoids structure and compromised photosynthesis capacity compared with VIGS-GPF plants (a negative control). The homeostasis between the generation of reactive oxygen species (ROS) and the scavenging capacity of antioxidant defense system was disturbed so that the ROS accumulated in the leaves of VIGS-CHLI and VIGS-CHLD plants. The results of metabolite profiling indicated a tight correlation between Mg chelatase activity and chloroplast/mitochondria interorganellar metabolic pathways. In addition, CHLD feedback-regulated the transcription of photosynthesis-associated nuclear genes (PhANGs). CHLD and CHLI silencing resulted in a rapid reduction of light-harvesting chlorophyll-binding proteins and changed the contents of tetrapyrrole biosynthesis related proteins in leaves of these gene silencing plants.Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that pea thioredoxin F (PsTRX-F) physically interacts with PsCHLI but not with either of the other two subunits or GUN4. In vitro, reduced PsTRX-F activated the ATPase activity of PsCHLI and enhanced the activity of Mg chelatase reconstituted from CHLI, CHLD, CHLH and the regulator protein GUN4. In vivo, Silencing of the PsTRX-F gene (VIGS-TRX-F) in pea plants did not show any significant phenotype, altered chlorophyll content, Mg chelatase activity and in vivo redox state of PsCHLI compared with the VIGS-GFP plants. However, simultaneous silencing of the PsTRX-F and PsTRX-M genes (VIGS-TRX-F/TRX-M) resulted in22%oxidized PsCHLI in vivo and30%,60%and20%reduction in Mg chelatase activity, chlorophyll content and ALA synthesizing capacity, respectively. The altered redox state in the chloroplast of VIGS-TRX-F/TRX-M plants led to the accumulation of ROS, which may serve as a retrograde signal for the transcriptional regulation of PhANGs. TRX-F and TRX-M also modulate the in vivo stability of the tetrapyrrole biosynthesis related proteins.Taken together, Mg chelatase is not only a key regulator of tetrapyrrole biosynthesis but its activity is also involved in ROS homeostasis, primary metabolism and retrograde signaling in plant cells. The chloroplast thioredoxins regulate the Mg chelatase activity in redox, transcriptional and post-translational levels. All these results contribute to more insight in the overall biofunction and activity regulatory mechanism of Mg chelatase.