Abstract: Plant biomass is determined by photosynthesis efficiency, which in turn is affected directly by the chlorophyll concentration. Chlorophyllide a oxygenase (CAO) is one of the three speed-limiting enzymes in the chlorophyll synthesis pathway, and catalyzes chlorophyllide a into chlorophyllide b. In this study, the open reading frame of Medicago truncatula CAO (MtCAO), which encodes a protein of 531 amino acid residues, was cloned. Phylogenetic analysis demonstrated that MtCAO was closer to its dicot homologs than to those in more distantly related plant species. Different from CAOs in lower plants, CAOs in higher plants contain nine exons. MtCAO was preferentially expressed in green tissues, especially leaves. When transiently expressed, the MtCAO-GFP recombinant protein was localized in the chloroplast of Arabidopsis protoplast cells. The transcript of MtCAO was repressed by the treatment with methyl jasmonate (100 μmol·L^-1) or darkness, but induced by NaCl (100 mmol·L^-1) or PEG (5%) within 4 h then repressed until 12 h from application. Also, the expression of MtCAO fluctuated with the circadian clock. These results are consistent with the cis-element analysis of the MtCAO promoter. MtCAO was ecotopically expressed in Arabidopsis. The analysis of two independent transgenic lines revealed no significant difference in the content of chlorophyll, the chlorophyll a∶b ratio or shoot weight from the wild type. The results are similar to those from AtCAO overexpression Arabidopsis. Therefore, we postulate that the non-abnormal phenotype of the transgenic Arabidopsis constitutively expressing MtCAO probably results from the fact that the conserved A domain contains a “degron” motif, which was proven to hamper the elevation of AtCAO in Arabidopsis overexpressing AtCAO.

Key words: Medicago truncatula, chlorophyllide a oxygenase, chloroplast-localization, circadian clock, transgenic Arabidopsis thaliana