Genome-wide survey of chlorophyllase (CLH) gene family in seven Rosaceae and functional characterization of MdCLH1 in apple (Malus domestica) leaf photosynthesis

Chlorophyll, a crucial pigment in plant photosynthesis, undergoes dynamic synthesis and degradation processes throughout the growth cycle of plants. Chlorophyllase (CLH) plays a crucial role in the degradation of chlorophyll by removing the phytol group from chlorophyll A to produce chlorophyllide A. However, Rosaceae species remain underexplored in terms of understanding the functional divergences among CLH gene family members (CLHs) involved in chlorophyll catabolism and photosynthesis. The apple (Malus domestica) CLHs also requires further systematic characterization and identification. In this study, 20 CLHs (MdCLH1-4, FvCLH1-2, PpCLH1-2, PcCLH1-3, PaCLH1-2, RrCLH1-4, RcCLH1-3) were identified from seven species belonging to the Rosaceae family. The chromosomal distribution of these gene family members is mostly separate across all species, except in Rosa rugosa. The number of amino acids encoded by these genes ranges from 171 aa to 391 aa, possessing a theoretical isoelectric point (PI) of 5.46-9.59, and a relative molecular weight of 18313.07D to 42413.21D. Secondary structure predictions highlight alpha-helix and random coil conformations as the dominant structural elements of CLH proteins present in Rosaceae species. Subcellular localization predictions indicate that all CLH proteins are expressed in chloroplasts, while MdCLH4 is uniquely localized to the nucleus. Phylogenetic analysis reveals high homology and close evolutionary relationships among the genes in three subfamilies. All these 20 CLHs contain elements responsive to phytohormones, environmental stress, and light. Furthermore, transcriptomic profiling using geneChip expression array coupled with qRT-PCR analyses revealed a heightened transcriptional activity of MdCLHs in leaf tissues and protective tissue of annual shoots as compared to other plant components. Additional experimental evidence specifically indicates MdCLH1 is located in the chloroplasts of tobacco leaves. Notably, MdCLH1 transient expression in apple leaves decreased chlorophyll a, carotenoids, total chlorophyll content, non - photochemical quenching coefficient (NPQ), and intercellular COQ concentration (Ci), while increasing chlorophyll b content, effective PSII quantum yield [Y(II)], net photosynthetic rate (Pn), stomatal conductance (Gs), and electron transport rate (ETR). These suggest MdCLH1 enhances light energy conversion in PSII by modulating chlorophyll degradation, potentially improving photosynthetic efficiency and reducing the potential for photoinhibition. This study lays a solid foundation for further exploration into the functional roles of CLHs in the Rosaceae family.