Functional Analysis Of A Chlorophyll Catabolic Gene CBD2 (Chlorophyll Biodegradation Deficiency 2)in Arabidopsis Thaliana

Leaves are one of the most indispensable parts of the plant and are the production factory of harvesting organic materials by photosynthesis.Leaf color mutation is a genetic phenomenon that occurs frequently and can easily be recognized.Leaf color mutants are important materials for chlorophyll biosynthesis and catabolism research,photosynthesis research,high photosynthesis efficiency breeding,and ornamental plant breeding.Chlorophyll is one of the most critical participating substances in photosynthesis.Thus,the study of chlorophyll-related processes with leaf color mutation can provide a theoretical basis for maximizing photosynthesis.The starting point of this research was based on the obtain of a T-DNA insertion mutant of Arabidopsis cbd1(chlorophyll biosynthetic deficiency 1).We attained the homologous gene of CBD1 as CBD2 and its functional deficiency mutant cbd2(chlorophyll biodegradation deficiency 2)and the double mutant cbd1cbd2 with a robust CRISPR/Cas9 gene editing system.The knowledge and methods of bioinformatics,genetics,biochemistry,and cell biology were employed for genetic,phenotypic,chlorophyll content,photosynthesis level,and intermediate substances of chlorophyll biosynthesis and degradation analysis with these three mutant lines.The function of the uncharacterized gene CBD2 and the relationship between CBD1 and CBD2 were investigated.The main results are as follows:1.CBD2,a homologous protein of CBD1,is conserved and localized in the stromaCBD1 and CBD2 are the only two members of Type ? CAAX superfamily in Arabidopsis with an amino acid sequence similarity of 58.39%.Bioinformatics analysis shows that CBD2 has three transmembrane domains,71 amino acids at the N-terminal predicted as cTP cleavage,and three evolutionarily conserved motifs,which are related to metal chelation.The transient transformation of Arabidopsis protoplasts revealed that CBD2 localized to the stroma of the chloroplast,where undergoing abundant enzymatic reactions.GUS chemically staining of transgenic plants showed that CBD2 expressed in various tissues such as roots,stems,leaves,flowers,and siliques,while the highest expression parts were the leaves.2.CBD2 is likely to be involved in the chlorophyll degradation pathwayThe cbd2 loss-of-function mutant had a typical phenotype after mutation of the gene involved in the chlorophyll degradation pathway,i.e.,had no apparent phenotype under normal cultural conditions,while had accelerated leaf senescence after dark induction.The key chlorophyll biosynthesis-related genes in cbd2 did not show expression level change after dark induction with qRT-PCR analysis.The accumulation of chlorophyll intermediate substances in cbd2 was approximately the same as in WT.However,some key genes of the chlorophyll degradation pathway in cbd2 were 2 to 6 fold up-regulated after dark induction.The chlorophyll non-green degradation intermediates RCC(Red Chlorophyll Catabolite)and FCC(Fluorescent Chlorophyll Catabolite)have different degrees of increased accumulation in cbd2 compared with WT after dark induction.The above results indicate that CBD2 does not participate in the chlorophyll synthesis pathway consistent with CBD1,and is more likely to be involved in the chlorophyll degradation pathway.3.The chd1cbd2 double mutant displays stunted plant growth and severely yellowish as well as derivative phenotypes compared with cbdlUnder normal growth condition,the cbd1 exhibited a pale-green phenotype,the Chl content was decreased by about 30%,and the ratio of Chl a/b was increased by about 26%;the cbd1cbd2 double mutant showed stunted plant growth and a more pronounced leaf color mutation,i.e.,the Chl content decreased by about 65%,and the Chl a/b ration approximately increased by 44%.While after three days of dark-induced incubation,cbd2 and cbd1cbd2 displayed a markedly accelerated leaf senescence and eventually died in advance,whereas cbd1 did not.The ultrastructure of cbd1cbd2's chloroplasts observed by transmission electron microscopy displayed a gerontoplast style,losing its normal shape as fusiform,with no thylakoid and starch granules accumulation,and rich of plastid globules.The photosynthesis intensity analysis represented by chlorophyll fluorescence detection showed that compared with wild type,cbd1 had higher photoreaction center openness and higher actual photochemical efficiency;cbd1cbd2 showed even higher photoreaction center openness,and actual photochemical efficiency.The accumulation of chlorophyll synthesis intermediates analysis can be tested with fluorescence ultraviolet spectrophotometer.Results showed that under the treatment of precursor in the biosynthetic pathway—ALA and Fe chelator—DP,cbd1cbd2 exhibited a higher degree accumulation of Mg-ProtoIX(ME)than cbd1 as well as WT,while the contents of the former step intermediate ProtoIX and the later step intermediate Pchlide showed no significant difference compared with WT.4.No physical interaction between the N-terminal of CBD1 and CBD2 is detected by Yeast two-hybrid experimentYeast two-hybrid experiment showed that there was no interaction between the 100 amino acids at N-terminal of CBD1 and CBD2.They might not exercise function as a combination form in the chlorophyll biosynthesis and biodegradation pathway.Based on the results above,we came to the conclusion that CBD2,expressing in the stroma,was likely to participate in the chlorophyll catabolism pathway.This research not only unveiled the uncharacterized CBD2 but also serendipitously found that CBD2 and its homologous protein CBD1 involved in respective branches of the chlorophyll cycle but inseparably led to intensified leaf color mutant.This discovery provided new insight into the research of the chlorophyll biosynthesis and biodegradation pathway.