Characterization And Functional Analysis Of Arabidopsis Chloroplast Protein For Growth And Fertility1 (CGF1) And CGF2

Chloroplasts are essential organelles of plant cells which contain three membrane systems:the outer envelope,the inner envelope and the thylakoid membrane,as well as three main compartments:the membrane spatium between the outer envelope and the inner envelope,the stroma and the thylakoid cavity,in addition to having its own nucleoid structure.Chloroplasts are derived from proplastids.They are the main sites of photosynthesis and provide energy for plant growth and development.In addition,photosynthesis in chloroplasts also plays a role in material metabolism and signal transmission.However,the role of chloroplasts are not limited to photosynthesis.Chloroplasts influence many aspects of plant growth and development such as embryogenesis,leaf development,gravimetric responses,temperature responses,and plant microbial interactions and so on.Proper function of chloroplasts relies on the coordination between chloroplast proteins,most of which are encoded in the nuclear genome and have yet to be functionally characterized.In this study,we identified and characterized two nuclear-encoded chloroplast proteins in Arabidopsis,Chloroplast protein for Growth and Fertility1?CGF1?and CGF2.We report that CGF1 and CGF2 play crucial roles in vegetative and reproductive growth and are important for chloroplast integrity.Results and conclusions are followed:?1?CGF1 and CGF2 are constitutively expressed.Quantitative real-time PCR results and histochemical staining showed that they were expressed in leaves and inflorescences,mature pollen and ovules,and roots.?2?CGF1 and CGF2 are associated with chloroplasts depending on their N-terminal signal peptides.CGF1 and CGF2 both contain a chloroplast transit peptide in its N-terminus based on TargetP1.1 prediction.We examined their localization in the protoplasts of mesophyll cells of the Pro_UBQ10:CGF1-GFP or Pro_UBQ10:CGF2-GFP transgenic plants,and found that they were associated with chloroplasts.By truncating the protein sequences of CGFs,we found that the signal sequences were sufficient to drive the targeting of GFP to chloroplasts,while proteins lacking the signal peptides were associated with the cytoplasm or the plasma membrane.These results suggested that the targeting of CGFs to chloroplast depends on their N-terminal signal sequence.?3?Mutations of CGF1 and CGF2 affected the vegetative growth of plants.Mutants of CGF1 and CGF2 generated by CRISPR/Cas9 exhibited vegetative defects,including reduced leaf size,dwarfism,and abnormal cell death.?4?Mutations of CGF1 and CGF2 affected female gametogenesis.We observed wrinkled and white ovules in the developing siliques of the CGF1 and CGF2 double mutants by optical section.These abnormal ovules did not contain embryo sac structure although integuments seemed normal,indicating that CGFs play roles in the formation of female gametophytes.?5?Mutations of CGF1 and CGF2 affected embryogenesis.We examined embryos development during time course by whole-mount clearing assays and found that the embryos of the homozygous CGF double mutants were arrested at the globular stage even when wild-type embryos develop to form embryonic cotyledons,suggesting that CGF1 and CGF2are involved in embryogenesis.?6?CGF1 and CGF2 mediate leaf development.Double mutants of CGFs showed large yellow patches,and mass cell death of mesophyll cells,especially in the palisade layer and the sponge layer.In addition,the size and thickness of leaves decreased significantly in the CGF double mutants.?7?CGF1 and CGF2 are involved in maintaining chloroplast integrity.Mutations of CGFs caused the alteration of chloroplast morphology with its membrane system disintegrated.Disrupted chloroplast integrity further led to the decrease of chlorophyll contents and starch contents,thus reducing the carbon source supply of plants.Indeed,exogenous sucrose compensated for the growth defects of mutant seedlings.