| Chloroplasts serve as the central site for photosynthesis,providing energy essential for plant growth.When chloroplast development is hindered,structural alterations occur,or chlorophyll content changes,leaves may exhibit abnormal coloration,subsequently impacting yield.In this study,two naturally occurring rice mutant lines(named wsl214 and gra117)were used as research materials to conduct map-based cloning and functional analysis of the mutated genes.The main research findings of WSL214 are summarized as follows:1.In this study,we obtained a rice white stripe leaf mutant,wsl214,which was generated by natural mutation.Under field conditions,the leaves of wsl214 showed white stripes from the fifth leaf stage to maturity.Compared with WT,wsl214 showed significantly reduced SPAD value,chlorophyll and its precursor content,damaged chloroplast structure,significantly increased plant height,and significantly reduced effective tiller number and grain yield.2.Genetic analysis reveals that the wsl214 mutant phenotype is controlled by a single recessive nuclear gene.The WSL214 is localized between molecular markers Indel3 and Indel5 on chromosome 1,with a physical distance of 33.9 kb.Sequencing uncovered a C-to-T substitution in the third exon of the Os01g0109300,leading to the replacement of the 343 rd amino acid Arg with Trp.Complementation tests and CRISPR/Cas9 knockout experiments confirmed that the wsl214 mutant phenotype is caused by the mutation in Os01g0109300.3.The WSL214 encodes a HD domain phosphohydrolase,which is homologous to the human gene SAMHD1 and belongs to the HD domain phosphohydrolase protein family.The enzyme is subcellularly localized in chloroplasts and widely expressed in various tissues of rice,especially at the highest level in leaves.4.The study found that the accumulation level of ROS was significantly increased while the activity of ROS scavenging enzymes was significantly decreased in the leaves of wsl214.This led to DNA damage and programmed cell death in leaf cells.In addition,the high level of ROS was one of the causes of the white-striped leaf phenotype in wsl214.Furthermore,the high level of ROS also enhanced the defense response of wsl214 against exogenous pathogens.5.The regulatory mechanism of WSL214 on the ROS homeostasis in rice leaf cells involves two aspects.On the one hand,WSL214 interacts with the peroxidase protein Os CATC,enhancing Os CATC expression and increasing intracellular ROS scavenging enzyme activity.On the other hand,WSL214 promotes chloroplast development,preventing the reduction and overexcitation of photosynthetic reaction centers,thus facilitating normal photosynthesis and reducing ROS production.The main research findings of GRA117 are summarized as follows:1.In this study,we obtained a rice green-revertible albino mutant,gra117,generated by natural mutation.Under field conditions,gra117 exhibited apparent whitening during the seedling stage and gradually recovered to normal green color until the five-leaf stage,with newly formed tillers showing similar phenotypes.Compared to the WT,gra117 showed significantly decreased SPAD values,chlorophyll and its precursors content,delayed chloroplast development,decreased yield,and decreased resistance to stress during the seedling stage.2.According to genetic analysis,the phenotype of the gra117 mutation is controlled by an recessive nuclear gene.Based on genetic mapping and cloning,we have located the GRA117 gene between molecular markers Indel3 and Indel5 on chromosome 3,with a physical distance of 33.8 kb.Further sequencing analysis revealed the presence of a 665 bp insertion in the promoter region of the Os03g0602600 gene,resulting in reduced transcriptional activity of GRA117.Complementation experiments confirmed that the mutation in the promoter region of Os03g0602600 is responsible for the phenotype of the gra117 mutation.3.GRA117 encodes a Pfk B-type fructokinase like 2,which belongs to the Pfk B-type carbohydrate kinase protein subfamily.This enzyme is subcellularly localized in chloroplasts and is widely expressed in various rice tissues,particularly at high levels in leaf tissues.4.The transcription of GRA117 is regulated by a 1029 bp core region upstream of its start codon.Using quantitative RT-PCR and Western blot techniques,the results of the detection indicate that the gene product of GRA117 promotes the expression and translation of photosynthesis genes.5.Studies have found that the leaf tissues of gra117 plants have significantly lower net photosynthetic carbon assimilation rates.At the same time,the enzyme activity of Rubisco,as well as levels of RUBP,PGA,carbohydrates,protein content,and dry matter accumulation in the leaf tissues of this plant are also lower.RNA-Seq analysis revealed that GRA117 plays an important role in photosynthetic carbon fixation,carbohydrate metabolism,and chloroplast ribosome-related pathways.6.The expression of GRA117 in rice can promote the Calvin-Benson cycle by regulating chloroplast development,thereby increasing the carbon assimilation efficiency of rice.In summary,this study conducted map-based cloning and functional analysis on two naturally occurring rice mutant lines,wsl214 and gra117.The research findings have enriched our understanding of rice photosynthesis and offer potential targets for future rice cultivar improvement. |
