| Tetrapyrrole plays a vital role in plant metabolic processes such as plant photosynthesis,respiration and nitrogen/sulfur assimilation.Tetrapyrrole metabolism is an important pathway of chlorophyll precursor and chlorophyll synthesis.The disorder of tetrapyrrole metabolism affects chlorophyll biosynthesis,chloroplast development and photosynthesis.Although the molecular mechanisms of tetrapyrrole biosynthetic have been reported in rice,Arabidopsis,corn and other crops,few studies have been reported in soybean.In this paper,the genetic and gene functions were studied in Glycine max lesion mimic mutant 2-1?Gmlmm2-1?and Glycine max pale green leaf 2-1?Gmpgl2-1?,and the effects of the metabolic pathway of tetrapyrrole on chlorophyll synthesis and plant immunity was also further analyzed.It provides an important theoretical basis and genetic material for soybean breeding,and also provides new insights into chlorophyll synthesis,cell death,and chlorophyll regulation network.The major results of the Gmlmm2-1 mutant are described as following:1.The leaves of the Gmlmm2-1 mutant displayed etiolation and lesion mimic in the whole life cycle.The contents of Chl a,Chl b,and Car in the Gmlmm2-1 mutant were approximately 51%,50%,and 40%of those in‘Williams 82',respectively.The number of nodules,hundred-grain weight,number of grains per plant,and plant height were also reduced in the Gmlmm2-1 mutant compared to‘Williams 82'.The ROS accumulated in lesion mimic leaves of the Gmlmm2-1 mutant,indicating programmed cell death appeared in the Gmlmm2-1 mutant.Genetic analysis revealed that the necrotic phenotype the Gmlmm2-1 mutant was controlled by a single recessive nuclear gene.2.Based on the map-based cloning method,the candidate region was restricted to the top of Chromosome 14 between 203,085–426,404 bp,and whole genome sequencing revealed that only an A to G point mutation occurred in Glyma.14G003200gene in the candidate region of Gmlmm2-1 mutant,which caused a nonsynonymous substitution of Y-192 to C-192.Either gene complementation or CRISPR/cas9 knockout experiments confirmed that Gm LMM2?Glyma.14G003200?controls the phenotype of the Gmlmm2-1 mutant.3.Gm LMM2 encodes a coprophyrin III oxidase?CPO?.The phylogenetic tree and synteny analysis revealed that Gm LMM2 is a single copy gene in the genome e?Glycine max Wm82.a2.v1?.Transient expression of GFP-Gm LMM2 in Arabidopsis protoplast indicated that Gm LMM2 locates in the chloroplast.The Gmlmm2-1 mutant contained fewer granal thylakoid membranes compared with those of‘Williams 82'using a transmission electron microscopy.4..The higher intensity light rapidly and broadly induced severer necrosis and chlorophyll deficiency in Gmlmm2-1 mutant,while no lesion had formed on shaded leaflets of‘Williams 82'.The levels of chlorophyll precursors in the Gmlmm2-1 mutant were significantly reduced except Coprogen III.Therefore,the light-dependent cell death in the Gmlmm2-1 mutant may attribute to the accumulation of Coprogen III.5.The expression profile of resistance-related genes in the Gmlmm2-1 were significant higher than those in‘Williams 82'.The mean area of lesion in the Gmlmm2-1 was significantly decreased compared with that in‘Williams 82',after inoculated with Phytophthora sojae strain P7076.It indicated that the disease resistance response and pathogen resistance in the Gmlmm2-1 was greatly activated and enhanced.The major results of the Gmpgl2-1 mutant are described as following:1.The leaves of the Gmpgl2-1 mutant presented visible albinism leaf with yellow color in the early vegetative growth stage.The leaves began to turn green color at the early stage of reproductive growth,and all the leaves restored green color as‘Williams82'at the late stage of reproductive growth.We also isolated two pale green leaf mutants similar as the Gmpgl2-1 mutant,named as Gmpgl2-2 and Gmpgl2-3.Genetic analysis revealed that these three mutants are allelic to each other and the mutant phenotype is controlled by a single recessive nuclear gene.2.Based on the whole-genome sequencing-based bulk segregant analysis?BSA?and map-based cloning method,the Gm PGL2 locus was located on chromosome 12between 38,096,702–38,406,000 bp,which is a large fragment deletion in this interval containing 30 annotated genes.The analysis of BSA and whole genome sequencing showed that a G-A substitute was found in the second exon of Glyma.12G222200 in the Gmpgl2-2 mutant,and a same mutation was found in the fourth exon of Glyma.12G222200 in the Gmpgl2-3 mutant.Therefore,Glyma.12G222200 is Gm PGL2that controls the mutant phenotype.3.Gm PGL2 encodes the protochlorophyllide oxidoreductase?POR?.Gm PGL2 has three copies in the‘Williams 82'genome and locates in the chloroplast.Gm PGL2expressed constitutively in dark-and light-grown leaf and decreased significantly in high intensity light.Meanwhile,the expression of Gm PGL2 significantly is affected by the duration of light significantly.The etioplasts of the Gmpgl2-1 mutant hardly formed prolamellar bodies,and the uniformly lattice of prolamellar bodies were smaller and disordered.The mature chloroplasts of the Gmpgl2-1 mutant had fewer starch granules and prothylakoid membranes compared with those in‘Williams 82'.4.The greening speed of dark-grown Gmpgl2-1 mutant was significantly slow down after light compared to that of‘Williams 82';the contents of total Pchlide and photoactive Pchlide of dark-grown Gmpgl2-1 mutant were significantly reduced compared with those of‘Williams 82'indicating that Gm PGL2 mutation affected the greening of soybean seedlings and the reduction of Pchlide. |
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