| Leaf color mutation is a common mutation trait.There are many reasons for leaf color mutation,which involve multiple physiological and biochemical processes during plant growth and development.The flag leaf and its neighboring leaves are important functional leaves of the gramineous crop,which contribute a lot to the yield,so it is of great significance to study the mechanism of flag leaf function.Setaria has been proposed as an ideal model of Panicoideae grasses,and it is also an important cereal crop,the study of flag leaf function has not been reported.In this study,a stable and inherited yellow leaf mutant T182,screened by ethyl methyl sulfonate(EMS)mutagenesis,was studied for its physio-ecological mechanism,map-based cloning of mutant genes,functional identification and mechanism of action.It has been clarified that the S-adenosylmethionine synthetase(SAMS)gene plays a key role in regulating the chlorophyll synthesis of millet flag leaf and neighboring leaves by affecting the absorption and transport of iron and ethylene synthesis.The main results are as follows:(1)Morphological observations revealed that T182 gradually yellowed from the flag leaf to the top 5th leaf during the later stage of elongation,and continued to the whole growth stage.Further analysis found that the chlorophyll content in T182 was significantly reduced,the chloroplast membrane structure was disintegrated,the structure of chloroplast was abnormal,and the photosynthetic capacity of the mutant was significantly reduced after the mutant turned yellow.Investigation of agronomic traits found that plant height,panicle length and panicle width of T182 were significantly reduced compared to wild type Yugu1.This indicates that the T182 mutation affects the function of the upper leaves of the plant and leads to the inhibition of plant growth.(2)Used the Mut-Map+ and map-based cloning,the target mutant gene was mapped to a region of about 400 Kb on chromosome 3,the mutated gene was identified as Seita.3G053400 which named SiSAMS1.Amino acid alignment results showed that the gene encodes S-adenosylmethionine synthetase,the 2495 bp C to T mutation on the exon resulted in thechange of the 272 th amino acid from alanine(Ala)to valine(Val).Explain that this site is crucial for the function of SiSAMS1.(3)Transient transformation of tobacco leaves and millet protoplasts showed that SiSAMS1 were localized to chloroplast.(4)Analysis of the expression pattern showed that SiSAMS1 was constitutively expressed,the main expression organ was root,followed by stems and leaves,and the expression level in panical was high.(5)SiSAMS1 participates in iron absorption and transport.Compared with Yugu1,the iron content in the yellowed leaves of T182 was significantly reduced,and there was no difference in the iron content in the normal leaves of T182.The expression of iron-deficient response genes is significantly affected in T182 and mostly induced expression.Phenotypic analysis showed that SiSAMS1 knockout lines were more sensitive to iron deficiency.It indicates that SiSAMS1 is involved iron absorption and transport process in the millet.(6)Ethylene treatment accelerated yellowing of upper leaves of T182.Compared with Yugu1,the chlorophyll content in the upper leaves of T182 was significantly reduced after treatment,indicating that SiSAMS1 may be involved in ethylene-mediated leaf senescence.(7)Transcriptome analysis showed that the differentially expressed genes of Yugu1 and T182 are mainly involved in photosynthesis-related pathways such as photosystem assembly,chlorophyll biosynthesis,photosynthetic antenna proteins and carbon fixation of photosynthetic organisms,followed by secondary metabolite synthesis and decomposition pathways.It indicates that the mutations of SiSAMS1 affect multiple metabolic pathways. |
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