Studies On Photosynthetic Characteristics And Chlorophyll Synthesis Of Chlorophyll-reduced Mutant In Brassica Napus L.

Leaf color Leaf yellow is a common type of leaf color mutants,which can be used as a marker trait in hybrid breeding and used for purity identification as a high-quality material for studying chloroplast development,chlorophyll biosynthesis,photosynthesis,nucleoplasmic gene interaction and leaf color gene genetic control.The yellow mutant crm1 was previously isolated from Brassica napus 2B seeds by EMS mutagenesis.In this study,the photosynthetic characteristics and chlorophyll synthesis of the mutant were analyzed,and the main research results were as follows:（1）By observing the growth period and measuring the dynamic plant height,it was found that the initial flowering,full flowering and final flowering periods of the yellowing mutant crm1 were 3-5 days later than those of the wild type,and the growth period was about 4 days longer than that of the wild type.After lichen extraction,the plant height of the mutant was shorter than that of the wild type.At the mature stage,the plant height of the wild type was about 172.4 cm,while that of the mutant was about 147.4 cm,and the plant height was about85.5%of that of the wild type.These results indicate that the mutation of crm1 gene caused the mutant to prolong the growth period and weaken the growth of the plant.（2）By cytological observation of the leaves in the five-leaf stage,it was found that compared with the wild type in the same period,the structure of the flat leaves of the yellowing variant crm1 was abnormal.Specifically,the palisade tissue and spongy tissue of crm1 leaves were loosely arranged,the mesophyll cell space was enlarged,the number of starch grains in the cells was significantly reduced,and the cells were arranged irregularities and irregular,but the microscopic structure of leaf veins was not significantly different from that of the wild type in the same period.The results showed that the development of crm1 leaf structure was affected by the mutation of leaf color（3）The net photosynthetic rate,stomatal conductance and transpiration rate of the yellow mutant crm1 flat leaves were significantly lower than those of the wild type,while the intercellular carbon dioxide concentration was not significantly different,indicating that stomatal factors were the main reason for the decrease of the net photosynthetic rate of crm1.The chlorophyll fluorescence kinetic parameters were measured.The F₀,Fm,F₀’of the yellow mutant crm1 were significantly lower than that of the wild type.The Fv/Fm,Fv’/Fm’,ΦPSII,ETR and q_pof the yellow mutant crm1 were not significantly changed,but the NPQ was significantly increased by about 20.5%.These results indicated that crm1 photosystem II exhibited high heat dissipation efficiency,which affected the photosynthesis of crm1.（4）The contents of chlorophyll biosynthesis intermediate metabolites were determined.Ala,PBG and Urogen III in leaves were not significantly different from those of wild type,but the content of Coprogen III was 49.3%higher than that of wild type.The synthesized Proto IX,Mg-proto IX and Pchlide were significantly lower than the wild type.Therefore,combined with the previous results of significantly reduced chlorophyll content in the mutant,it is speculated that its chlorophyll biosynthesis may be blocked by the process of Proto IX formation of Coprogen III.（5）The expression levels of chlorophyll-biosynthesis pathway related genes in chlorophyll-biosynthesis mutant crm1 and wild-type crm1 were detected by real-time quantitative PCR assay.The expression levels of most chlorophyll-biosynthesis related genes in crm1 including HEMA1,GSA1,HEMB1,HEMF1,CRD1,PORA and CAO were significantly down-regulated.Therefore,The above results indicate that the crm1 gene mutation affects the expression of most genes related to chlorophyll biosynthesis in the mutant.（6）Through leaf color genetic analysis,it was found that the F₁generation of crm1 and wild-type 2B were normal green leaves.The self-crossing of F₁generation resulted in F₂generation,and the separation ratio between green leaf plants and yellow-leaf plants was15:1,indicating that the crm1 phenotype may be controlled by two pairs of recessive nuclear genes.