Reserch On The Physiological And Photosynthetic Characteristics And Mutation Mechanism Of The Chlorophyll-deficient Mutant

Melon(Cucumis melo L.)is a very popular and widely cultivated vegetable for its nutritional and flavour quality.Heilongjiang Province is an important commodity base of China's thin-skinned melon,the production area is about 70,000 hm².Melon quality,especially the content of sugar,is the key factor affecting economic benefits.The sugar in fruits is the production of photosynthesis,and inhibition of photosynthesis will affect the formation of sugar.Because leaves are photosynthetic apparatus,leaf color mutations will directly influence the accumulation of photosynthetic productions.Leaf color mutations are of special value in the researches of chlorophyll biosynthesis,photosynthesis mechanism,chloroplast structure,function and physiological and biochemical processes,plastid genetic,plastid-nuclear signaling,protein-protein interactions.Leaf color mutation can be a morphological marker that simplify crossbreeding.The melon chlorophyll-deficient mutant is a spontaneous mutation of cultivars.The mechanism of leaf color variation and the effect on photosynthesis is unclear.Proteomics,molecular techniques,and physiological analysis were performed on the mutant and wild-type.The results will provide bases for the application of the mutant in breeding to improve the quality of melon,studying metabolic pathways and physiological changes,and understanding the molecular mechanism of mutation.The main results are as follows:1.The mutant was smaller size than the wild-type during the whole growth stage,the fruit size,sugar content,and the fruit numbers per plant were not significantly different from the wild-type.The content of chlorophyll a,chlorophyll b and total chlorophyll in the mutant was significantly lower than the wild-type,respectively.The content of carotenoid was not significant difference between the mutant and the wild-type.It indicated the change of leaf color was mainly caused by the decrease of chlorophyll.The chlorophyll precursor Urogen?was significantly reduced in the mutant melon.It was speculated that the decrease of Urogen?content resulted in chlorophyll decrease.The ALA(Urogen?precursor substance)was accumulated in the mutant,which could be related to the oxidation resistance of the mutant.2.The net photosynthetic rate,stomatal conductance and transpiration rate decreased,the light compensation point and CO₂saturation point increased,and light saturation point and CO₂ compensation point decreased in the mutant.The daily change of net photosynthetic rate and the values of F₀,F_m,F_v/F_m and q P also decreased significantly.It was showed that PS?was protected by increasing q N in the mutant.The photosynthetic efficiency was improved by reducing photoinhibition,increasing the intercellular CO₂concentration and increasing the CO₂saturation point.3.The mutant had higher conductivity,higher concentration of reactive oxygen molecules(O₂^·-,H₂O₂),and the lower LOX activity.The antioxidant enzyme activities including SOD,POD,CAT and APX all significantly increased,respectively.It indicated that the mutant melon was under oxidative stress.The proline content increased to maintain the permeability that was changed by the active oxygen molecules.4.Comparative liquid chromatography-mass spectrometry was performed for protein profiling of wild-type and mutants.It was identified 475 differentially expressed proteins,of which 189 were upregulated and 32 were downregulated.Differentially expressed proteins were involved in the following biological processes:binding,catalytic,structural,transporter,and antioxidant activities.In addition,pathways enriched with these proteins included porphyrin/chlorophyll metabolism;carbon metabolism,ribosome,glyoxylate,and dicarboxylate metabolism;and carbon fixation.The mutant had 6.08-fold higher expression of glutamate-1-semialdehyde 2,1-aminomutase(GSAM),an enzyme that synthesizes the chlorophyll precursor 5-aminolevulinic acid,along with a 5.02-fold higher expression of pyridoxal biosynthesis protein,a GSAM coenzyme.However,psa D,psa A,psa C,psa B and psb L were downregulated by 2.01-,1.59-,1.43-,1.11-and 1.06-fold,respectively.Substantially altered expression of multiple enzymes associated with carbon fixation.Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit,Glyceraldehyde 3-phosphate dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase,transketolase were all differentially expressed.A catalase found in microtubules connecting chloroplasts to the cell nucleus increased in expression by 6.22-fold.An RNA recognition motif-containing protein(RRM)decreased in expression by 5.22-fold,while lipoxygenase—an enzyme related to hydrogenperoxide production—decreased by2.95-fold.5.The content of phosphoenolpyruvate carboxylase in the mutant was higher expressed by 3.8-fold than wild-type,and the activities of PEPC,NADP-ME and NAD-ME all increased in mutant melon.The phosphoenolpyruvate carboxylase was involed in C₄pathway and crassulacean acid metabolism,and other enzymes involved in C₄ pathway,such as malate dehydrogenases,were all increased in the mutant.And except for Calvin cycle,the pathways changes in KEGG were related to the production of CO₂.It indecated in melon might exist C₄ pathway.At normal growth environment melons fixed CO₂ by C₃cycle,and melons fixed CO₂by C₄ pathway in chlorophyll deficient or under stress status.6.To further validate the results from LC-MS/MS,the expression of RRM and GSAM was examined by Western blot analysis.The results showed increased protein levels of GSAM and decreased level of RRM in the mutant were with similar to MS results.It indicated the results of mass spectrum was reliable.7.Two down-regulated and seven up-regulated proteins were selected for analysis using quantitative real-time polymerase chain reaction.The results indicated that,except for RRM and actin,the regulated expression of GSAM,CAT,RCA,psa D,AOR,aminotransferase and pyridoxal biosynthesis protein occurred at the transcriptional level.8.The RRM was the factor that led to the decrease of chlorophyll in the mutant.It was speculated RRM was related to GSAM.9.The Crrispr/Cas9 expression vector of RRM gene was constructed,and the RRM gene was knocked out successfully.It was showed that RRM was related to leaf color change.