Characterization And Proteomics Of A Novel Xantha Mutant In Muskmelon

Muskmelon (Cucumis melo L.) is an important annual and economic crop, widely cultivatedin home and abroad, and its fruit is with good flavor, taste and color. Heilongjiang province is oneof the traditional sources of muskmelon, riches in types of resources and variation of germplasmfor breeding and biologic research. The leaf color mutant is an ideal material for study on thephotosynthetic system structure, chlorophyll metabolism, chloroplast development, photosynthesis,hormone and a series of physiological metabolic processes, which is an important material forstudy the genetic and breeding. Previous reports mainly focus on the laws of the occurrence andgenetics, but the mechanism of leaf color mutation in muskmelon is not clear.The novel xantha mutant9388-1of muskmelon was used in this study. The main agronomiccharacteristics, photosynthetic, chloroplast ultrastructure, chlorophyll biosynthetic and geneticcharacteristics of mutant were studied. Furthermore, the use of two-dimensional electrophoresisand mass spectrometry techniques obtained the differentially expressed proteins between themutant and parent leaf. All the results were to support the mechanism of transformation of theyellow leaf color. The main results are as follow:1. A novel xantha mutant of muskmelon arose spontaneously and displayed a distinctivephenotype of yellow leaf across its whole growth period. Compared to baishami1hao, the growthcycle is relatively long about7-10d, and the plant is normal growth. The main agronomic traitswere no significant differences, so the mutant can blossom and bear fruit, but setting ratedecreased. According to the flow cytometry analysis, the number of chromosome is no differencebetween xantha mutant and baishami1hao, indicating that the mutant did not variation ofchromosome. The vigor of pollen in mutant is viability.2. The photosynthetic pigment contents, photosynthetic rate and key enzymes, chlorophyllfluorescence kinetics parameters were determined. The results showed that the photosyntheticpigment content was significantly lower than the baishami1hao, chla, chab, total chl and carodecreased by79.5%,90.3%,81.5%,70.7%, and no significant differences between hybrid F1andbaishami1hao plant. Caro/chl8.67, higher than that of baishami1hao plant in4.10, the reductionof chl content was greater than caro. The net photosynthetic rate was significantly lower, were only50.23%and66.28%. The cond and Trmmol have significant difference, and change trend is sameto Pn. The activity of Rubisco was significantly lower than baishami1hao, while PEPCase andNADP-ME were higher, respectively,119.23%and118.91%. The value of F0and Fmweresignificantly lower than those of hybrid F1and baishami1hao, but there was no significant difference in Fv/Fm. qP is no difference with baishami1hao, and qN is significantly higher thanhybrid F1and baishami1hao, ФPSⅡ and ETR are different.hybrid F1plants have no significantdifferences with photosynthetic pigment contents, photosynthetic rate and key enzymes,chlorophyll fluorescence kinetics parameters from baishami1hao.3. Comparative study on the structure characteristics of different growth period leaf anatomicbetween the mutant and baishami1hao. Under the scanning electron microscope, the leafmesophyll tissue of baishami1hao is developed and high differentiation, and palisade tissuethickness is lager than that of mutant, closely spaced and rules arranged.The palisade tissue ofmutant underdeveloped, arranged no rules, cell spacing are further than that of normal, containingless chloroplasts. Under the transmission electron microscope, the chloroplast of normal plants aredeveloped, maintaining highly differentiated form of thylakoid grana, stromal lamellae clearlyvisible, and arranged neatly, containing much more starch.The chloroplast development of mutanthas obvious flaw, maintaining irregular chloroplast, no intact thylakoid and fewer granum, a linestrip, and most grana has only few pieces of layer, containing a small amount of starch.4. In order to understand the mechanism of chlorophyll biosynthesis, the precursors oftetrapyrrole synthesis content and key enzyme was measured to evaluate the mutant. The resultshowed that PBG accumulated in mutant leaf, and UrogenⅢ content decreased significantly, andthe activity of PBGD, COPX, PPOX were also significantly decreased, but activity of ALADhigher than that of baishami1hao leaf, indicating that the mutation of chlorophyll biosynthesis isblocked, which is in the reaction steps of PBG and UrogenⅢ.5. Endogenous hormones and amino acid content was determined in different growth stage,the result showed that the change of IAA, GA3and ABA of mutant are basically the same trend asbaishami1hao in the whole growth period. IAA and GA3content were no significant differencefrom baishami1hao, but the ABA content was significantly lower than baishami1hao. The mutantand baishami1hao leaf has the same amino acid component but not content, one of the mostsignificant differences are Glu and Asp, indicating that the ABA content decreased ABAbiosynthesis affect caused by chloroplast underdeveloped. Glu and Asp were in the upper reachesof entire amino acid metabolic pathway, its conversion blocked in mutant leaves resulting indownstream of amino acid metabolism declined.6. The activity of SOD, POD, CAT and ATP enzyme were measured in different growth stageof leaf in mutant and baishami1hao, the result showed that the activity of SOD in tendril and agingstage were significantly higher than that of baishami1hao, and in cotyledon, seedling stageshowed no significant difference. Compared to the baishami1hao plant, activity of CAT in mutantwas not obvious in cotyledon and seedling stage, but higher in straw stage. Activity of PODshowed a same trend, first increased and then decreased soon. In cotyledon and seedling stage,activity of POD increased slowly, and the change amplitude was significantly lower than that ofbaishami1hao. In tendril stage, reached the highest activity in mutant, while the arrival peak was delayed, and maintain the higher activity in fruit stage, and then decreased slowly. The activity ofMg++-ATP was significantly higher than the baishami1hao, and Ca++-ATP is relatively low.7. The xantha mutant in muskmelon was analyzed genetically after inbred and reciprocal,crosses with parental mutant, the result showed that progeny of inbred presenting a singlephenotype (yellow), and reciprocal test indicated that the progeny with a single phenotype (green).The test backcross and F2populations showed that the progeny present segregation ratio of1:1and3:1between baishami1hao green and yellow plant, indicating that xantha mutational trait iscontrolled by a single recessive nuclear gene designated as Yellow-plant (ypl) temporarily. SSRmakers linked to the leaf color mutant were identified, ypl is located between N41and MU4104-2,genetic distances are2.45cM and3.45cM，repectively.8. Proteomic analysis was performed to test the proteins expression was different in mutant andparent leaves, using2-DE and GC-MS, the result showed that chloroplast oxygen-evolving enhancerprotein1,phenylcoumaran benzylic ether reductase2, ribulose bisphosphate carboxylase largechain,chloroplast ribulose bisphosphate carboxylase/oxygenase activase1, ribulose-1,5-bisphosphatecarboxylase/oxygenase large subunit,chloroplast ribulose bisphosphate carboxylase/oxygenase activase1,Actin,Ribulose-1,5-bisphosphate carboxylase small subunit, phosphoribulokinase, rubisco activase wereidentified in18proteins.Compared to the baishami1hao plant,phosphoribulokinase, phenylcoumaranbenzylic ether reductase2, ribulose-1,5-bisphosphate carboxylase small and large subunit showed aup-regulated,chloroplast oxygen-evolving enhancer protein1,chloroplast ribulose bisphosphatecarboxylase/oxygenase activase1and Actin showed down regulated in mutant, which may be causedby chloroplast underdeveloped. According to the COG result, differentially expression of these proteins,protecting the photosynthetic mechanism not to be destroyed, and maintaining a series baishami1haophysiological processing of photosynthetic and energy metabolism and playing a key role in plantgrowth of mutant. The differential expression of these proteins might have occurred as a result inyellow color of xantha mutant.