Studies On The Molecular Mechanism Of Capsanthin Synthesis And Regulation In Pepper Fruits

Capsanthin is a natural edible red pigment, it is a metabolic end product in red pepperfruits carotenoid biosynthetic pathway, which account for60%of the content of mature fruitpigment. Capsanthin has the virtue of brilliant color and good staining function. It canenhance antistaling time of food, non-poisonous effects on God’s image. Thus, it will replacechemical synthesis of pigment by degrees in the near future, and will be applied widely in thefood processing department. Capsanthin/capsorubin synthase (Ccs) catalyzes zeaxanthin toform capsanthin. The expression of Ccs gene is undetectable in immature pepper fruit. Whenpepper fruits begain to being green mature, and Ccs gene began to express and graduallyincreased, capsanthin subsequently formed and continuously accumulate. Research indicatedthat capsanthin can not be synthesized because of mutation or deletion of Ccs gene in yellowor orange pepper fruits, so, the ripened fruits could not turn red.Currently, peppers (Capsicum) are widely cultivated as a natural pigment resource inparts of Chinese north-west regions. The lack of rain aggravated pepper production in theseareas. So, plastic greenhouse cultivation becomes a major planting mode of pepper production.But plastic films as the main component of greenhouses are often used for many years, andthe rate of light permeation of plastic films is declining by degrees, with poor light conditionsin plastic sheds. All of these will affect pepper pigment production.With the development of capsanthin research, the biosynthesis pathway of capsanthin isclear basically, but it was few concern how environmental factors affect the synthesis ofcapsanthin. The molecular mechanism of environment effect on capsanthin synthesis is notclear. From that point, and jointing predecessor research results, we researched capsanthin bylearning environmental regulation to find out the molecular mechanism of capsanthinsynthesis, and to explain how environment factors affect the synthesis of capsanthin. Themain research conclusions include seven parts, as below:1. Soil drought stress affected capsanthin accumulation and expression of capsanthinbiosynthesis-re-related genes. Mainly including: capsanthin was determined under differentdegree stress; and the expression of capsanthin synthesis pathway genes (Ccs, Psy, Lcyb andCrtz) was analyzed. The results showed that drought significantly affected capsanthin synthesis in pepper fruits; there was no significant difference between the control and LDgroups, however, moderate to severe drought had the greatest impact on capsanthinaccumulation. Analyzing the expression of key genes in the capsanthin biosynthesis pathwayindicated that soil drought inhibited key genes express; especially, soil drought has a greatereffect on the expression of the lycopene-β-cyclase (Lcyb) and β-carotene hydroxylase (Crtz)genes, resulting in a significant reduction in the fruit capsanthin content in pepper.2. Fruit bagging affected capsanthin synthesis in the pepper. Mainly including: effect offruit bagging on fruit surface temperature and capsanthin synthesis in the stage of pepper fruitdevelopment; effect of pepper fruits bagging on the expression of capsanthinbiosynthesis-re-related genes in the stage of pepper fruit development. These researchconclusions showed that bagging inhibited capsanthin synthesis in the stage of pepper fruitdevelopment; of note, bagging had the greatest effect on capsanthin during the mature-greenstage of the fruit. Bagging restrained the accumulation of capsanthin, and the chief reason wasthat bagging inhibited Crtz and Ccs genes expression over the entire course of pepper fruitdevelopment and ripening, which may be the main reason that fruits bagging affectedcapsanthin synthesis.3. Using cDNA-AFLP technique, some differently expressed genes that fruit shadingcaused fruit color change were screened. We obtained and identified80transcript derivedfragments (TDFs), these TDFs were sequenced and analyzed. According to the phenomenonthat shading caused peel faded green, a TDF associated with chlorophyll was found bysequencing alignment. The full-length gene was obtained by electronic cloning andsequencing. By means of homologous protein three-dimensional structure prediction method，it was found that the gene was quite similar to the ribulose1,5-diphosphate enzyme carbonicacid (rbcL) gene of Cv.Voucher Blackcluster (GenBank: KF917582.1), and the protein of thisgene is a monomeric protein. The cluster analysis also showed that the gene and rbcL geneare in the same group. Bioinformatics analysis indicated that the gene may be concerned withthe synthesis of chlorophyll. And named CarbcL, and registered in GenBank (KM489073).Using VIGS technology to analyse CarbcL gene function, identification results showed thatpepper fruits faded green early and turn red when the gene was silenced, and CarbcL geneexpression level decreased strikingly. Finally, we researched CarbcL gene expression level byusing light-interrupted fruits. The results indicated that CarbcL gene expression level wasvery low in the shading; the color of light-interrupted fruits changed from dark green to thingreen; and the content of chlorophyll reduced considerably. These data further confirm thatCarbcL gene was indeed concerned with the synthesis of chlorophyll in pepper fruits, topertain to light-sensitive gene. These experimental results were obtained to clarify the phenomena that pepper fruit color faded green early and turn red under the condition of weaklight, to explain the regulatory mechanism of weak light influencing fruit color in the stage ofpepper fruit development.4. The appropriate concentration of ABA could improve the content of capsanthin inpepper fruits. Mainly including: optimizing optimal exogenous ABA concentration impacttingon capsanthin content; applying exogenous ABA effect on key genes of capsanthinbiosynthetic pathway. The expression level of key genes in ABA biosynthetic pathway whenexogenous ABA was sprayed on pepper fruits. The main conclusions were: the fruitcapsanthin content was increased highly than that of the control by using ABA150mg/Lconcentration. And the expression of capsanthin biosynthesis-re-related genes (Psy, Lcyb,Crtz and Ccs) is increased. And key genes (AO, Zep and NCED) which regulate ABAmetabolic pathway had an elevated expression level too. That lead to capsanthin content wasincreased. The resistant of pepper fruits to abiotic stresses had been improved with theincreasing of endogenous ABA content, it indirectly guaranteed the normal growth of pepperfruits.5. Further explanation was done about the relationship of genes and red fruit colors inpepper. Including concretely: Ccs gene was detected in Capsicum with different fruit colors;Ccs gene expression was analyzed in fruit color-changed Period; comparing the sequences ofCcs gene open reading frame in Capsicum with different fruit color. Our results showed Ccsgene not only exist and express normally in red pepper fruit (cv. R15), but also present andexpress normally in parts of yellow(cv.CK7) and orange (cv. R37-1) pepper fruit. Throughanalyzing the related genes expression of red (cv.R15), orange (cv.R37-1) and yellow (cv. CK7)Capsicum cultivars in capsanthin synthesis pathway，it was found that the synthesis ofcapsanthin was regulated not only by Ccs gene, but also by other genes. It suggested thatcapsanthin biosynthesis is very complex pathway in pepper fruits colors formation and theabnormal expression of any key genes will affect red color formation and capsanthinsynthesis.6. Establishing the VIGS detection system for fruit color-re-related genes in detachedpepper fruits. On the basis of previous studies, in detached pepper fruit was selected as theresearch matierials in this study. We constructed viral vectors (pTRV2-Psy, pTRV2-Lcyb,pTRV2-Crtz, pTRV2-Ccs), using the tobacco rattle virus (TRV) carrying target gene (Psy,Lcyb, Crtz and Ccs genes) to infect detached pepper fruits. By using VIGS technology, onegene of carotenoids synthesis in pepper fruit was silenced or several genes were silencedsimultaneously, and fruit color variation was observed. Explain the role of different genes in the formation of fruit red color. This provided us a technical support to better realize themolecular mechanism of pepper fruit color formation.7. Functions of specific repeat sequences of Ccs gene promoter were discussed. Byligating L0, L1, L2, L2-1and L3DNA fragments and plant expression victor PBI121containing CaMV35s promoter individually to form recombinant vectors PL0, PL1, PL2,PL2-1and PL3. These recombinant vectors (PL0, PL1, PL2, PL2-1and PL3) were transfectedto agrobacterium tumefaciens GV3101. The repeat fragments were analysed by analyzing theactivity of GUS gene. Compared with the control, PL0, PL1, PL2, PL2-1repeat unit had someactivities, and there were no significant differences among PL0, PL1, PL2, PL2-1promoterrepeat units; while PL3had a little activity. It should be preliminary concluded thatCis-element in the promoter of Ccs gene was not a dosage effect but a position effect. That isto say, a repeating unit of Ccs gene promoter can play a transcription and regulation role inCcs gene expression.