Researches On The Fruit Quality Formation Mechanism Of Youxi Glossy Kumquat

Kumquat（Citrus japonica,once named as Fortunella japonica）,an ever-green shrub or small tree belonging to genus Citrus of Rutaceae,is originated from China.Its fruit flavor is very special and is popular for many consumers.In our previous study,a seedling mutant of Youxi kumquat was discovered in Youxi city of Fujian Province.Its main mutation characteristics include the significantly deceased number or even disappearance of oil vacuoles in fruit peels and much more compact flesh.Therefore,it was tentatively named as Youxi glossy kumquat.To investigate the underlying fruit quality mutation mechanism of Youxi glossy kumquat,several fruit quality-related parameters of Youxi glossy kumquat and normal Youxi kumquat（as control）were determined and compared.The carotenoids and flavonoids metabolism differences between the two kinds of kumquat fruits were further explored by determining their contents and the expression of related structural genes in fruits at different ripening stages.To explore the underlying mutation mechanism of Youxi kumquat,chloroplast genomes of Youxi glossy kumquat and Youxi kumquat were assembled and compared,and comparative transcriptome analysis was also applied to identify the differentially expressed genes（DEGs）and their significantly enriched pathways between Youxi glossy kumquat and Youxi kumquat.Moreover,based on the comparative transcriptome analysis data,many biochemical indexes related to the pathways significantly enriched by DEGs,such as plant hormones,cellulose and total calcium content,were determined.The main results obtained in this study are as follows:1.Fruit quality comparison results between Youxi glossy kumquat and Youxi kumquatThe edible rate of Youxi glossy kumquats was 97.09%with the total sugar content of 36.41 mg/g,and soluble solid content of 12.68%,which were all significantly higher than those of the Youxi kumquat.The L and b values of Youxi glossy kumquat were significantly higher than those of the Youxi kumquat,while the a value of Youxi glossy kumquat was significantly lower than Youxi kumquat.The fruit texture indexes of the two kinds of kumquat fruits also differed a lot.The hardness,brittleness,springiness,gumminess and chewiness of Youxi glossy kumquat fruits were significantly higher but its adhesiveness value was significantly lower than Youxi kumquat.2.The carotenoid and flavonoids metabolism differed greatly between Youxi glossy kumquat and Youxi kumquat.The carotenoid content in the peels and pulps of Youxi glossy kumquat and Youxi kumquat both increased as fruit ripening.Moreover,the expression levels of seven carotenoid biosynthesis related structural genes in the peel of two kumquats,including PSY,PDS,ZDS,CRTISO,LCYe,HYb and ZEP,were found to be significantly positively correlated with carotenoid content.The flavonoids content in peels of the two kinds of kumquats decreased as fruit ripened,but the flavonoid content in pulp both showed a‘rise-fall’change pattern.The expression of flavonoid metabolism-related genes in the two kinds of kumquats peel were significantly different.In fruits at 90 days after flowering,the contents of carotenoids and flavonoids in the peel of Youxi glossy kumquat were significantly higher than those of Youxi kumquat.In fruits at 180 days after flowering,the contents of carotenoids and flavonoids in the peel of glossy kumquat were respectively 0.221 mg/g and 22.08 mg/g,accounting for 1.66-and 1.85-times of Youxi kumquat,respectively.3.Molecular mechanism underlying the fruit mutation of Youxi glossy kumquatThe chloroplast genomes of Youxi kumquat and Youxi glossy kumquat were assembled.Sequence comparison results showed that there was no difference between the two chloroplast genomes,indicating that the glossy fruit mutation is not caused by the chloroplast genome sequence differences.RNA-Seq analysis was further applied to investigate the transcriptome profiling differences in fruits of Youxi kumquat and Youxi glossy kumquat at three different ripening stages（90,120 and 150 days after flowering）.In total,we identified 1823 differentially expressed genes（DEGs）between fruits of Youxi glossy kumquat and Youxi kumquat.Most of them were found to be down-regulated in Youxi glossy kumquat.GO enrichment analysis results showed that these DEGs were significantly enriched in biological process,metabolic process,oxidation-reduction process,organic acid metabolic process,carboxylic acid metabolic process and oxoacid metabolic process and so on.KEGG enrichment analysis showed that DEGs were mainly involved in biosynthesis of secondary metabolites,biosynthesis of amino acids,plant hormone signal transduction,plant-pathogen interaction and other pathways.Notably,140 DEGs were found to be common DEGs between Youxi glossy kumquat and Youxi kumquat at three different stages.Mapman annotation results revealed that these DEGs are mainly in the cell wall biosynthesis,calcium ion binding,programmed cell death,plant hormone synthesis,flavonoid and carotenoid synthesis,lipid metabolism,stress and so on,indicating that they play important roles in the formation of the glossy mutation.4.Validations of physiological and biochemical indexes related to the pathways significantly enriched by DEGsBased on the results obtained from the transcriptome data,physiological and biochemical indexes related to the DEG enriched pathways were further determined.Results showed that endo-β-1,4-glucanase（Cx）,peroxidase（POD）,polyphenol oxidase（PPO）,glutathione S-transferase（GST）and chitinase activities showed similar change patterns in the two kinds of kumquats.In fruits at 150 days after flowering,the difference of GST activity between Youxi glossy kumquat and Youxi kumquat was the largest,and the GST activity in Youxi glossy kumquat is about 2.31-times of Youxi kumquat.The change patterns of pectinesterase（PE）and phenylalanine ammonia lyase（PAL）in the two kinds of kumquats were opposite.In fruits at 150 days after flowering,except for total calcium content,the contents of soluble protein,cellulose,flavonoids and carotenoids in Youxi glossy kumquat were significantly higher than the Youxi kumquat.There was little difference in auxin（IAA）and zeatin（ZT）between the two kinds of kumquats at 90,120 and 150days after flowering,while their abscisic acid（ABA）,gibberellin（GA₃）,zeatin riboside（ZR）,salicylic acid（SA）and jasmonic acid（JA）contents were significantly different.The accumulation of SA,ZR,ABA and JA in Youxi glossy kumquat were significantly higher than Youxi kumquat.Notably,the JA content in Youxi glossy kumquat fruit was always at a high level,and its content in fruits at 90 days after flowering was the highest,accounting for approximately 3.49-times of Youxi kumquat.Moreover,the GA₃content in Youxi glossy kumquat was low in fruits at different ripening stages and was significantly lower than that in Youxi kumquat at all ripening stages.The results obtained in this study showed that the overall fruit quality of glossy kumquat was better than Youxi kumquat.The disappearance of oil vacuoles in glossy kumquat might be closely related to the down-regulation of PCD-related genes.The Cx and PE activities differences might contribute greatly to the oil vacuole formation by influencing the cell wall degradation during secretory cavity formation.Moreover,these two enzymes might be close related to the fruit texture differences between Youxi glossy kumquat and Youxi kumquat.Some stress resistance related indexes in fruits of Youxi glossy kumquat,such as SA,JA,PAL contents and GST activity,were significantly higher than Youxi kumquat,which might be related to the fruit stress resistance（such as storage ability）of Youxi glossy kumquat.