The Coloring Mechanism Of Pitaya Fruit Based On Cytohistological Observation And Omics Analysis

Pitaya,belonging to the genus Hylocereus(Berger)Britton and Rose(Cactaceae),being species of vine cactus,are perennial tropical fruit trees,and well-known for their unique shape,color as well as rich nutrition.Also,it is the major fruit tree in karstic and rocky desertification area of Guizhou Province,P R China.Red pulp pitaya(Hylocereus.polyrhizuz)and white pulp pitaya(Hylocereus.undatus)are main types grown in Guizhou.Color is an important quality character of the fruit.Betalains is the main coloring substance of pitaya,with antioxidant function,widely used in food field.Therefore,red pulp pitaya is more appealing than the white.Meanwhile,betalains had been attracting the attention for more scientific researchers.Even so,the available information on the pitaya betalains is still limited.In this study,'Zihonglong'(with red peel and pulp)and'Jinghonglong'(with red peel and white pulp)were used as materials,histocytological characteristics of the fruits between two cultivars were observed during the developmental stages,and the comparative analysis of metabolome,proteome and transcriptome were further carried out for the pulp of the two cultivars,so as to illustrate the coloring mechanism of pitaya fruit.The main result are as follows:1.Increase in pigmented cells of pulp promoted fruit coloringRed pigment appeared in ripe pitaya pericarp,and the emergence of red pericarp indicate fruit maturation.Red pigment in pitaya pericarp mainly appeared in the vacuoles of epidermal cells and 2-3layers of cell under epidermis,which distributed in a linear status in flattened cells.In pulp,the accumulation of red pigment was observed as scattered spots during the color-breaking period,meanwhile,some red tissues appeared as short lines.The red pigment first concentrated in the core of the pulp,as the fruit developed,the red pigment spread radially all around.The microscopic observation in cell coloration showed that the red pigment appeared mainly in the form of sporadic red dot in pulp cells firstly,as meanwhile,the red pigment dispersed in cell tissue with the cell development,wherease,the pigmentation of different pulp cells was not synchronized.In mature pulp,cells mainly presented as individuals and the coloring process of each cell were different,neverthelessthe,cells pigmented were relatively concentrated.2.Color parameters varied in fruit types and tissuesThe L*value,representing the brightness of fruit surface of peel and pulp showed a decreasing trend as fruit development.At the early stage of fruit development,the L*value of pulp was higher than that of pee,while the L*value of pulp decelined dramatically in mature fruit and was slightly lower than that of peel.The C*parameters in peels did not varied significantly during the fruit development stages,while in pulp,which exhibited a mixed and dim colors before coloring,but a few color type and brighter color when pigmentated.During the fruit developmental stages,there was no significant change in L*,a*,b*,C*and h~0 values of‘Jinghonglong'pulp,whererase,a significant change was observed in L*,a*,b*,C*and h~0 values of‘Zihonglong'.There was no significant difference in color parameters of pulp between‘Zihonglong'and‘Jinghonglong'before coloring,while the a*,C*and h~0 values of‘Zihonglong'pulps increased significantly along with the appearance of red pigment,however,the L*values decreased gradually as the formation of red pigment.The vary of color parameters in pitaya tissue preceded that of pigment contents.3.Four potential differential metabolites were justified to be related to betalais synthesisUsing the non-targeted metabonomics platform,the metabolic spectrum of‘Zihonglong'pulp and peel in nine development stages was analyzed by GC-MS and LC-MS methods,and a total of 65metabolites were identified.The PCA analysis were performed,we found that the two different fruit parts and the nine development stages of pulp were obviously separated,but the nine development stages of peel were relatively concentrated on PCA diagram.According to the heat map of metabolites,the clustering results of samples were consistent with the fruit development stages,indicating that identified metabolites could represent the characteristics of each sample.The contents of amino acids,sugars and organic acids of pulps were higher than that of peels.In pulps,the content of amino acid decreased with the development of the fruit,whereas,the content of sugar and organic acid were lower in fruit early stages and increased dramaticaly while the fruit was approaching to be mature.The correlation between major metabolites and betalains,the significant positive correlateions were investigated between the contents of fructose,glucose,sorbose,glycoside as well as citric acid and the contents of betalains in the pulp.The metabolites comparation between peel and pulp of‘Zihonglong'was carried out,four common metabolites,e.g.tyramine,citramalic acid,tryptophan and tryptamine were screened as potential biomarker metabolites.4.The main enrichment pathway of differential metabolites between H.polyrhizuz and H.undatus was discoveredA total of 55 metabolites were identified in the pulp samples of‘Zihongllong'and‘Jinghonglong'from four developmental stages.The two cultivars and the four developmental stages were well separated in PCA graph,indicating that metabolites varied obviously in different cultivars and developmental stages.In general,as the fruit developed,the content of most amino acids decreased,while the content of sugar and acid increased,and the content of betalains increased rapidly in'Zihonglong'fruit approaching maturity.The characteristics of different metabolites between red and white pitaya pulp were compared,it was found that the different metabolites were mainly enriched in metabolism of phenylalanine and tyrosine,and the metabolic pathways involved in tyrosine metabolism,aminoacyl biosynthesis,isoquinoline alkaloid biosynthesis and nitrogen metabolism.5.Seventy-eight candidate proteins involved in betalains regulation were identifiedLC-MS/MS-based label-free proteomics approach was used to assess the differential proteome between red and white pulps.We deep sequenced the c DNA from pitaya pulp to obtain transcript libraries,which were used in the mass spectrometry-based identification of the pulp proteins during fruit development.A total of 5,994 pulp proteins were identified,including 5,235 in‘Zihonglong'and5,295 in‘Jinghonglong'respectively,amongwhich,78 DEPs were identified as the candidate proteins involved in betalains regulation,and the DPEs were classified according to their putative function and known biosynthetic pathways.The majority of the most DEPs were categorized as plant stress resistance,pigment synthesis,product metabolism(amino acid metabolism,N metabolism)and fatty acid metabolism.The present study appears to define well some of the biochemical and physiological events that characterize the biosynthesis of pitaya betalains,and provides a basis for further study on revealing betalains synthesis through proteome approach.6.The full-length transcriptome data of pitaya were establishedThe SMRT(Single Molecule Real-Time Sequencing)corrected with RNA-Seq technology was carried out to explore gene sequence on pitaya fruit for the first time.65,312 genes from the pulp of H.polyrhizuz and 91,638 genes from the pulp of H.undatus were obtained,the average length of the genes were 1,175 nt and 1,337 nt respectively.111,650 and 11,113 lnc RNA were predicted from the pulp of H.polyrhizuz and H.undatus respectively.Among which,104 genes involved in betalains synthesis were identified.The obtained transcriptome provides a practical pipeline for further exploration of the genetic data and biosynthesis of betalains in pitaya fruit.The correlation between gene expression level and color indexs were analyzed,two new pitaya genes Hp CYP76AD4(I1?HQ?R?C 13003/F5P0/1979)and Hp DODA(I1?LQ?R?C 96099/f1p0/1004)involved in betalain synthesis were found,furthermore,three new potential genes(Hp GSTs,Hp NAC and Hp CYP704C1)that may be involved in the regulation of betalains synthesis in pitaya pulp were identified.Based on the combined analysis of metabolome,proteome and transcriptome,it was found that the expression of DODA gene was inhibited in the pulp of H.undatus,which resulted in the inability to synthesize betalamic acid and the absence of betalains.