Transcriptome profiling reveals the mechanism of ripening and epidermal senescence in passion (Passiflora edulia Sims) fruit

Passion fruit (Passiflora edulia Sims), an important tropical and sub-tropical species, is classified as a respiration climacteric fruit, the quality deteriorates rapidly after harvest. To reveal the mechanisms involved in ripening and rapidly fruit senescence, the phytochemical characteristics and RNA sequencing were conducted in the purple passion fruits with different (1-MCP and PF) treatment. Comprehensive functional annotation and KEGG enrichment analysis showed that the starch and sucrose metabolism, plant hormone signal transduction, phenylpropanoid biosynthesis, flavonid biosynthesis, carotenoid biosynthesis were involved in fruit ripening. Applying with PF and 1-MCP significantly affected transcript levels of passion fruit after harvest storage. A large number of differently expressed unigenes (DEGs) were identified significantly enrichen in starch and sucrose metabolism, plant hormone signal transduction and phenylpropanoid biosynthesis at postharvest stage. The preservative film (PF) and 1-Methylcyclopropene (1-MCP) treatments increased superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) gene expression and enzyme activities, accelerated the lignin accumulation, decline β-galactosidase (β-Gal), polygalacturonase (PG) and cellulose activities and gene expression to delay cell wall degradation during fruit senescence. The RNA sequencing data of cell wall metabolism and hormone signal transduction pathway related unigenes were verified by RT-qPCR. The results indicated that the cell wall metabolism and hormone signal pathways were notably related to passion fruit ripening. PF and 1-MCP treatment might inhibited ethylene signaling and regulated cell wall metabolism pathways to inhibited cell wall degradation. Our results reveal ripening and senescence related networks during passion fruit ripening, which can provide a foundation for understanding the molecular mechanisms underlying PF and 1-MCP treatment on fruit ripening.

raw data containing adaptors and poly-N and low-quality reads were removed. Then sequence 123 duplication level of the clean reads were assembled into expressed sequence tag clusters (contigs) 124 and de novo assembled into transcript and the Q20, GC content were calculated by using Trinity [25] .

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Blastx (E-value< 0.00001) was employed to search for homologues of our assembled unigenes and 126 annotated in protein databases including NR, KOG, SwissProt and PFAM and database. The best 127 results were used to determine the sequence orientations of the unigenes. The functional annotation 128 by GO terms (http://www.geneontology.org) was analyzed using the program Blast2GO. The COG 129 and KEGG pathway annotations were performed using Blastall software against the COG and 130 KEGG databases, respectively [26] .

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To verify the usability of the transcriptomic data, the relative transcript levels of 18 genes that 133 were either significantly up-or down-regulated were determined using quantitative real-time

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Effect of PF and 1-MCP treatment on physiological biochemical index of passion fruit 1-MCP-treated and control in the passion fruit at 0D-10D. And respiratory rate of passion fruit that 153 treated with 1-MCP and PF were significantly lower than CK group at 4D, 8D and 10D (Figure1B).

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The weight loss (shrinkage rate) of the control and 1-MCP-treated passion fruit showed a rapid 155 increase from 0D to 10D, while it notably suppressed by PF treatment (Figure1C). The control group 156 showed notably higher shrinkage rate than the 1-MCP and PF-treated passion fruit, flowing storage 157 for 2D-10D. The results of membrane permeability, respiratory rate and weight loss in PF treated 158 fruit were significantly lower than in 1-MCP and CK groups at 2D-10D.

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We determined ROS-scavenging enzymes activities, including SOD, POD and CAT. SOD

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However, the expression levels of DEGs in D group (CK for 4D) was not significantly changed 205 comparing with F (PF treatment for 4D) and H (1-MCP treatment for 4D) group ( Figure S1B).

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Therefore, we speculated that the 8D was a key stage to study the underlying mechanism of 207 senescence in passion fruit with PF and 1-MCP treatment.  Figure 5B). The expression level of these unigenes in B and C group were higher 236 more than 10-fold comparing with A group. Therefore, the results suggesting that these unigenes 237 may play an important roles to regulate the coloration of passion fruit during its growth and ripening.

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In addition, we also found that certain DEGs were significantly enriched into 'Phenylpropanoid the stage of B and C, compared with A. Among them, CCR (Cluster-29126.13688) and F5H

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(Cluster-29126.6512) were up-regulated significantly, which involved in the regulated degree of 244 lignification in passion fruit during its development ( Figure 5C).Thus, we inferred that the degree 245 of lignification was also related to ripening or senescence in passion fruit. senescence. And we also found that PF and 1-MCP treatment can affected the expression of these 280 cell wall metabolism related unigenes, to delay the senescence of passion fruit.

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To validate the transcriptome sequencing data, eighteen unigenes with different expression 283 patterns were selected for qRT-PCR analysis ( Figure S2). These genes included three 'Carotenoid

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PCR data showed a positive correlation in this study (R 2 =0.564, p<0.01, Figure S2). It indicated 294 that the RNA_Seq data was reliable to further analysis. Climacteric fruit usually suffers from rapid senescence after harvest, which exhibits increase 307 in weight loss, respiratory rate, ethylene production rate and physiological disorders at room 308 temperature storage. The results will lead to fruit quality deterioration, tissue softening, dehydration 309 and flavor changing [29,30] . Purple passion fruit exhibited a typical climacteric pattern during 310 postharvest ripening [31] . Therefore, in order to study the mechanism of fruit senescence and 311 preservation during storage process, we determined the physiological and biochemical indexes in 312 passion fruit by PF and 1-MCP treatment at room temperature storage. Firstly, weight loss and 313 respiratory rate were determined in different storage time points with PF or 1-MCP treatment.

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Respiratory burst was elicited through aerobic and anaerobic respiration, which was regulated 315 specifically by related signal transduction. In this study, weight loss and respiratory rate increased 316 progressively and peaked at the later stage of storage at room temperature. However, this increased 317 trend was significantly inhibited by 1-MCP and PF treatment, and lowest weight loss and respiratory 318 rate were shown in PF-treated fruit. The respiratory burst was mainly related to increased production 319 of reactive oxygen species (ROS), to enhanced peroxidase activity and stimulating lipoxygenase 320 pathway [32] , which were closely related to accelerated senescence of fruit.

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ROS accumulation level was controlled by the balance between capacity of ROS production 322 and scavenging [33] . The cell membrane, maintain relative stability in the internal environment, 323 might be damaged by excess ROS level which also cause peroxidization and accelerates senescence 324 in fruit [34] . Therefore, the enzymatic and non-enzymatic ROS scavenging systems to scavenge the 325 potential damage to the cells [35,36] . The antioxidant enzymes of ROS scavenger including 326 superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) etc [37] . Our result have 327 revealed that applying 1-MCP and PF postharvest could enhance SOD, POD, and CAT activities in 328 passion fruit, and the related gene expression pattern were also consist with RNA_seq analysis. In Besides ROS, other signaling pathways or molecular are also involved in plant senescence,

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including plant hormones transduction, ethylene, auxin, jasmonic acid and salicylic acid [39] . Plant 334 hormones are indispensable to regulate fruit ripening and senescence, which controlled fruit color,

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sugar, flavor and aroma during ripening and senescence [40] . And previous study reported that 336 ethylene and auxin were plays a major role in the ripening and senescence process of climacteric 337 fruits [41] . In present study, the result revealed that a large number of DEGs were enriched in plant The process of fruit senescence after harvest will leading to the irreversible destruction of 345 membrane integrity and to accelerated leakage of ions [42] . Loss of membrane integrity may lead to 346 subcellular de-compartmentalization which resulting in enzymatic browning catalyzed by 347 peroxidase and polyphenol oxidase in postharvest fruits [43] . The membrane integrity damage is 348 reflected by membrane permeability, which can be determined by the relative electrical conductivity 349 (REC) [44] . In the present study, REC increased under 1-MCP and control conditions after storage,

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Consist with our results, previous study reported that melatonin inhibited membrane phospholipid 354 degradation and maintained the degree of unsaturation fatty acids, which contribute to the 355 preservation of membrane integrity in tomato fruit [45] . The degradation of unsaturated fatty acids 356 results in destruction of the cell membrane integrity of the fruit peel [46,47] . PF and 1-MCP treatment 357 inhibit membrane lipid peroxidation that was beneficial for maintaining unsaturated degradation to 358 saturated fatty acids, which can maintain passion fruit cell membrane integrity.
structure and components were depolymerized by the action of cell wall hydrolases [49] . Senescence degrading enzymes in fruits [52] , which leading to cell wall loosing and fruit softening [53] . β-Gal was 372 a pectin-debranching enzyme which capable to simultaneously modify pectin and hemicellulose [54].

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And cellulase was widely regarded to cause the degradation of cellulose matrix in the cell walls of 374 fruits [55] . In the present results, the integrity of the cell wall was well maintained by PF and 1-MCP 375 treatment to inhibit the fruit softening and PF-treated showed better results than 1-MCP. Therefore,

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our results suggesting that PF treatment can alleviate passion fruit quality deterioration and suppress 377 the enzyme activities of β-Gal, PG and cellulase to inhibit cell wall components degradation.

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The expression of sucrose and cell wall degradation related DEGs during postharvest storage

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The content of sugar, fructose and glucose are the key factors in the formation of fruit quality, 380 which were significantly accumulated during the fruit ripening process [56,57] . However, the sucrose 381 and fructose content of passion fruit decreased during postharvest storage [7] . In this study, the DEG 382 expression level of starch and sucrose metabolism pathway was significantly up-regulated at 383 ripening stage in passion fruit, while these DEGs were all down-regulated in G and J group by 1-

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MCP and PF treatment. Therefore, we speculate that PF and 1-MCP can delay the senescence of 385 passion fruit.

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Starch, hemicelluloses, cellulose, and pectin are the major factors of cell wall polysaccharides 387 [58] . In present study, these related pathways were identified by RNA_seq, including sucrose 388 metabolism and lignin metabolism. Most of these unigenes were involved in the cell wall that the activities of POD, PAL, C4H, and 4CL were positively correlated with lignin accumulation 393 in loquat fruit [60] . In the present study, the lignin metabolism-related unigenes in 'Phenylpropanoid 394 biosynthesis' pathway were identified by RNA_seq analysis, such as POD, PAL, C4H, and 4CL.

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The results has consist with the reported that several postharvest approaches, such as 1-MCP and

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In general, these results consist with previous studies that exogenous 1-MCP, melatonin or

Conflicts of interest/Competing interests 424
The authors declare that they have no competing interests. There was no competing Interests in this work.

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Authors' contributions 426 CBL, MX and JS provided experimental design and plant material. CBL performed experiments and RNA_Seq data