Akebia trifoliata (Thunb.) Koidz may have applications as a new potential source of biofuels owing to its high seed count, seed oil content, and in-field yields. However, the pericarp of A. trifoliata cracks longitudinally during fruit ripening, which increases the incidence of pests and diseases and can lead to fruit decay and deterioration, resulting in significant losses in yield. Few studies have evaluated the mechanisms underlying A. trifoliata fruit cracking.
In this study, by observing the cell wall structure of the pericarp, we found that the cell wall became thinner and looser and showed substantial breakdown in the pericarp of cracking fruit compared with that in non-cracking fruit. Moreover, integrative analyses of transcriptome and proteome profiles at different stages of fruit ripening demonstrated changes in the expression of various genes and proteins after cracking. Furthermore, the mRNA levels of 20 differentially expressed genes were analyzed, and parallel reaction monitoring analysis of 20 differentially expressed proteins involved in cell wall metabolism was conducted. Among the molecular targets, pectate lyases and pectinesterase, which are involved in pentose and glucuronate interconversion, and β-galactosidase 2, which is involved in galactose metabolism, were significantly upregulated in cracking fruits than in non-cracking fruits. This suggested that they might play crucial roles in A. trifoliata fruit cracking.
Ripened Peach Crack
Our findings provided new insights into potential genes influencing the fruit cracking trait in A. trifoliata and established a basis for further research on the breeding of cracking-resistant varieties to increase seed yields for biorefineries.
However, the pericarp of A. trifoliata cracks longitudinally along the ventral suture when matured. Fruit cracking is a serious problem that increases the incidence of pests and diseases, leading to fruit decay and deterioration, affecting the utilization rate of fruit and seeds and causing significant losses in yields and commercial value [14, 15]. Studies have indicated that the dry mass and oil contents of insect-infested fruits (33.1 mg/fruit and 3.9%, respectively) are lower than those of healthy fruits (67.4 mg/fruit and 39.9%, respectively); moreover, the observed fruit and oil yields (2.9 and 0.6 kg/tree, respectively) are lower than the excepted yields (4.7 and 1.9 kg/tree, respectively) [16]. Fruit ripening and cracking is a complex, genetically programmed process that is accompanied by the synthesis of large amounts of proteins and the transcription of many genes [17, 18]. Studies have indicated that different genetic accessions exhibit major differences in cracking resistance, suggesting that genetic factors play important roles in sweet cherry fruit cracking resistance [19]. Studies on fruit cracking have been performed in many species, including tomatoes, litchis, durians, and apples; these studies have suggested that cell wall-modifying proteins, such as polygalacturonases (PGs), pectinesterase (PE), β-galactosidases (β-GALs), expansins (EXPs), and xyloglucan endotransglycosylase proteins [14, 20,21,22], are associated with fruit cracking. The basic helix-loop-helix (bHLH) gene INDEHISCENT regulates Lepidium campestre fruit dehiscence [23]. Additionally, Dong et al. [24] found that pod shattering resistance in soybeans was mediated by the NAC gene. Sorefan et al. [25] indicated that a regulated auxin minimum was required for seed dispersal in Arabidopsis. Consequently, a better understanding of the genetic analysis of fruit cracking is necessary to prevent cracking phenotypes.
Although many studies have evaluated fruit cracking, little progress has been made with regard to our understanding of the molecular mechanisms underlying A. trifoliata fruit cracking, and only two studies have performed transcriptome sequencing in this species [1, 26]. Our limited knowledge of the molecular characteristics of A. trifoliata has made it difficult to recommend preventive measures for fruit cracking. Next-generation sequencing methods, such as transcriptome and proteome technologies for measuring gene expression and protein abundance, have become powerful tools for the discovery of novel genes and their functions in regulating fruit ripening and cracking [27, 28].
Therefore, in this study, an integrative analysis of the transcriptome and proteome was performed to enhance our understanding of A. trifoliata fruit cracking during ripening at the molecular level. Our data provide important insights into omics resources and candidate genes responsible for fruit cracking traits in A. trifoliata.
First, we evaluated the dynamic structures of fruit pericarps during different stages of ripening (Fig. 1). In the non-cracking stage (PS), the pericarp cells and cuticles were densely arranged, with small intercellular spaces and continuous distribution (Fig. 1a, d, g). However, in the initial cracking stage, the cell wall thinned, the cell volume increased, the number of cell layers decreased, and the cells were loosely arranged with poor integrity; moreover, the spacing between the cells increased, and the cell of the exocarp and mesocarp began to degrade in the initial cracking stage (PM; Fig. 1b, e, h). Irregularly arranged layers with continuously reduced numbers and larger spaces were observed as an evidence of cell degradation throughout the cracking stages (PL; Fig. 1c, f, i).
Summary of some of the biological pathways involved in A. trifoliate fruit ripening and cracking. TRINITY_DN143250_c1_g6 (PL); TRINITY_DN196976_c0_g1 (PG), TRINITY_DN142042_c0_ g2 (PG3); TRINITY_DN142943_c1_g1 (PG2); TRINITY_DN143028_c0_g1 (PE); TRINITY_ DN141074_c0_g1 (PE2); TRINITY_DN137437_c3_g1 (BGLU33); TRINITY_DN141880_c0_g1 (ENBG); TRINITY_DN142424_c1_g1 (F26G); TRINITY_DN141432_c1_g2 (BXL). Red indicates significantly upregulated proteins, and blue indicates significantly downregulated proteins. White indicates proteins with no significant changes
Fruit cracking occurs when the stress exerted on the pericarp from the enlarged aril is greater than the strength of the fruit skin, and the mechanical strength of the pericarp depends largely on its cell wall [29]. Studies have shown that jujube fruit cracking may be related to structural changes and rearrangement of the cell wall during the later stages of fruit ripening [30]. Arrangement of the subcutaneous layers of cells was found to be relatively regular, and cell layers had a closer arrangement in the cracking-resistant tomato genotype [31]. In this study, the cell wall of the pericarp had poor integrity, loose structures, deformed and reduced cell layers, and larger spaces and began to degrade in cracked fruits during ripening, consistent with previous results in grapes and oranges [32, 33]. This suggests that structural changes in the cell wall might play key roles in the occurrence of A. trifoliata fruit cracking.
Some inevitable passive processes will occur in the cracked pericarp following fruit cracking, such as oxidative stress and microbial invasion [27]. Therefore, differences in the expression of genes and proteins among PS, PM, and PL alone cannot accurately reflect the cause of fruit cracking. In this study, our comparative analysis of protein and gene expression levels indicated that more DEGs and shared DEGs than DAPs were identified in cracked fruits than in non-cracking fruits. This could be explained by the technical limitations of MS-based proteomics, such as low amount of readily available samples and low MS scanning rate for quantitative data acquisition, and the need for extensive fractionation, limiting the capacity to identify proteins [38]. The PPI analysis indicated that the interactions of proteins were infrequent and weak, which was associated with the results that the interactions are often weak for many cellular processes, which are regulated by post-translational modifications that are recognized by specific domains in protein binding partners [39]. Moreover, correlation analyses showed negative correlations between the proteome and transcriptome, indicating a discordance between the transcript levels and protein abundance. These results were similar to those observed in previous reports, suggesting that post-transcriptional and post-translational regulation, reversible phosphorylation, splicing events in cells, and translation efficiency play key roles in the regulation of fruit ripening [40, 41]. Thus, gene translation and post-translation processes could be important regulatory methods in fruit ripening and cracking.
GO and KEGG functional enrichment provide prediction information of inner-cell metabolic pathways and of the genetic and biologic behaviors of genes. GO terms associated with oxidoreductase activity and structural molecule activity were mainly enriched in the PM_PS and PL_PM groups, respectively, similar to previous studies showing that the target genes could be classified into different categories based on their functions, such as peroxidase and cell wall polysaccharide [20, 42]. Cell polysaccharides are degraded by cell wall hydrolases, and whereas phenolic crosslinking of cell wall structural components is catalyzed by cell wall peroxidase. These modifications reduce the strength of the fruit pericarp, resulting in changes in pericarp development and fruit cracking [43]. KEGG analysis indicated that cell wall-related pathways, including pentose and glucuronate interconversions, the galactose metabolism pathway, and the phenylpropanoid biosynthesis pathway, were common pathways shared by DEGs and DAPs, suggesting that cell well metabolism may have important roles in A. trifoliata fruit ripening and cracking. Moreover, these results revealed that the proteomic data and transcriptome data were complementary, and that the proteome could confirm the transcriptome data; in addition, genes perform the same function at the transcriptome and proteome levels [44]. Functional classification of the transcriptome and proteome could improve our understanding of the molecular physiology of fruit ripening and cracking. 2ff7e9595c
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