Tomato Fruit Softening Research Articles

Double CRISPR knockout of pectin degrading enzymes improves tomato shelf‐life while ensuring fruit quality

Social Impact StatementTomato fruit is an important and popular commodity producing $95.62 billion worldwide. Tomato fruit losses in the supply chain vary between 25% and 42% depending on the production area and the availability of postharvest technologies. For many decades, conventional tomato breeding programs have focused on extending the shelf‐life of fresh‐market varieties. However, in many instances, consumer‐based quality traits were not considered a priority. Consumers are now demanding safe, nutrient‐rich, high‐flavor, and convenient fruit. Here, we demonstrate the use of gene editing to improve fruit shelf‐life and positively impact quality, which can help significantly reduce tomato fruit losses and meet consumer expectations.Summary Finding alternative ways to extend tomato fruit shelf‐life without reducing the quality is critical to ensure the accessibility and likeability of this commodity worldwide. Improving fruit firmness in tomato fresh‐market varieties directly impacts their shelf‐life potential. We simultaneously knocked out two pectin‐degrading enzymes, polygalacturonase (SlPG2a) and pectate lyase (SlPL), key for tomato fruit softening. We expected this gene‐editing approach to result in longer‐lasting fruit without negatively impacting consumer‐based quality attributes. By generating the double clustered regularly interspaced short palindromic repeats (CRISPR) knockout PGPL, we evaluated the combined functions of SlPG2a and SlPL on fruit quality, including shelf‐life attributes like firmness and water loss, fruit marketability, and disease incidence. We also assessed additional attributes impacting consumer acceptance, such as taste and aroma. We revealed that the enzymes SlPG2a and SlPL act additively, significantly impacting fruit firmness and shelf‐life, with the double CRISPR knockout PGPL outperforming the wild‐type fruit. Additionally, fruit quality traits, such as sugar: acid ratio, aroma volatiles, and skin color, were improved or not affected in the double CRISPR knockout PGPL compared to the wild‐type. The discoveries of this research provide new insights into the influence of pectin backbone degradation on fruit physiology and postharvest quality, which can be used in crop improvement programs to make fruit more resilient in the supply chain without compromising quality.

Gene-specific silencing of SlPL16, a pectate lyase coding gene, extends the shelf life of tomato fruit

Pectate lyase plays an important role in fruit development and ripening. Although earlier studies showed SlPL, a pectate lyase encoding gene, functions dominantly in tomato fruit softening, the functions of many other pectate lyase encoding genes involved in tomato fruit development and softening remain unknown. Here, gene-specific silencing of SlPL16 (Solyc06g083580) by RNA interference (RNAi) resulted in reduced pectate lyase enzyme activity, pericarp thickness, and soluble sugar content, and increased both the firmness and shelf life of tomato fruit. Through microscopic observation, it was found that the SlPL16-RNAi pericarp had reduced cell size, in comparison to the wild-type (WT). Moreover, gene silencing of SlPL16 resulted in the down-regulation of genes involved in cell wall degradation and pectin synthesis at various fruit developmental stages. Together, these results indicate that SlPL16 provides an important supplement of pectate lyase activity in tomato fruit softening, along with a fundamental role in cell expansion during early fruit development. Additionally, this study provides a potential gene locus in genetic engineering to improve fruit storage tolerance.

Elucidating the role of SlXTH5 in tomato fruit softening

Fruit softening in tomato (Solanum lycopersicum) is closely associated with cell wall disassembly, which is brought about through the action of a range of cell wall structure-related enzymes and other proteins such as expansins. Xyloglucan endotransglucosylase/hydrolase (XTH) (EC 2.4.1.207 and/or EC 3.2.1.151) has been proposed to be key player involved in xyloglucan metabolism. SlXTH5 showed the highest expression level among all SlXTHs during tomato ripening. In this study, the role of SlXTH5 involved in tomato softening was investigated in CRISPR-based knockout mutants of SlXTH5. Loss-of-function of SlXTH5 in transgenic tomato lines resulted in slightly firmer fruit pericarp, but significantly decreased their color index compared with azygous wild type (WT) control fruits. Increased paste viscosity was detected in CRISPR mutants, indicating that the activity of SlXTH5 is responsible for maintaining cell wall structural integrity. Immunocytochemistry studies were performed using the monoclonal antibody probe LM25 to examine the localization and distribution of xyloglucan in the pericarp cells of the CRISPR mutant fruits. The data indicated more xyloglucan was retained in the pericarp of CRISPR mutant fruit than in WT control fruit. This study revealed the link between SlXTH5 and xyloglucan metabolism and indicated the potential of manipulating SlXTH5 to regulate fruit softening. .

CRISRP/Cas9-Mediated Targeted Mutagenesis of Tomato Polygalacturonase Gene (SlPG) Delays Fruit Softening.

Polygalacturonase (PG) gene has been documented as a key candidate for the improvement of fruit firmness, which is a target trait for tomato production because it facilitates transportation and storage. To reduce the expression of the PG gene, most of the elite commercial tomato varieties were obtained by RNA interference technology. However, this approach of producing commercialized tomatoes by integration of the exogenous gene is controversial. In this work, CRISPR/Cas9 technology was used to induce the targeted mutagenesis of the SlPG gene to delay the softening of tomato fruit. Results showed that the SlPG gene was frameshift mutated by 4 bp deletion, 10 bp deletion, and 1 bp insertion, which generated premature translation termination codons. Compared with wild-type (WT), homozygous T1-generation tomato plants exhibited late fruit softening under natural conditions. Consistent with this phenomenon, the firmness value of WT fruit was lower in slpg mutant fruit, and the physiological loss of water was higher. Collectively, these data demonstrate that the mutation of the SlPG gene delays tomato fruit softening. More importantly, 8 out of 20 transgene-free tomato plants, which were homozygous for null alleles of SlPG, were separated in the T3-generation of line slpgT2-#2. This transgene-free slpg may provide materials for more in-depth research of SlPG functions and the molecular mechanism of fruit softening in tomatoes.

Control of fruit softening and Ascorbic acid accumulation by manipulation of SlIMP3 in tomato.

SummaryPostharvest deterioration is among the major challenges for the fruit industry. Regulation of the fruit softening rate is an effective strategy for extending shelf‐life and reducing the economic losses due postharvest deterioration. The tomato myoinositol monophosphatase 3 gene SlIMP3, which showed highest expression level in fruit, was expressed and purified. SlIMP3 demonstrated high affinity with the L‐Gal 1‐P and D‐Ins 3‐P, and acted as a bifunctional enzyme in the biosynthesis of AsA and myoinositol. Overexpression of SlIMP3 not only improved AsA and myoinositol content, but also increased cell wall thickness, improved fruit firmness, delayed fruit softening, decreased water loss, and extended shelf‐life. Overexpression of SlIMP3 also increased uronic acid, rhamnose, xylose, mannose, and galactose content in cell wall of fruit. Treating fruit with myoinositol obtained similar fruit phenotypes of SlIMP3‐overexpressed fruit, with increased cell wall thickness and delayed fruit softening. Meanwhile, overexpression of SlIMP3 conferred tomato fruit tolerance to Botrytis cinerea. The function of SlIMP3 in cell wall biogenesis and fruit softening were also verified using another tomato species, Ailsa Craig (AC). Overexpression of SlDHAR in fruit increased AsA content, but did not affect the cell wall thickness or fruit firmness and softening. The results support a critical role for SlIMP3 in AsA biosynthesis and cell wall biogenesis, and provide a new method of delaying tomato fruit softening, and insight into the link between AsA and cell wall metabolism.

Slltpg3, a non-specific lipid transfer protein, acts on the cuticle synthetic pathway to delay water loss and softening of tomato fruit

Non-specific lipid transfer proteins (nsLTPs) are a class of small, omnipresent proteins that are involved in the transport of cuticular wax and other cell wall precursors. Although the cuticle serves as an outer barrier that protects plants from abiotic and biotic stresses and maintains postharvest storage quality, the regulatory role of nsLTPs on the cuticle remains unclear. Here, we isolated the Slltpg3 gene (a member of the tomato nsLTPs family) from tomato (Solanum lycopersicum) and overexpressed Slltpg3 in Micro-Tom tomato cultivars. We found that Slltpg3-overexpression optimized cuticle characteristics by increasing cuticular wax, cutin content, and cuticle thickness and by decreasing cuticle permeability. Our results suggested that Slltpg3 gene is involved in dynamic cuticle synthesis by increasing the content of cuticular wax components and delaying fruit softening and dehydration. Thus, this study revealed a direct relationship between cuticle characteristics and fruit softening and dehydration and provided new insights into tomato adaptation under drought conditions.

Fungus Polygalacturonase-Generated Oligogalacturonide Restrains Fruit Softening in Ripening Tomato.

Fruit softening exacerbates mechanical damage incurred during shipping and handling and the increase in pathogen susceptibility. Here, oligogalacturonides (OGs) produced by fungal polygalacturonase (PG) delayed fruit softening in tomato and maintained fruit firmness at 8.37 ± 0.45 N at 13 d of storage, which was consistent with the fruit firmness level of 5 d in the control groups. From RNA sequencing data in line production of phytohormones, we confirmed ethylene and jasmonic acid signals, the MAPK signaling cascade, and calmodulin involved in the OG-mediated firmness response of whole fruit. SlPG2, SlPL3, and SlPL5 were the major contributing factors for fruit softening, and their expression decreased continuously upon OG application. Suppression of the expression of ethylene response factors using a virus-induced gene-silencing strategy revealed that SlERF6 was negatively involved in OG-restrained fruit softening. Taken together, these results indicated that fungal PG-generated OGs have potential application value in controlling tomato fruit softening.

Cloning and Functional Identification of SlPG49 in Solanum lycopersicum

The modification and degradation of pectin in cell walls are necessary for the fruit softening process, which involves a series of pectin-modifying enzymes. Polygalacturonases (PGs) are a major group of pectin-hydrolyzing enzymes, which participate in fruit maturation, organ shedding, pollen development, and other processes by catalyzing the degradation of polygalacturonic acid. However, their function in plants has not yet been fully elucidated. In this paper, a full-length cDNA encoding SlPG49 was cloned from a tomato. Sequence alignment and phylogenetic analysis demonstrated that SlPG49 contains four typical conserved domains and belongs to clade E in PG classification. Quantitative real-time PCR analysis showed that SlPG49 was highly expressed in fruits during the softening stage, indicating that SlPG49 may be involved in fruit softening. Subcellular localization results revealed that SlPG49 was located in the cell membrane and the cell wall. In addition, an in vitro enzymatic activity assay confirmed that SlPG49 does have the ability to catalyze the hydrolysis of polygalacturonic acid. These results indicate that SlPG49 is a newly discovered PG gene involved in tomato fruit softening, and provide an experimental basis for elucidating the biological functions of plant PGs during fruit softening.

SlBES1 promotes tomato fruit softening through transcriptional inhibition of PMEU1

SummaryFruit softening indicated by firmness determines the texture, transportability, and shelf life of tomato products. However, the regulatory mechanism underlying firmness formation in tomato fruit is poorly understood. Here, we report the regulatory role of SlBES1, an essential component of brassinosteroid hormone signaling, in tomato fruit softening. We found that SlBES1 promotes fruit softening during tomato fruit ripening and postharvest storage. RNA-seq analysis suggested that PMEU1, which encodes a pectin methylesterase, might participate in SlBES1-mediated softening. Biochemical and immunofluorescence assays indicated that SlBES1 inhibited PMEU1-related pectin de-methylesterification. Further molecular and genetic evidence verified that SlBES1 directly binds to the E-box of PMEU1 to repress its expression, leading to fruits softening. Loss-of-function SlBES1 mutant generated by CRISPR-Cas9 showed firmer fruits and longer shelf life during postharvest storage without other quality alteration. Collectively, our results indicated the potential of manipulating SlBES1 to regulate firmness without negative consequence on visual and nutrition quality.

Mutagenesis of <i>SlNAC4</i> by CRISPR/Cas9 alters gene expression and softening of ripening tomato fruit

Softening is one of the key fruit quality traits, which results from the selective expression of cell wall metabolism genes during ripening. The identification of transcription factors (TFs) that regulate fruit softening is an important field in order to understand and control fruit softening. In tomato, NAC (NAM, ATAF, and CUC) TFs members have been demonstrated to be involved in fruit ripening regulation, including NAC-NOR (non-ripening), NOR-like1, SlNAC4, SlNAC1. Here, we generated slnac4 mutant knockout (CR-SlNAC4) tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system (CRISPR/Cas9) and SlNAC4 overexpressing (OE-SlNAC4) plant. In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening, we found that SlNAC4 has a strong effect on tomato fruit softening. Compared with the control fruit, fruit softening was inhibited in slnac4 fruit and conversely was accelerated in OE-SlNAC4 tomato fruit. Through RNA-sequencing (RNA-seq) analysis, we found that expression levels of SlEXP1 (expansin) and SlCEL2 (endo-β-1,4 glucanase) genes involved in cell wall metabolism were significantly different in WT (wild type)/slnac4 and WT/OE-SlNAC4 fruit. Further study showed that these genes contained a NAC TF binding domain in their promoter regions. In vitro electrophoretic mobility shift assays (EMSA) and dual-luciferase reporter assays (DLR) demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation. The results enriched the function of SlNAC4 and provided a new dimension in understanding the regulation of tomato fruit softening.

Genome-wide identification and expression analysis of extensin genes in tomato

Extensins (EXTs) are major protein components in plant cell walls that play crucial roles in higher plants. The function of EXTs has been reported in several plants but is limited in tomato, especially in fruit ripening. In this study, we identified 83 EXTs in tomato, and divided them into seven groups. The gene intron-exon structure and protein-motif composition of SlEXTs were similar within each group but different among groups. SlEXT genes showed different expression patterns in roots, leaves, flowers and fruits, and some SlEXT gene expressions in flowers could be regulated by treatments of auxin, gibberellic acid and jasmonic acid. In particular, SlSEXT8 had higher and increased expression during tomato fruit ripening, and its expression could be induced by ethylene, suggesting SlSEXT8 may be involved in tomato fruit softening. The result provides insights into the function of EXTs, and will facilitate to further study EXT roles in tomato fruit ripening.

A relationship between tomato fruit softening, cuticle properties and water availability

While fleshy fruit softening has long been mechanistically linked to cell wall disassembly, the importance of the fruit cuticle in water relations and firmness has been suggested through studies of the long-shelf life delayed fruit deterioration (dfd) tomato genotype. We tested the hypothesis that dynamic cuticle properties and composition affect tomato fruit transpiration and firmness and are influenced by environmental water availability, using dfd and two normally softening fruit cultivars, Ailsa Craig (AC) and M82, grown under control and water stress (WS) conditions. The effect of WS was also assessed following fruit detachment. WS increased fruit firmness, cuticle load, and the expression of cuticle biosynthetic genes, while reducing cuticle permeability and fruit transpiration rate in AC and M82, but not in dfd fruit. This study supports a direct relationship between fruit cuticle properties, transpiration and firmness, and provides insights into the adaptation of tomato genotypes to environments where water can be scarce.

Fruit Softening: Revisiting the Role of Pectin.

Fruit softening, which is a major determinant of shelf life and commercial value, is the consequence of multiple cellular processes, including extensive remodeling of cell wall structure. Recently, it has been shown that pectate lyase (PL), an enzyme that degrades de-esterified pectin in the primary wall, is a major contributing factor to tomato fruit softening. Studies of pectin structure, distribution, and dynamics have indicated that pectins are more tightly integrated with cellulose microfibrils than previously thought and have novel structural features, including branches of the main polymer backbone. Moreover, recent studies of the significance of pectinases, such as PL and polygalacturonase, are consistent with a causal relationship between pectin degradation and a major effect on fruit softening.

Effects and Mechanism of 1-Methylcyclopropene and Ethephon on Softening in Ailsa Craig Tomato Fruit

Studies were conducted to determine the efficacy and mechanism of 1-methylcyclopropene (1-MCP) and ethephon on softening of Ailsa Craig tomato fruit. Ethylene production, reduction of fruit firmness and protopectin content were suppressed in fruit exposed to 1.0 µL/L 1-MCP. Comparing with control fruit, the climacteric ethylene peak was delayed by approximately 12 d and polygalacturonase activity was strongly inhibited in 1.0 µL/L 1-MCP treated fruit. On the other hand, ethylene production, reduction of fruit firmness, protopectin content and polygalacturonase activity of 100 µL/L ethephon treated fruit were strongly promoted compared with these indexes of untreated fruit. Moreover, electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay proved that the expression of PG and tomato fruit softening was regulated by the transcription activator RIN. Practical Applications The RIN transcription factor plays an indispensable role in fruit ripening and softening. According to the physiological index of tomato fruit under varied concentrations of 1-MCP and ethephon, optimal concentrations of 1-MCP and ethephon would be used for further studies and industrial applications. Furthermore our study clearly showed RIN transcription factor directly regulated the expression of PG. Since the importance of polygalacturonase (PG) activity and indispensable role of RIN during the process of fruit softening, it suggested that RIN affects tomato fruit softening via directly acting on PG. It provides new clues to understand the molecular mechanism of fruit softening. To sum, these results will help to further clarify the mechanism of tomato fruit softening, and to maintain the quality and prolong the shelf life of tomato fruit.

Influences of 1-MCP on AC Tomato Fruit Softening

The influences of 1-MCP on AC tomato fruit softening were investigated. Ethylene production, flesh firmness, protopectin content, soluble pectin content and polygalacturonase (PG) activity were used to evaluate the quality of tomato fruit. It was shown that 1.0 and 1.5 μL/L 1-MCP treatment significantly decreased the levels of ethylene production, and inhibited flesh firmness and protopectin content decline and polygalacturonase activity increase. At last, it was concluded 1.0 μL/L 1-MCP treatment on AC tomato fruit was the best determining economy.

Regulatory specialization of xyloglucan (XG) and glucuronoarabinoxylan (GAX) in pericarp cell walls during fruit ripening in tomato (Solanum lycopersicum).

Disassembly of cell wall polysaccharides by various cell wall hydrolases during fruit softening causes structural changes in hemicellulose and pectin that affect the physical properties and softening of tomato fruit. In a previous study, we showed that the changes in pectin during tomato fruit ripening were unique in each fruit tissue. In this study, to clarify the changes in hemicellulose in tissues during tomato fruit ripening, we focused on glucuronoarabinoxylan (GAX) and xyloglucan (XG). GAX was detected only in the skin and inner epidermis of the pericarp using LM11 antibodies, whereas a large increase in XG was detected in all fruit tissues using LM15 antibodies. The activity of hemicellulose degradation enzymes, such as β-xylosidase and α-arabinofuranosidase, decreased gradually during fruit ripening, although the tomato fruits continued to soften. In contrast, GAX and XG biosynthesis-related genes were expressed in all tomato fruit tissues even during ripening, indicating that XG was synthesized throughout the fruit and that GAX may be synthesized only in the vascular bundles and the inner epidermis. Our results suggest that changes in the cell wall architecture and tissue-specific distribution of XG and GAX might be required for the regulation of fruit softening and the maintenance of fruit shape.

Tissue specific localization of pectin-Ca²⁺ cross-linkages and pectin methyl-esterification during fruit ripening in tomato (Solanum lycopersicum).

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca2+) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.

Postharvest UV-C irradiation inhibits the production of ethylene and the activity of cell wall-degrading enzymes during softening of tomato (Lycopersicon esculentum L.) fruit

Mature green cherry tomato fruit were harvested and treated with ultraviolet-C (UV-C) irradiation at a predetermined dose of 4.2kJm−2 and stored at 18°C for 35 days. The effects of UV-C treatment on firmness, cell wall compositions (pectin and cellulose), cell wall ultra-structure, levels of ethylene production, and activities of cell wall degrading enzymes in the fruit were investigated during storage. Major genes involved in cell wall degradation (PME 2.1, Cel 1, PG cat and Exp 1) were also checked. Fruit firmness was better maintained in UV-C treated fruit corresponding with higher contents of cellulose and acid-soluble pectin. Transmission electron microscopy indicated that UV-C irradiation retarded cell wall disassembly in the pericarp. Ethylene production was significantly inhibited by UV-C treatment. The UV-C treatment also suppressed the transcriptional expression of PME2.1, Cel1, PGcat and Exp1 genes, and inhibited pectin methylesterase (PME; EC 3.1.1.11), polygalacturonase (PG; EC3.2.1.15) and cellulase (EC3.2.1.4) activities during storage. These results suggested that the inhibition of ethylene production, which in turn down-regulated expression of genes encoding cell wall degrading enzymes, might be one of the possible mechanisms of UV-C delaying tomato fruit softening.

Effect of ethylene on polygalacturonase, lipoxygenase and expansin in ripening of tomato fruits

Fruit ripening is a complex process and is regulated by many factors. Ethylene and polygalacturonase (PG), lipoxygenase(LOX), expansin(EXP) are all critical regulating factors in fruit ripening and softening process. With antisense ACS tomato, Nr mutant tomato and cultivated tomato as materials, Northern blot hybridization showed that PG, LeEXP1 and LOX expressed differently in different parts of cultivated tomato fruit during ripening, which was related to fruit ripening. The ripening process of columella and radial pericarp was faster than pericarp. In both Nr mutant and antisense ACS transgenic tomato fruit, expression levels of PG, LeEXP1 and LOX were generally lower than those in cultivated fruit but still related to fruit ripening. The expression levels of PG, LeEXP1 and LOX increased in the mature green tomato fruits after 0.5 h treatment with ethylene (100 μL/L). These results indicate that gene expression of PG, LeEXP1 and LOX were positively regulated by ethylene. The time and cumulative effect of the concentration exists in the expression of PG regulated by ethylene. The regulation of LOX expression mainly depended on the fruit development after great amount of ethylene was produced. PG played a major role in ripening and softening of tomato fruit, and cooperated with the regulation of EXP and LOX.

Xyloglucan endotransglucosylase/hydrolases (XTHs) during tomato fruit growth and ripening

Depolymerization of cell wall xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endotransglucosylase/hydrolases (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151) have been proposed to have a dual role integrating newly secreted xyloglucan chains into an existing wall-bound xyloglucan, or restructuring the existing cell wall material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SlXTHs were studied and grouped into three phylogenetic groups to determine which members of each family were expressed during fruit growth and fruit ripening, and the ways in which the expression of different SlXTHs contributed to the total XET and XEH activities. Our results showed that all of the SlXTHs studied were expressed during fruit growth and ripening, and that the expression of all the SlXTHs in Group 1 was clearly related to fruit growth, as were SlXTH12 in Group 2 and SlXTH6 in Group 3-B. Only the expression of SlXTH5 and SlXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SlXTHs were expressed at the red ripe stage. Both total XET and XEH activities were higher during fruit growth, and decreased during fruit ripening. Ethylene production during tomato fruit growth was low and experienced a significant increase during fruit ripening, which was not correlated either with SlXTH expression or with XET and XEH activities. We suggest that the role of XTH during fruit development could be related to the maintenance of the structural integrity of the cell wall, and the decrease in XTHs expression, and the subsequent decrease in activity during ripening may contribute to fruit softening, with this process being regulated through different XTH genes.