VvPL15 Is the Core Member of the Pectate Lyase Gene Family Involved in Grape Berries Ripening and Softening

The process of ripening and softening in grape begins at veraison and is closely related to the depolymerization of pectin components. A variety of enzymes are involved in pectin metabolism and one class of enzyme, pectin lyases (PLs), have been reported to play an important role in softening in many fruits; however, little information is available on the VvPL gene family in grape. In this study, 16 VvPL genes were identified in the grape genome using bioinformatics methods. Among them, VvPL5, VvPL9, and VvPL15 had the highest expression levels during grape ripening, which suggests that these genes are involved in grape ripening and softening. Furthermore, overexpression of VvPL15 affects the contents of water-soluble pectin (WSP) and acid-soluble pectin (ASP) in the leaves of Arabidopsis and significantly changes the growth of Arabidopsis plants. The relationship between VvPL15 and pectin content was further determined by antisense expression of VvPL15. In addition, we also studied the effect of VvPL15 on fruit in transgenic tomato plants, which showed that VvPL15 accelerated fruit ripening and softening. Our results indicate that VvPL15 plays an important role in grape berry softening during ripening by depolymerizing pectin.


Introduction
Grape (Vitis vinifera L.) is one of the most economically important fruit crops in the world and is also an important genetic resource for woody fruit crops [1]. Grapes are widely popular because of their nutritional value and health benefits. However, fruit softening after ripening is a major contributor to postharvest deterioration, which directly affects storage logistics and shelf life [2]. Therefore, identifying fruit softening-specific regulatory genes, balancing texture and other fruit quality traits, and obtaining germplasm varieties that show delayed softening while maintaining good fruit quality have become the goal of grape breeders and scientists [3].
Fruit softening is an important characteristic of fruit ripening, which occurs at veraison (the onset of ripening) and mainly involves significant changes in the properties of cell wall polysaccharides [4] and turgor pressure [5,6]. The structural changes that occur during fruit softening are mainly caused by changes in the mechanical strength of the cell wall and intercellular adhesion, and pectin depolymerization is considered to be one of the most important factors that causes fruit softening in different species [7]. The plant cell wall is composed of the middle lamella, the primary cell wall, and the secondary cell wall. The primary cell wall consists of pectin, hemicellulose, and cellulose [8]. Pectin is an important component of the cell wall in higher plants, and it is mainly present in the primary wall and middle layer [9]. It is a complex polysaccharide that is mainly composed of three domains, including Homo-galacturonan (HG), Rhamno-galacturonan I (RG-I), and Rhamno-galacturonan II (RG-II) [10]. Pectin is involved in the adhesion between cell wall cells mainly through Ca 2+ cross-linking with partially de-esterified galacturonic acid glycans in the middle layer [11]. In general, the main causes of fruit softening are cell wall disassembly and reduction in the cell-to-cell adhesion, which is the result of middle lamella dissolution [12]. While the dissolution of pectin is one of the most significant changes that occurs during fruit softening, with the dissolution of the pectin layer, the intercellular space becomes larger and the fruit softening intensifies. During fruit ripening and softening, the changes in pectin in the intercellular layer and the cell wall mainly include the degradation of insoluble protopectin into soluble pectin, the removal of methyl groups in polymers, and the decrease in the contents of neutral sugars, such as galactose and arabinose [13].
Pectin metabolism in plants involves a variety of enzymes, including pectin methylesterase (PME, EC3.1.1.11), polygalacturonase (PG, EC3.2.1.15), and pectin lyase (PL, EC4.2.2.2) [14][15][16]. In particular, pectate lyase (PL) is a key component of the enzyme mixture involved in maturation-related cell wall remodeling, and it plays an important role in the softening process of succulent fruits [17]. A study of transgenic lines of strawberry (Fragaria × ananassa) expressing antisense FaPL (njjs25) provided the first experimental evidence of the role of PL in fruit firmness [18]. The high expression levels of the MaPel I and MaPel II genes during fruit ripening has also been reported in banana [19]. PpePL1 and PpePL15 are involved in fruit ripening and softening in peach [20], and VvPL11 plays a role in ripening and softening in grape berries [21]. Recently, the mechanism of action of PL has been studied in detail; for example, silencing SlPL inhibited fruit softening and reduced the water soluble pectin (WSP) content in tomato [22], and transcription factors, such as SlLOB1 and SlERF.F12 can play a role in fruit ripening by regulating expression of SlPL [23,24]. In strawberry fruits, FvPL appears to act against tightly bound pectin [25], and FvWRKY48 binds to the pectate lyase FvPLA gene promoter to control fruit softening in Fragaria vesca [26]. However, the function of PL in grape fruits is still less reported.
In this study, we identified genes encoding PL family members that are expressed during ripening and softening in grape berries. We analyzed and predicted PL family members using bioinformatics methods, and the results provide valuable clues to predict their function. Genetic transformation and virus-induced gene silencing technology (VIGS) were used to clarify the function of VvPL15 in grape berry softening. Our research provides a foundation for further investigation of the function of PL family members in the process of ripening and softening in grape berries.

Determination of Pectin Content in 'Kyoho' Grape Fruit Flesh during Ripening
Veraison is a critical period in the grape life cycle as it signals the onset of fruit ripening. We collected 'Kyoho' grape berries at five stages of development. The skin color began to change at E-L 35 and gradually became darker, indicating that E-L 35 is the onset of veraison ( Figure 1A). Moreover, we found that grape flesh firmness decreased rapidly from E-L 35 to E-L 36, after which the rate slowed ( Figure 1B). At the same time, the WSP content increased gradually ( Figure 1C), but the ASP content decreased continuously ( Figure 1D). These results indicated that the firmness of grape berries could be related to the changes in pectin components.

Identification of VvPLs in Grape
A total of 16 genes were identified from the grapevine genome through orthologues analysis with Arabidopisis thaliana and Solanum lycopersicum. These genes were designated as VvPL1-VvPL16 for VvPLs according to their corresponding location on each chromosome (from top to bottom). A brief summary of basic information for VvPLs in grapevine is listed in Table 1, where the basic features include the corresponding gene names, gene length, chromosome location, protein properties and subcellular localization. In general, the gene length of VvPLs varied from 1581 to 7549 bp, while the amino acid length ranged from 320 to 496 amino acid (aa), respectively. Moreover, the molecular weight (kDa) ranged from 36.1 to 53.9 for VvPLs, while the theoretical isoelectric points (pIs) varied from 4.98 to 9.66 for VvPLs. Furthermore, VvPL family members were unevenly distributed among the ten chromosomes of the grape genome and three, two and one members were distributed on each chromosome ( Figure 2). The tandem duplication sites (VvPL1 and 2, VvPL9 and 10) were found on chromosomes 1 and 13. In addition, according to web-based prediction of VvPLs location in cells, different members of VvPL gene family were mainly found in the cell wall.

Identification of VvPLs in Grape
A total of 16 genes were identified from the grapevine genome through orthologues analysis with Arabidopisis thaliana and Solanum lycopersicum. These genes were designated as VvPL1-VvPL16 for VvPLs according to their corresponding location on each chromosome (from top to bottom). A brief summary of basic information for VvPLs in grapevine is listed in Table 1, where the basic features include the corresponding gene names, gene length, chromosome location, protein properties and subcellular localization. In general, the gene length of VvPLs varied from 1581 to 7549 bp, while the amino acid length ranged from 320 to 496 amino acid (aa), respectively. Moreover, the molecular weight (kDa) ranged from 36.1 to 53.9 for VvPLs, while the theoretical isoelectric points (pIs) varied from 4.98 to 9.66 for VvPLs. Furthermore, VvPL family members were unevenly distributed among the ten chromosomes of the grape genome and three, two and one members were distributed on each chromosome ( Figure 2). The tandem duplication sites (VvPL1 and 2, VvPL9 and 10) were found on chromosomes 1 and 13. In addition, according to web-based prediction of VvPLs location in cells, different members of VvPL gene family were mainly found in the cell wall.

Phylogenetics Analysis of VvPLs
The phylogenetic relationship of 16 VvPLs of grape and three other plants were obtained using MEGA7.0 via the neighbor-joining approach. Based on the classification of AtPLLs and SlPLs, the phylogenetic tree revealed that VvPLs and PLs of three other plants evolved from a common ancestor and VvPLs can further be divided into five major groups ( Figure 3). In phylogenetic tree of VvPLs, we observed that group 1 contained the most number of genes compared to group 2 and 3 and consisted of four members of the VvPLs family. Meanwhile, VvPL7 and VvPL13 were clustered into group 4, in which VvPL13 showed a closer relationship with AtPLL3. In addition, VvPL8 was only one member of the VvPLs family in group 5, suggesting a specific function of VvPLs in grape.
The phylogenetic relationship of 16 VvPLs of grape and three other plants were obtained using MEGA7.0 via the neighbor-joining approach. Based on the classification of AtPLLs and SlPLs, the phylogenetic tree revealed that VvPLs and PLs of three other plants evolved from a common ancestor and VvPLs can further be divided into five major groups ( Figure 3). In phylogenetic tree of VvPLs, we observed that group 1 contained the most number of genes compared to group 2 and 3 and consisted of four members of the VvPLs family. Meanwhile, VvPL7 and VvPL13 were clustered into group 4, in which VvPL13 showed a closer relationship with AtPLL3. In addition, VvPL8 was only one member of the VvPLs family in group 5, suggesting a specific function of VvPLs in grape. Figure 3. Phylogenetic tree of VvPLs from grape and other species. All PL proteins were divided into five groups, and different groups were labelled and distinguished with each other by different branch color. Furthermore, PL proteins from different species were labelled with different shape types.

Protein and Gene Structure Analysis of VvPLs
The phylogenetic tree among VvPLs showed VvPL proteins are divided into five major groups due to variations in tree topologies ( Figure 4A). We analyzed the composition of motifs for VvPLs and obtained ten conserved motifs using the online server, Multiple Em for Motif Elicitation (MEME). The results revealed that the motifs six, two and one were dominantly found among PL members and 9 PL members contained ten motifs . Phylogenetic tree of VvPLs from grape and other species. All PL proteins were divided into five groups, and different groups were labelled and distinguished with each other by different branch color. Furthermore, PL proteins from different species were labelled with different shape types.

Protein and Gene Structure Analysis of VvPLs
The phylogenetic tree among VvPLs showed VvPL proteins are divided into five major groups due to variations in tree topologies ( Figure 4A). We analyzed the composition of motifs for VvPLs and obtained ten conserved motifs using the online server, Multiple Em for Motif Elicitation (MEME). The results revealed that the motifs six, two and one were dominantly found among PL members and 9 PL members contained ten motifs except members 16,9,8,13 and 7 ( Figure 4B). These results suggest that proteins on the same branches may have broadly similar functions. Furthermore, based on the coding sequence (CDS) and untranslated regions (UTR) of VvPL genes in grape, gene structures were resolved using the online tool GSDS2.0. The results revealed that VvPL members exhibited high divergence and largely conserved compared to each other, again suggesting that genes from the same branch may have retained similar functions during plant evolution ( Figure 4C). In addition, 3D models of the VvPL proteins were created using Phyre. The individual 3D models indicated that VvPLs also shared a common structure ( Figure 5). except members 16, 9, 8, 13 and 7 ( Figure 4B). These results suggest that proteins on the same branches may have broadly similar functions. Furthermore, based on the coding sequence (CDS) and untranslated regions (UTR) of VvPL genes in grape, gene structures were resolved using the online tool GSDS2.0. The results revealed that VvPL members exhibited high divergence and largely conserved compared to each other, again suggesting that genes from the same branch may have retained similar functions during plant evolution ( Figure 4C). In addition, 3D models of the VvPL proteins were created using Phyre. The individual 3D models indicated that VvPLs also shared a common structure ( Figure 5).

Collinearity Analysis of the VvPLs
To reveal the expansion mechanism of the VvPLs family, intergenomic duplication data filers of grape and Arabidppsis were filtered using TBtools. Microsynteny between species can be used to identify the location of orthologous genes. In total, we identified 23 orthologous gene pairs between grape and Arabidppsis ( Figure 6). This indicates that numerous grape and Arabidppsis counterparts may have evolved from a common ancestor This result suggests that the grape orthologous genes functions may be predicted by the Arabidppsis orthologous genes.

Collinearity Analysis of the VvPLs
To reveal the expansion mechanism of the VvPLs family, intergenomic duplication data filers of grape and Arabidppsis were filtered using TBtools. Microsynteny between species can be used to identify the location of orthologous genes. In total, we identified 23 orthologous gene pairs between grape and Arabidppsis ( Figure 6). This indicates that numerous grape and Arabidppsis counterparts may have evolved from a common ancestor. This result suggests that the grape orthologous genes functions may be predicted by the Arabidppsis orthologous genes.

Cis-Regulatory Element Analysis in VvPL Genes
The cis-acting elements present in the VvPL gene promoters were analyzed using the online PlantCARE database. The number of cis-elements identified in the promoter re gions of the VvPL genes ranged from 6 to 26 (Table S2). Our analysis included hormone related responsive elements, such as those for abscisic acid (ABA), gibberellin (GA), auxin salicylic acid (SA), methyl jasmonate (MeJA), and ethylene [27]; stress-related responsive elements, such as those for low temperature and drought; and also typical binding sites for transcription factors from families, such as MBS and W-box (Table S1) [28,29]. We also used TBtools software to construct distribution maps of the promoter sequences showing the number and positions of the cis-elements (Figure 7). A total 16 members of the VvPL family were analyzed, among which 14 of the genes had ERE elements and 11 genes had ARBEs (Table S2). Almost all of the gene promoters contained multiple MYB elements, and most o the promoters contained TCA-elements, CGTCA-motifs, and W-box elements.

Cis-Regulatory Element Analysis in VvPL Genes
The cis-acting elements present in the VvPL gene promoters were analyzed using the online PlantCARE database. The number of cis-elements identified in the promoter regions of the VvPL genes ranged from 6 to 26 (Table S2). Our analysis included hormonerelated responsive elements, such as those for abscisic acid (ABA), gibberellin (GA), auxin, salicylic acid (SA), methyl jasmonate (MeJA), and ethylene [27]; stress-related responsive elements, such as those for low temperature and drought; and also typical binding sites for transcription factors from families, such as MBS and W-box (Table S1) [28,29]. We also used TBtools software to construct distribution maps of the promoter sequences showing the number and positions of the cis-elements (Figure 7). A total 16 members of the VvPL family were analyzed, among which 14 of the genes had ERE elements and 11 genes had ARBEs (Table S2). Almost all of the gene promoters contained multiple MYB elements, and most of the promoters contained TCA-elements, CGTCA-motifs, and W-box elements.

Identification of VvPL Gene Family Members Related to Fruit Flesh Softening
To characterize the putative function of VvPL members, we examined the expression levels of 16 VvPL genes via RT-qPCR during ripening and softening in 'Kyoho' grape berries. The results showed that all the 16 members were expressed, but only 6 members (VvPL3, VvPL4, VvPL5, VvPL9, VvPL13, and VvPL15) showed upregulated transcription at E-L 35. The expression of three genes (VvPL5, VvPL9, and VvPL15) changed significantly from E-L 35 to E-L 38 ( Figure 8B), and VvPL15 showed the highest relative expression level during grape berry ripening and softening ( Figure 8A,B). Therefore, it is inferred that VvPL15 (VIT_217s0000g09810) may play an important role in the process of ripening and softening in grape berries.

Identification of VvPL Gene Family Members Related to Fruit Flesh Softening
To characterize the putative function of VvPL members, we examined the expression levels of 16 VvPL genes via RT-qPCR during ripening and softening in 'Kyoho' grape berries. The results showed that all the 16 members were expressed, but only 6 members (VvPL3, VvPL4, VvPL5, VvPL9, VvPL13, and VvPL15) showed upregulated transcription at E-L 35. The expression of three genes (VvPL5, VvPL9, and VvPL15) changed significantly from E-L 35 to E-L 38 ( Figure 8B), and VvPL15 showed the highest relative expression level during grape berry ripening and softening ( Figure 8A,B). Therefore, it is inferred that VvPL15 (VIT_217s0000g09810) may play an important role in the process of ripening and softening in grape berries. VvActin served as a reference gene. Each value represents the means ± SE of three independent biological replicates. Significant differences (p < 0.05) between means are indicated by different letters.

VvPL15 Overexpression Affects Pectin Content and Plant Growth in Transgenic Arabidopsis
To further study the effect of VvPL15 on pectin content, a vector was constructed to overexpress VvPL15 in transgenic Arabidopsis Col-0 plants. To verify that the transgenic Arabidopsis plants carried the VvPL15-overexpression cassette, DNA was extracted from mature rosette leaves for PCR VvPL15 gene amplification. DNA fragments with lengths identical to that of the VvPL15 coding region were amplified ( Figure S1), indicating that VvPL15 had been transferred into the Arabidopsis genome and was successfully expressed in T0-generation plants. Three transgenic lines (VvPL15-OX #2, VvPL15-OX #4, and VvPL15-OX #6) were selected for phenotypic analysis. The results showed that the leaves of the overexpression lines were significantly smaller than those of the wild type and had a curly shape ( Figure 9A). In addition, the overexpression lines were significantly thinner and shorter ( Figure S2). We next measured the pectin content of Arabidopsis leaves. The results showed that compared with the wild-type, the WSP content in the Arabidopsis overexpression leaves increased significantly, while the ASP content decreased ( Figure 9B,C). These results show that overexpression of VvPL15 affects the growth of Arabidopsis plants, which may be related to pectin degradation. VvActin served as a reference gene. Each value represents the means ± SE of three independent biological replicates. Significant differences (p < 0.05) between means are indicated by different letters.

VvPL15 Overexpression Affects Pectin Content and Plant Growth in Transgenic Arabidopsis
To further study the effect of VvPL15 on pectin content, a vector was constructed to overexpress VvPL15 in transgenic Arabidopsis Col-0 plants. To verify that the transgenic Arabidopsis plants carried the VvPL15-overexpression cassette, DNA was extracted from mature rosette leaves for PCR VvPL15 gene amplification. DNA fragments with lengths identical to that of the VvPL15 coding region were amplified ( Figure S1), indicating that VvPL15 had been transferred into the Arabidopsis genome and was successfully expressed in T 0 -generation plants. Three transgenic lines (VvPL15-OX #2, VvPL15-OX #4, and VvPL15-OX #6) were selected for phenotypic analysis. The results showed that the leaves of the overexpression lines were significantly smaller than those of the wild type and had a curly shape ( Figure 9A). In addition, the overexpression lines were significantly thinner and shorter ( Figure S2). We next measured the pectin content of Arabidopsis leaves. The results showed that compared with the wild-type, the WSP content in the Arabidopsis overexpression leaves increased significantly, while the ASP content decreased ( Figure 9B,C). These results show that overexpression of VvPL15 affects the growth of Arabidopsis plants, which may be related to pectin degradation.

Silencing VvPL15 Expression Affects the WSP and ASP Contents in Grape Leaves
To further investigate the role of VvPL15 in grape, we performed both VIGS and transient overexpression of VvPL15 in grape leaves. Softening phenotypes were analyzed, and pectin content in antisense expression and overexpressed grape leaves were studied in detail ( Figure 10). VvPL15-TRV infiltration was the RNAi treatment in leaves, and TRV infiltration was the control. VvPL15-PRI infiltration was used to overexpress VvPL15 in the leaves, and PRI infiltration was used as the control. VvPL15 expression was inhibited in VvPL15-TRV grape leaves compared with the control. In VvPL15-PRI grape leaves, VvPL15 expression was significantly up-regulated ( Figure 10A). The WSP content in VvPL15-TRV leaves was lower than in the control, while the ASP content was significantly

Silencing VvPL15 Expression Affects the WSP and ASP Contents in Grape Leaves
To further investigate the role of VvPL15 in grape, we performed both VIGS and transient overexpression of VvPL15 in grape leaves. Softening phenotypes were analyzed, and pectin content in antisense expression and overexpressed grape leaves were studied in detail ( Figure 10). VvPL15-TRV infiltration was the RNAi treatment in leaves, and TRV infiltration was the control. VvPL15-PRI infiltration was used to overexpress VvPL15 in the leaves, and PRI infiltration was used as the control. VvPL15 expression was inhibited in VvPL15-TRV grape leaves compared with the control. In VvPL15-PRI grape leaves, VvPL15 expression was significantly up-regulated ( Figure 10A). The WSP content in VvPL15-TRV leaves was lower than in the control, while the ASP content was significantly higher than in the control ( Figure 10B). On the contrary, the WSP content of VvPL15-PRI leaves was lower than it was in the control, and the ASP was higher than in the control ( Figure 10C). These experiments demonstrate that pectin degradation was delayed after VvPL15 expression was downregulated, and that pectin degradation was accelerated by VvPL15 overexpression. higher than in the control ( Figure 10B). On the contrary, the WSP content of VvPL15-PRI leaves was lower than it was in the control, and the ASP was higher than in the control ( Figure 10C). These experiments demonstrate that pectin degradation was delayed after VvPL15 expression was downregulated, and that pectin degradation was accelerated by VvPL15 overexpression.

VvPL15 Overexpression Accelerates Fruit Softening in Tomato
To elucidate the function of VvPL15 during fruit ripening, we overexpressed VvPL15 in tomato plants ( Figure 11). We obtained three transgenic tomato lines overexpressing VvPL15 (VvPL15-OX # 1, VvPL15-OX #2, and VvPL15-OX #3) and showed that they were transgenic via PCR ( Figure S3). Under suitable growth conditions, there was a significant difference between the VvPL15-overexpressing (OX) lines and the wild-type (WT) at the seedling stage. The seedlings of the VvPL15-OX lines showed weaker growth compared to WT plants ( Figure 11A), which was the same as we observed in the Arabidopsis Col-0 plants overexpressing VvPL15 ( Figure S2). In addition, the changes in the characteristics of the fruits of the transgenic lines were also obvious. We observed that the fruits of the VvPL15-OX tomato lines turned red earlier than they did on WT plants, suggesting that the breaker stage (BR) occurred earlier in the transgenic tomato fruits ( Figure 11B). The firmness of the transgenic tomato fruits was decreased significantly compared with WT fruits ( Figure 11C). These results indicate that overexpression of VvPL15 in tomato can accelerate tomato fruit ripening and softening. Furthermore, we analyzed the changes in fruit pectin content. In the transgenic tomato fruits, the ASP content was greatly reduced ( Figure 11E), and the WSP content was higher in all stages and all three OX lines compared with the WT (Figure 11D). The firmness and pectin content showed the largest change in MG and BR. Therefore, overexpression of VvPL15 in tomato results in tomato fruits that mature earlier. Furthermore, overexpression of VvPL15 promoted fruit softening, which might be attributed to the degradation of ASP.

VvPL15 Overexpression Accelerates Fruit Softening in Tomato
To elucidate the function of VvPL15 during fruit ripening, we overexpressed VvPL15 in tomato plants ( Figure 11). We obtained three transgenic tomato lines overexpressing VvPL15 (VvPL15-OX # 1, VvPL15-OX #2, and VvPL15-OX #3) and showed that they were transgenic via PCR ( Figure S3). Under suitable growth conditions, there was a significant difference between the VvPL15-overexpressing (OX) lines and the wild-type (WT) at the seedling stage. The seedlings of the VvPL15-OX lines showed weaker growth compared to WT plants ( Figure 11A), which was the same as we observed in the Arabidopsis Col-0 plants overexpressing VvPL15 ( Figure S2). In addition, the changes in the characteristics of the fruits of the transgenic lines were also obvious. We observed that the fruits of the VvPL15-OX tomato lines turned red earlier than they did on WT plants, suggesting that the breaker stage (BR) occurred earlier in the transgenic tomato fruits ( Figure 11B). The firmness of the transgenic tomato fruits was decreased significantly compared with WT fruits ( Figure 11C). These results indicate that overexpression of VvPL15 in tomato can accelerate tomato fruit ripening and softening. Furthermore, we analyzed the changes in fruit pectin content. In the transgenic tomato fruits, the ASP content was greatly reduced ( Figure 11E), and the WSP content was higher in all stages and all three OX lines compared with the WT (Figure 11D). The firmness and pectin content showed the largest change in MG and BR. Therefore, overexpression of VvPL15 in tomato results in tomato fruits that mature earlier. Furthermore, overexpression of VvPL15 promoted fruit softening, which might be attributed to the degradation of ASP.

The VvPL Gene Family and Its Evolutionary Analysis in Grape
The first gene encoding a PL enzyme (also known as Pectate lyase-like, PLL, or PEL) in higher plants was cloned from tomato pollen [16]; in our study, we identified 16 VvPL family members in the grape genome. PL genes have been found in other plants; for example, there are 26 AtPLL genes in Arabidopsis [30], 12 OsPLL genes in rice [31], 46 BrPLL genes in Brassica rapa [32], and 30 PtPL1 genes in the Populus trichocarpa genome [33]. There are also 20 PpePL genes in peach and 22 SlPL genes in the tomato genome [20,22], and 53, 42, and 83 PEL family genes have been identified in the genomes of three cotton species, Gossypium raimondii, G. arboretum, and G. hirsutum, respectively [34]. The reason for the small number of grape VvPL family genes could be that they did not undergo genomewide duplication during the evolution of V. vinifera. Gene duplication is considered to be one of the main drivers of evolution and the expansion of gene families, and segmental duplication and tandem duplication are two main mechanisms that can cause the expansion of plant gene families [35,36]. For example, PpePL5, 6, 7, and 8 have been identified as arising from tandem repeats, which may have resulted in the expansion of the PpePL gene family, leading to changes in the structure and function of family members [20]. This is consistent with previous studies on tomato [22], poplar [32], and cotton [34]. The PL phylogenetic tree were clustered into five groups. Moreover, a multiple protein sequence alignment showed that all plant PL proteins contain a conserved Pec_Lyase_C domain, and that some PL proteins have a signal peptide and a Pec_Lyase_N domain located at the N-terminus [37]. This indicates that PL proteins are highly evolutionarily elements conserved in plants. In this study, the conserved nature of the VvPL gene family was further demonstrated via the collinearity analysis of VvPL genes from the grape and

The VvPL Gene Family and Its Evolutionary Analysis in Grape
The first gene encoding a PL enzyme (also known as Pectate lyase-like, PLL, or PEL) in higher plants was cloned from tomato pollen [16]; in our study, we identified 16 VvPL family members in the grape genome. PL genes have been found in other plants; for example, there are 26 AtPLL genes in Arabidopsis [30], 12 OsPLL genes in rice [31], 46 BrPLL genes in Brassica rapa [32], and 30 PtPL1 genes in the Populus trichocarpa genome [33]. There are also 20 PpePL genes in peach and 22 SlPL genes in the tomato genome [20,22], and 53, 42, and 83 PEL family genes have been identified in the genomes of three cotton species, Gossypium raimondii, G. arboretum, and G. hirsutum, respectively [34]. The reason for the small number of grape VvPL family genes could be that they did not undergo genomewide duplication during the evolution of V. vinifera. Gene duplication is considered to be one of the main drivers of evolution and the expansion of gene families, and segmental duplication and tandem duplication are two main mechanisms that can cause the expansion of plant gene families [35,36]. For example, PpePL5, 6, 7, and 8 have been identified as arising from tandem repeats, which may have resulted in the expansion of the PpePL gene family, leading to changes in the structure and function of family members [20]. This is consistent with previous studies on tomato [22], poplar [32], and cotton [34]. The PL phylogenetic tree were clustered into five groups. Moreover, a multiple protein sequence alignment showed that all plant PL proteins contain a conserved Pec_Lyase_C domain, and that some PL proteins have a signal peptide and a Pec_Lyase_N domain located at the N-terminus [37]. This indicates that PL proteins are highly evolutionarily elements conserved in plants. In this study, the conserved nature of the VvPL gene family was further demonstrated via the collinearity analysis of VvPL genes from the grape and Arabidopsis genomes ( Figure 6). These research results indicate that the evolution of PL gene family in V. vinifera is very conservative.

Functional Analysis of the VvPL Gene Family in Grape
Analysis of VvPL gene promoter sequences showed that there are many types of ciselements present that allow gene expression to respond to different environmental stimuli, and this may be closely related to the multiple functions of VvPL genes in plant species. A total of 16 members of the VvPL family were identified, among which 14 gene promoters had ERE elements and 11 had ARBEs (Table S2). These results indicate that members of this gene family could play roles in grape fruit ripening and abscission. Almost all of the VvPL gene promoters were found to contain multiple MYB elements, suggesting that the transcription of these genes may be regulated by MYB transcription factors. Most of the promoters contain TCA-elements, CGTCA-motifs, and w-box elements, suggesting that the gene family may be involved in plant defense responses. In addition, many known PL genes involved in growth and development were included in the phylogenetic tree. In group 1, VvPL15 showed a close relationship with SlPL, which encodes an important pectinase associated with tomato cell wall decomposition and fruit softening [22]. This indicates that VvPL15 may play a similar role in regulating grape softening. In the first group, VvPL5 is highly homologous to OsPLL4, and OsPLL4 is associated with pollen and grain development [31]. VvPL9 belongs to group 3 and is on the same branch with AtPLL12. AtPLL12 is expressed in the phloem and promotes the development of the cambium and xylem, which is necessary for the growth of vascular bundles in Arabidopsis inflorescence stems [38]. This suggests that VvPL9 may be involved in cell expansion. Moreover, in our study, transgenic Arabidopsis and tomato plants have shown that VvPL15 affects plant growth (Figures 9 and 11). Therefore, VvPL15 may have divers functions, which still need further study.

VvPL15 Is a Key PL Family Member Associated with Fruit Softening in Grape
Ripening and softening of fruits is the main factor that determines their shelf life and commercial value. Many studies have shown that plant PL proteins participate in the ripening and softening process in fruits by degrading demethylated pectin in cell walls [10]. PL genes have been shown to be specifically expressed during fruit ripening, such as Pm65 (pectin lyase) in Chinese plum (Prunus mume) [39], banana (Musa acuminata) [40,41], and mango (Mangifera indica) [42]. Our study showed that VvPL15 is highly expressed during stages E-L35 and E-L36 in grape flesh (Figure 8), and that overexpression of VvPL15 in transgenic tomato accelerated ripening, which suggests that VvPL15 may play an important role during ripening and softening in grape berries. The content of ASP in transgenic tomato fruit decreased and the content of WSP increased, showing that degradation of protopectin accelerates fruit softening and texture changes. These results are also consistent with those of previous studies in species, such as strawberry [18,43], peach [20], and tomato [22]. Recently, Li et al. identified 18 VvPL genes and found that VvPL11 (VIT_213s0019g04910) is a key member of the pectin lyase gene family associated with fruit softening [21]. Compared with the study of Li et al., we identified two fewer VvPL genes in our study. The reason is that the extremely short sequences of VvPL4 (VIT_201s0010g03340) and VvPL18 (VIT_200s0317g00150) located on chromosome 0 were excluded in our analysis. In addition, differences were found in the key genes involved in fruit softening. We found that VvPL15 (VIT_217s0000g09810) is a key gene involved in fruit ripening and softening in 'Kyoho' grape, whereas Li et al. found that VvPL11 (VIT_213s0019g04910) is a key gene that affects fruit softening in 'Hanxiangmi' grape. Therefore, VvPL family members that play key roles in fruit ripening and softening may differ between grape varieties.

Plant Materials and Growth Conditions
Four-year-old rooted cuttings of grapevine (Vitis vinifera cv. 'Kyoho') grown under standard field conditions were selected from the Shandong Agricultural University Horticultural Experiment Station (Taian, China). The berries were collected at five developmental stages; E-L 33, E-L 35, E-L 36, E-L 37, and E-L 38 based on the standard system for clas-sifying grapevine growth stages (E-L system). The grape flesh firmness was determined immediately after sampling, and the samples were then frozen in liquid nitrogen and stored at −80°C prior to their use in the experiments.
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild-type. Seeds of Col-0 were sterilized with 70% alcohol and 1% sodium hypochlorite (NaClO) and sown on 0.5X MS semisolid medium (supplemented with 8 g·L −1 agar and 30 g·L −1 sucrose). Seeds were vernalized by incubation at 4 • C for three days and then transferred to a growth chamber under a 16 h/8 h (light/dark) cycle (200 µmol·m −2 ·s −1 ) at 21 • C/18 • C (day/night) for nine days. Seedings were then cultivated in nutrient soil and grown in agreenhouse under the same conditions as in the growth chamber.
Solanum lycopersicum 'Ailsa Craig' (AC) was used in transformation experiments. Transgenic cultures were grown in a greenhouse under a 16 h/8 h (light/dark) photoperiod at temperatures of 25 • C/18 • C (day/night). Tomato plants in the first generation (T 0 ) came directly from tissue culture, and plants of the second generation (T 1 ) were derived from seeds harvested from T 0 -generation transgenic plants. The flesh firmness of T 1 -generation tomato fruits was determined immediately after sampling. The samples were picked at four developmental stages; mature green (MG), breaker stage (BR), yellow red stage (YR), and red ripening stage (RR) based on the study of Liu et al. [44]. The remaining intact samples were immediately frozen in liquid nitrogen and stored at −80 • C for further analysis.

Measurement of Flesh Firmness in Grape Berries
The firmness was measured using a TA.XT plus texture analyzer (Stable Micro Systems, Surrey, UK), a P/2 probe (2 mm diameter), and a puncture method with a puncture depth of 7 mm. Fifteen berries were randomly selected for each biological replicate, and two parts above and below the maximum transverse diameter of each fruit were measured and averaged. There were three biological replicates for each of the five fruit stages.

Measurement of Water-Soluble Pectin (WSP) and Acid-Soluble Pectin (ASP) Contents
The test used was the carbazole colorimetry method described by He et al. (2018) [45]. The standard curve was drawn with the amount of galacturonic acid as the abscissa and the absorbance value as the ordinate. One-gram samples of grape berry and tomato fruit flesh were used to prepare the extracts. The WSP was extracted with distilled water in a 50 • C water bath, and the ASP was extracted with 0.5 mol/L sulfuric acid solution in a boiling water bath. The absorbances of 1 mL samples of the WSP and ASP extracts were measured using a spectrophotometer at 530 nm, and the concentrations were determined from the standard curve.

Genome-Wide Identification of VvPLs
The accession numbers and amino acid sequences of AtPLLs and SlPLs were collected using for grape VvPLs genes family analysis according to the previous study [30]. A total of 26 AtPL proteins and 22 SlPL proteins were used as queries to search the JGI database (https://phytozome-next.jgi.doe.gov/ (accessed on 26 November 2021)). The homologous protein alignment was performed using a Blastp research with 26 AtPLL and 21 SlPL protein sequences as the query sequence. All grape VvPL proteins are identified in the grape proteome.

Phylogenetic Tree Analysis
Twelve OsPLLs gene family numbers were collected from Zheng et al. (2018) [31]. All protein sequences of PL family members were collected from Arabidopisis thaliana, Solanum lycopersicum, Oryza sativa and Vitis vinfera, and used to construct phylogenetic tree via the MEGA7 software. The multiple sequence alignment result was obtained by the built-in Cluster W with default parameters. The construction method was selected Neighbor-joining and 1000 bootstrap replicates. The phylogenetic tree was modified iToL online tool (https://itol.embl.de/ (accessed on 1 August 2021)).

Conserved Motif, Gene and Protein Structure Analysis
The conservative model analysis was conducted via the online tool MEME (https:// meme-suite.org/meme/tools/meme/ (accessed on 1 August 2021)) with the following parameters: maximum number of motifs, 10; and optimum motif width, between 6 and 50. Additionally, the result file was visualized via TBtools. The VvPL gene structure was analyzed to understand the gene characterization of transcription factors via the visualization server GSDS2.0 (http://gsds.gao-lab.org/index.php/ (accessed on 5 August 2021)). The three-dimensional structures of VvPL proteins were predicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index/ (accessed on 11 August 2021)) and 3D model visualization was performed according to protein homology modeling by PyMoL.

Collinearity Analysis
The whole genome files of grape and Arabidopsis were obtained on the website (http://plants.ensembl.org/index.htm/ (accessed on 17 February 2022)), and the collinearity between grape and Arabidopsis was analyzed using the TBtools software [46].

Promoter Analysis
The 2000-bp sequences upstream of transcription factor of VvPL gene were used as the promoter sequence. The promoter cis-acting elements were predicted using the online site PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 August 2021)).

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted using the TIANGEN kit (Polysaccharides & Polyphenolicsrich; Tiangen Biotech, Beijing, China). cDNA was synthesized using a reverse transcriptase kit (Takara, Dalian, China). Primers for real-time quantitative PCR (RT-qPCR) were available in qPrimerDB-qPCR Primer Database (https://biodb.swu.edu.cn/qprimerdb/ accessed on 6 September 2021) and VvActin was used as a reference gene. Gene expression analysis was performed using system (BIO-RAD iQ5, Hercules, CA, USA) by three independent biological replicates for each sample. The relative expression level was calculated using the 2 −∆∆Ct method. The specific primers are listed in Table S2.

Vector Construction and Plant Transformation
The coding sequence of VvPL15 was cloned using kyoho grape flesh CDNA as a template. To get the transgenic lines, the coding sequence of VvPL15 was inserted into pRI101 vector, named as 35S::VvPL15-OX. 35S::VvPL15-OX transgenic Arabidopsis, was obtained using the Agrobacterium tumefaciens strain GV3101 transformation method following the steps described in Kang et al. (2021) [47]. 35S::VvPL15-OX transgenic tomato was obtained using the cotyledon disc method, as previously described [48]. The specific cloning primers were designed via the CE Design software (Table S2).

Transient Transformation in Grape Leaves
The TRV vector was used to construct an antisense expression vector [49]. The specific 300 bp fragments of VvPL15 were inserted into TRV vector in the antisense orientation, named as TRV-VvPL15. TRV1 was used as an auxiliary carrier. The antisense vectors were transformed using the Agrobacterium tumefaciens strain GV3101. The empty TRV vector served as a control. The detailed experimental operations followed the method of Kang et al. (2020) [50]. The specific cloning primers are listed in Table S2. The gene expression, WSP and ASP contents of infected grape leaves were analyzed. Three biological replicates were designed, and each biological replicate was formed by mixing three grape leaves.

Statistical Analysis
Statistical differences were determined using single factor analysis of variance (ANOVA), and the least significant difference (LSD) at p < 0.05 by SPSS 26. The figures were prepared using GraphPad Prism9.

Conclusions
In this study, we identified and characterized the 16 members of the pectin lyase gene family in grape. Analysis of firmness and pectin content during grape berry ripening showed that changes in firmness were closely related to pectin composition. Analysis of gene expression patterns in berries from five stages of grape ripening revealed that VvPL15, a core member of the VvPL family, may play a major role in the onset of veraison in grapes. The role of VvPL15 in grape ripening was investigated using heterologous transformation and transient transformation experiments, and we found that the relative expression levels of VvPL15 promoted the softening of grape flesh through the depolymerization of pectin.