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Article

VvPL11 Is a Key Member of the Pectin Lyase Gene Family Involved in Grape Softening

by
Wenxin Li
†,
Chang He
†,
Hongli Wei
,
Jiakang Qian
,
Jiannan Xie
,
Zhiqian Li
,
Xianbo Zheng
,
Bin Tan
,
Jidong Li
,
Jun Cheng
,
Wei Wang
,
Xia Ye
* and
Jiancan Feng
*
College of Horticulture, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, China
*
Authors to whom correspondence should be addressed.
†
Those authors contributed equally to this work.
Horticulturae 2023, 9(2), 182; https://doi.org/10.3390/horticulturae9020182
Submission received: 12 December 2022 / Revised: 18 January 2023 / Accepted: 22 January 2023 / Published: 1 February 2023
Browse Figures
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Abstract

:
Fruit ripening includes several metabolic changes that lead to sweeter and softer fruit. Pectin depolymerization is one of major factors that softens developing grape berries. Pectin lyases (PLs) play important roles in pectin degradation in the grape berry. However, little is known about the temporal and spatial expression of grapevine (Vitis spp.) pectin lyase genes (VvPLs) or their function during fruit ripening and softening. In this study, 18 individual VvPL genes were identified in the grape genome. All VvPL genes were sorted into group I and group II, except VvPL12 which demonstrated higher and similar expression trends in different tissues and organs. In grape berry, VvPL1, 5, 7, 11 and 16 were highly expressed, whereas VvPL18, 15, 2, 13, 10, 14, 17, 6 and 8 showed lower expression levels at different berry developmental stages. Expression of VvPL11 firstly increased and then decreased, and the highest expression was shown at 6 weeks after full bloom (WAFB) during berry development. Over-expression of the VvPL11 gene in tomato caused higher ethylene production and lower firmness compared to wild-type fruit. Moreover, decreased propectin and increased water-soluble pectin (WSP) levels were observed in VvPL11 transgenic tomato fruit. Consistent with this result, the expression levels of SlPG2, SlEXP, and SlPME1, all of which are genes involved in fruit softening, were up-regulated in VvPL11-OE tomato fruit, which supported the idea that VvPL11 plays an important role in fruit ripening and softening. This study provided a comprehensive analysis of the grapevine PL family and advanced our knowledge of the functions of VvPLs during fruit softening.

1. Introduction

Grapevine is one of the most efficient and economical horticultural crop plants around the world. Recently, grape has become very popular among consumers because of its beautiful color, distinct flavors, and rich nutrients. It is widely cultivated in the Henan Province in China which significantly contributes to local agricultural industry development [1]. However, grapevines are at risk of losing soft berries through fruit drop and decay. Grape softening is an important process before ripening, and the berry softening rate directly affects grape berry firmness [2]. Rapid softening at veraison results in extremely short storage and shelf life after harvest, which causes great losses to the grape industry [3,4,5]. Previous studies indicate that grape berry softening is a complex process [6] that is associated with changes in the fruit cell wall structure [7,8], cell morphology [9,10,11], cell spatial arrangement, and cell integrity during berry development [11,12].
The fruit cell wall consists mainly of polysaccharides, which are classified as pectin, hemicellulose, and cellulose, and proteins. Pectin is one of the most complex polysaccharides and is the main component of the primary cell wall and the intermediate layer between the cell wall and the cell membrane. Pectin consists of homogalacturonic acid (HG), rhamnogalacturonic acid (RG), and xylogalacturonic acid [13]. Previous studies suggest that altering the structure of pectin through depolymerization or breaking of side chains is the major factor determining fruit texture during softening [14]. Pectin degradation is synergistically carried out by various enzymes, including pectin methylesterases (PMEs), polygalacturonases (PGs), galactosidases, and pectin lyases (PLs) [15,16,17].
PLs are the only known pectin-degrading enzymes and are capable of cleaving glycosidic bonds [18]. PLs belong to a family of polysaccharide lyase enzymes, whose members have been identified in tomato [8], strawberry [19], and peach [20]. Many studies have shown that PL plays a role in a number of physiological and biochemical pathways, including stomatal opening and closing [21], petal abscission [22], pollen development [23], fruit softening [7], leaf senescence [24], and disease resistance [25,26,27]. The role of PL in regulating fruit softening has been shown in both climacteric and nonclimacteric fruit [28]. During ripening of strawberry and tomato, fruit softening is accompanied by an increase in PL activity [29,30]. Silencing or knocking out PL genes leads to a slow rate of fruit softening and increasing fruit firmness, and SlPL was verified to be an excellent candidate for improving tomato firmness [13,29,30]. These results suggest that there is an inextricable link between fruit ripening and softening and the PL genes.
During grape berry softening, the cell-wall components are important factors, and degradation of pectin polysaccharides has been commonly associated with fruit softening [3,31]. Grapes with higher firmness levels have a lower rate of cell-wall disassembly, and VvPG and VvPL are involved in the difference in firmness between hard and soft berries [31,32]. There are relatively few studies on pectin lyases in grape, and the expression patterns and functions of the VvPLs in grape during fruit softening are not known. Based on these shortcomings in our knowledge, this report first identifies and analyzes the pectin lyase family in the entire grape genome and analyzes the conserved motifs and the evolutionary relationships of the grape pectin lyase proteins. VvPL11 showed a higher expression in mature berry, and it was over-expressed in tomato. The results of this study provide a preliminary theoretical basis for understanding the function of VvPL11 in berry softening.

2. Material and Methods

2.1. Plant Materials

Grape (Vitis labrusca × Vitis vinifera ‘Hanxiangmi’) was planted in a commercial vineyard in Zhengzhou of Henan province. ‘Hanxiangmi’ produces an extremely soft berry. The samples of grape berry clusters were collected at different weeks after full bloom (WAFB), namely 2 W, 4 W, 6 W, 8 W (veraison when almost 50% berries per cluster were colored), and 10 W (ripening). At every sampling time, nine grape clusters were harvested randomly from different vines of the cultivar ‘Hanxiangmi’ grown in the Yellow River Plain with an average temperature of 14.62 °C and an average precipitation of 744.14 mm [33]. After being subjected to instrumental analyses, the berries from grape clusters were stored at −80 °C for RNA extraction.

2.2. Identification and Analysis of VvPL Family Members

To identify grape pectin lyase genes, 26 Arabidopsis (AtPL) proteins were downloaded from a search of the TAIR10 database (https://www.arabidopsis.org/index.jsp (accessed on 16 July 2022)) [34], and then AtPL proteins were used as queries to search the JGI database (https://phytozome-next.jgi.doe.gov/ (accessed on 16 July 2022)). Eighteen VvPL proteins were obtained using BLASTP in the JGI database, and each VvPL gene was manually checked to confirm whether a conserved PL domain existed. In addition, gene sequences, mRNA, CDS, amino acid sequences and annotation information of the grape PL members were obtained from the JGI database. These data were used in the relevant analyses that follow. Twenty tomato PL genes (SlPLs) were also downloaded from the JGI database using the same method according to protein accession numbers [30].

2.3. Phylogenetic Analysis and Gene Structure of VvPLs

To investigate the evolutionary relationships between VvPLs and other PLs, the protein sequences of the 18 VvPLs, 26 AtPLs, 22 SlPLs, FaPLc, and FvPLA were aligned to construct a phylogenetic tree by MEGA (7.0) software. Based on the protein sequence alignment results, phylogenetic analysis was performed by using the neighbor-joining (NJ), 1000 bootstrap method. The evolutionary tree file was initially obtained and then plotted using the online website ITOL (https://itol.embl.de/ (accessed on 25 July 2022)). The amino acid sequences of all proteins in the phylogenetic tree are listed in Supplementary Table S1.
The amino acid sequences of the VvPLs were uploaded to SMART (http://smart.embl-heidelberg.de/ (accessed on 11 August 2022)), and information on length of amino acids, relative molecular weight, and theoretical isoelectric point was obtained. These sequences were then uploaded to Cell-Ploc (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 11 August 2022)) for prediction of protein subcellular localizations. The motif analysis was performed using MEME (https://meme-suite.org/meme/tools/meme (accessed on 12 August 2022)). All of these analyses were performed using default parameters. Gene structure and chromosome location of the VvPLs were analyzed using Tbtools methods (www.tbtools.com/ (accessed on 12 August 2022)).

2.4. Tissue-Specific Expression Analysis of the VvPL Genes

The expression levels of the VvPLs were analyzed in 54 different tissues/organs at different developmental stages of grapes from transcriptome data in the NCBI database (GSE36128) [35]. The identified VvPLs were searched in the GEO database (GSE36128), and the expression profile data of these genes were obtained. Heatmaps of the expression levels were created using the log2-RPKM normalized values +1 using OmicStudio (https://www.omicstudio.cn/tool/4/ (accessed on 11 August 2022)).

2.5. Expression Analysis of VvPL Genes during Berry Development

The expression levels of 14 VvPL genes were obtained by analyzing our previous transcriptome data of the ‘Hanxiangmi’ berry at four developmental stages (non-published data). The expression of the VvPLs was analyzed by clustering, and the heat map was constructed using OmicStudio.
To verify the reliability of the data, reverse transcriptase-quantitative PCR (RT-qPCR) of the VvPL genes was performed at different developmental stages of the ‘Hanxiangmi’ berries. Total RNA was extracted using a plant RNA extraction kit (Huayueyang Co. Ltd., Beijing, China). RT-qPCR was performed as previously described [36]. All PCR primers are listed in Table S2. For each sample, quantifications were made in triplicate. Three biological replicates and three technical replicates were conducted to verify the accuracy of the expression data.

2.6. Overexpression of VvPL11 Gene in Tomato

The CDS sequence of VvPL11 without its stop codon was cloned into pSAK277, and the pSAK277 plasmid was transformed into Agrobacterium tumefaciens strain GV3101. Transgenic tomato (Solanum lycopersicum cv. Micro-Tom) plants were obtained according to the reference [37]. Transgenic fruits from the T1 generation were collected at breaker, breaker + 3d, breaker + 5d, breaker + 7d, and breaker + 15d, respectively. Fruit firmness was measured using a TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, U.K.) and slightly modified as described by Tan to analyze the ethylene production of fruits [38]. Expression of VvPL11 and pectin-degradation genes in transgenic tomato fruit were evaluated by the RT-qPCR method.

2.7. Analysis of Propectin and WSP Contents

The propectin and water-soluble pectin (WSP) contents of transgenic tomato fruit were determined using the carbazole colorimetric method according to Guan et. al. [39]. The enzymatic activity of pectin lyase was analyzed using a colorimetric enzyme activity kit (Kemin Co., Ltd., Suzhou, China).

3. Results

3.1. Identification of Grapevine PL Family Members

A total of 18 genes were identified as candidate PL family members in the grapevine genome (Table 1). These 18 PL family members were named VvPL1 to VvPL18 according to their chromosomal position (from top to bottom). VvPLs are unevenly distributed on 12 of the chromosomes of the grapevine genome (Figure 1). Chromosome 1 contains four VvPL members, whereas chromosomes 5, 7, 10, 16, and 19 have one member each. Chromosomes 8, 13, 14, and 17 have two VvPL members each. The inferred peptide sequence lengths of the VvPL proteins ranged from 78 to 543 amino acid residues, and the predicted molecular weights ranged from 36,103.92 to 57,104.76 (kDa). The predicted isoelectric points of the VvPL proteins were between 4.92 and 9.44 (Table 1).
A phylogenetic tree of the 18 VvPL proteins, 26 AtPL proteins, 22 SlPL proteins, FaPLc, and PvPLA clustered into five groups (Figure 2). Group I contained VvPL5 and VvPL6, which were closely clustered with FvPLA and FaPLc from strawberry, respectively. VvPL1, VvPL16, and VvPL12 proteins clustered in group II together with SlPL, which is a candidate gene for tomato firmness. VvPL17, VvPL8, VvPL10, and VvPL11 proteins were clustered in group III, with VvPL11 showing a closer relationship with AtPLL12, which may suggest that VvPL11 is an orthologous proteinof AtPL12. In groups IV and V, there were four (VvPL15/2/3/13) and five VvPL (4/9/14/7/18) proteins, respectively.
The conserved motifs of the VvPL protein family members were identified using Tbtools (Figure 3). Most VvPL members contained 10 motifs, whereas VvPL14 was missing motif 7 and 9, and VvPL4 was missing motifs 1, 2, 4, 5, 7, 8, 9, and 10. Results of intron/exon structural analysis showed that eleven VvPL members contained three exons, two VvPL members had four exons, two VvPL members had two exons, 2 VvPL members had six exons, and VvPL4 contained one exon (Figure 3).
Multiple sequence alignment of the grape, tomato and strawberry pectin lyase genes showed that most of the protein sequences contained conserved motifs I (WIDH), II (DGLIDAIMASTAITISNNYF) and III (LIQRMPRC RHGYFHVVNNDY), with the exception of VvPL4, for which the protein sequence lacked three conserved motifs.

3.2. Tissue-Specific Expression Analysis of the VvPL Genes

For V. vinifera, the available transcriptomic data (GSE36128) were used for the expression profiles of VvPL genes. The expression levels of the 18 VvPLs in 54 different tissues/organs at different fruit developmental stages indicated that most of the VvPLs were expressed in all tissues (Figure 4). Higher expression of VvPL7 was shown in all tissues and organs, whereas VvPL5/1/6/11/16, from different groups in the phylogenetic tree, which are showed similar expression patterns of higher expression levels in inflorescence, flower, and tendril. After berry veraison, expression of VvPL11 and VvPL1 were generally down-regulated, which may imply important roles before berry ripening. VvPL2/3/4/5/8/9/10/12/14/15/17/18 showed scattered expression in different tissues. VvPL2/3/15/9 showed lower expression in different tissues, except that VvPL2/3/15 showed higher expression levels in the flowers.

3.3. Expression Analysis of VvPL Genes during Berry Development

Our transcriptome data in cultivar ‘Hanxiangmi’ (Table S3) indicated that the VvPL genes in groups I and II were highly expressed during berry growth and development (Figure 5). The expression levels of VvPL1/5/7/11/16 were higher, and the levels of VvPL18/15/2/13/10/14/17/6/8 were lower across the berry developmental stages (Figure 5A). The highest expression levels for VvPL5 and 16 were observed at 4 WAFB, whereas VvPL11 and VvPL1 were highly expressed at 6 WAFB. RT-qPCR also supported the higher expression levels of VvPL11 and 1 before berry ripening (Figure 5B). In addition, the expression levels of VvPL11 and 1 showed a positive correlation with the content of WSP during berry development (data not shown). Together, these results prompted the choice of VvPL11 for further experimentation.

3.4. Overexpression of VvPL11 in Tomato Decreased Fruit Firmness

To elucidate any roles of VvPL11 during fruit ripening, VvPL11 was overexpressed in tomato plants (Figure 6). A total of 11 VvPL11-overexpressing (OE) transgenic lines were obtained and verified by RT-qPCR (Figure 6B). The ectopic VvPL11 was highly expressed in the leaves of transgenic lines 1, 2, and 3 (Figure 6A,B). There were no phenotypic differences in plant height, flowering time, or fruit set between the OE and WT tomato plants (data not shown). VvPL11-OE2 and -OE3 seemed to turn red earlier-specifically, at the Br + 3d and Br + 5d stages-compared to the controls (Figure 6A). After the breaker stage, firmness of the T1 transgenic tomato fruits was lower than that of the control at the 3, 5, 7, and 15 days after breaker, and ethylene production of the fruit was significantly increased compared to control fruits at the same stage (Figure 6C,D). These results suggested that over-expression of the VvPL11 gene in tomato accelerated tomato fruit ripening and softening.

3.5. Overexpression of VvPL11 Accelerated Cell Wall Degradation in Transgenic Tomato

To determine how overexpression of VvPL11 alters the pectin content, the propectin and WSP content in transgenic tomato fruits were analyzed (Figure 7). The propectin content in the VvPL11-OE2 and -OE3 lines was greatly decreased at the Br + 3 and Br + 5 stages (Figure 7A), whereas the WSP content consistently increased after the breaker stage compared to controls (Figure 7B). Moreover, the transcript levels of key pectin degradation-related genes, namely SlPG2, SlPME1, and SlEXP, were up-regulated in transgenic tomato fruit at all stages after the breaker stage (Figure 7C–E).

4. Discussion

During grape berry ripening, degradation of cell-wall components leads to cell-wall structural disruption and fruit softening, and depolymerization of pectin in the primary wall is an important factor [3,32]. Until now, the identification and characterization of the PL family have been conducted in different plants, such as A. thaliana, S. lycopersicun, Fragaria ananassa, and Fragaria vesca, in order to identify the crucial PL genes associated with fruit development processes [34]. However, the expression pattern and function of VvPL genes, which code for the only known pectin-degrading enzymes cleaving glycosidic bonds [18], have remained poorly understood with regard to berry softening. In this study, we identified 18 VvPL genes, characterized them in term of their phylogenetic relationships, gene conservation patterns, and gene structure, and analyzed the possible function of the VvPL11 gene in fruit softening.

4.1. Most of VvPL Genes in Groups I and II were Highly Expressed in Different Tissues and Organs

In a search of the grape genome database, 18 VvPL genes were identified and were classified into five major groups. Most of VvPL members exhibit the conserved motifs of PL proteins in S. lycopersicun, F. ananassa, and F. vesca [34], whereas VvPL4 has the shortest predicted amino acid sequence and seems to lack most motifs and conserved domains, suggesting that VvPL4 is an incomplete gene. However, the VvPL4 gene showed differential expression among the tissue and organ samples, indicating that this conflicting result needs to be further investigated. The proteins in the same group have similar exon–intron structures, except for group 5. The difference in exon numbers indicated that the VvPL members possibly have their own diversified function [40].
The expression analysis performed by GEO Datasets (GSE36128) and our previous transcriptome profiles showed that all VvPL genes in group I and group II except VvPL12 demonstrated higher and similar expression trends in different tissues and organs. In group I, expression levels of VvPL5 and VvPL6 were higher, and the amino acid sequences of VvPL6 and VvPL5 were highly similar to FaPLc and FvPLA, which may imply VvPL6 and VvPL5 were orthologs of FaPLc and FvPLA, respectively. FaPLc and FvPLA have proven roles in ripening and softening of strawberry [41], suggesting that VvPL6 and VvPL5 may be associated with softening in grape berries. In group II, VvPL1 and VvPL16 clustered with SlPL12, SlPL15, and SlPL, which show dominant expression during fruit maturation; SlPL is a candidate gene for tomato firmness [30]. In the phylogenetic tree, VvPL12 was close to SlPL5, which is an important gene that contributes to fruit softening [42]. The clustering and expression results suggested that VvPL genes in groups I and II may be involved in fruit ripening and softening. Moreover, transcriptome analysis of berries at different developmental stages from the grape cultivar ‘Hanxiangmi’ showed that VvPL6/5 in group I and VvPL1/16 in group II had higher expression levels, which further implies that these VvPLs play vital roles in berry ripening and softening.

4.2. VvPL11 Plays an Important Role in Fruit Softening

As enzymes that take part in pectin degradation, PL genes have been proven to be highly expressed in mature fruit but not in unripe fruit, which indicates PLs are associated with fruit ripening and softening of banana and peach [43,44]. VvPL11 belonged to group III and was the only gene with higher expression during berry development. Because a positive correlation between VvPL11 gene expression and WSP content in berry was observed, the VvPL11 gene was overexpressed in tomato plants to elucidate its role in fruit softening. Overexpression of VvPL11 resulted in lower fruit firmness and higher ethylene production in transgenic tomato compared to WT, which suggested that the protein VvPL11 was an accelerator on fruit softening. Cell wall component analysis indicated that there was a lower propectin and higher WSP content in transgenic tomato fruit, which suggests that fruit softening and texture changes were advanced by accelerating degradation of propectin. This result is consistent with the findings in banana [45,46], strawberry [47,48], tomato [49], and peach [44], for which inhibition of fruit ripening by external treatments was often found to be accompanied by a decrease in pectin lyase expression. Furthermore, antisense expression of a pectin lyase gene delayed postharvest softening of strawberry [50].
The present study also showed that an overexpression of the VvPL11 gene resulted in increased transcript levels for the SlPG, SlPL, and SlPE genes, similar to results observed in tomato fruit [51]. In transgenic tomato plants overexpressing apricot PaPL9, genes related to ethylene biosynthesis, fruit softening, and chlorophyll degradation showed induced expression, which suggested that PL genes play a core role in fruit ripening and that their overexpression may modulate the expression of ripening-related genes. In this study, our research was mainly concentrated on the crucial function of the VvPL11 gene, which is associated with cell wall degradation, fruit softening, and ripening. Nevertheless, further work in molecular and genetic identification is necessary to explore the regulatory network of berry ripening and softening mechanisms in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9020182/s1. Figure S1: Characterisation of VvPL5/6, FvPLA and FaPLc. Table S1: The sequence of protein used in phylogenetic trees. Table S2: The information of the primers used for RT-qPCR and cloning. Table S3: Transcriptome data of VvPLs in the cultivar ‘Hangxiangmi’.

Author Contributions

Conceptualization, W.L., C.H., X.Y. and J.F.; methodology, W.L., Z.L., J.C. and X.Y.; software, W.L., H.W. and B.T.; validation, W.L., C.H. and J.L.; formal analysis, W.L., C.H. and X.Y.; investigation, W.L., C.H., J.Q. and J.X.; resources, X.Z., X.Y. and J.F.; data curation, W.L., W.W. and X.Y.; writing—original draft preparation, W.L. and C.H.; writing—review and editing, X.Y. and J.F.; supervision, B.T. and X.Y.; funding acquisition, X.Y. and J.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Natural Science Foundation of Henan Province (Grant No. 222300420457), the National Natural Science Foundation of China (Grant No. 32002017), and the Henan Province Outstanding Foreign Scholar Program (Grant No. GZS2020007).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used in the research are available within the article and the Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Chromosome distribution of pectin lyase (PL) genes in the grapevine genome. Chromosome numbers are shown at the top of each chromosome. Scale is in megabases (Mb).
Figure 1. Chromosome distribution of pectin lyase (PL) genes in the grapevine genome. Chromosome numbers are shown at the top of each chromosome. Scale is in megabases (Mb).
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Figure 2. Phylogenetic tree of pectin lyase (PL) proteins from grapevine and other species. The proteins clustered into five groups, which were labeled with different colors at the periphery. Grapevine proteins were labeled with red dots. The species shown are Arabidopsis thaliana (At), Vitis vinifera (Vv), Solanum lycopersicun (Sl), Fragaria ananassa (Fa), and Fragaria vesca (Fa).
Figure 2. Phylogenetic tree of pectin lyase (PL) proteins from grapevine and other species. The proteins clustered into five groups, which were labeled with different colors at the periphery. Grapevine proteins were labeled with red dots. The species shown are Arabidopsis thaliana (At), Vitis vinifera (Vv), Solanum lycopersicun (Sl), Fragaria ananassa (Fa), and Fragaria vesca (Fa).
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Figure 3. Characterization of PL family members in V. vinifera and other species. (A) Phylogenetic relationships, motifs, and exon/intron structures of VvPLs. The left part represents the phylogenetic tree of VvPL proteins. The middle part displays the conserved motifs of VvPLs. Different motifs are represented by blocks of different colors. The right part indicates the exon–intron distribution of VvPL genes. (B) Amino acid sequence alignment of V. vinifera VvPLs with other PLs. Orange shading indicates three typical conserved motifs of PLs, which were referred to as motif I, II and III respectively.
Figure 3. Characterization of PL family members in V. vinifera and other species. (A) Phylogenetic relationships, motifs, and exon/intron structures of VvPLs. The left part represents the phylogenetic tree of VvPL proteins. The middle part displays the conserved motifs of VvPLs. Different motifs are represented by blocks of different colors. The right part indicates the exon–intron distribution of VvPL genes. (B) Amino acid sequence alignment of V. vinifera VvPLs with other PLs. Orange shading indicates three typical conserved motifs of PLs, which were referred to as motif I, II and III respectively.
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Figure 4. Tissue-specific expression patterns of VvPLs. The color scale on the right represents the RPKM normalized log2+1. Blue indicates low expression levels, yellow indicates moderate expression levels, and red indicates high expression levels. Transcriptome data were from the NCBI database (GSE36128) and represent multiple studies. The tissue samples were as follows: Stamen-pool of stamens from 10% and 50% open flowers; BerryPericarp-FS-berry pericarp at fruit set; BerryPericarp-PFS-berry pericarp post-fruit set; BerryPericarp-V-berry pericarp véraison; BerryPericarp-MR-berry pericarp mid-ripening; BerryPericarp-R-berry pericarp ripening; Bud-S-bud swell; Bud-B-bud burst; Bud-AB-bud after-burst; Bud-L-latent bud; Bud-W-winter bud; BerryFlesh-PFS-berry flesh post fruit set; BerryFlesh-V-berry flesh veraison; BerryFlesh-MR, berry flesh mid-ripening; BerryFlesh-R, berry flesh ripening; BerryFlesh-PHWI-berry flesh post-harvest withering I (1st month); BerryFlesh-PHWII-berry flesh post-harvest withering II (2nd month); Berry Flesh PHWIII-berry flesh post-harvest withering III (3rd month); Inflorescence-Y-young inflorescence; Inflorescence-WD-well-developed inflorescence; Flower-FB-flowering begins; Flower-F-flower at flowering; Root-root in vitro cultivation; Leaf-Y-young leaf; Leaf-FS-mature leaf; Leaf-S-senescing leaf; Carpel-pool of carpels from 10% and 50% open flowers; Petal-pool of petals from 10% and 50% open flowers; BerryPericarp-PHWI-berry pericarp post-harvest withering I (1st month); BerryPericarp-PHWII-berry pericarp post-harvest withering II (2nd month); BerryPericarp-PHWIII-berry pericarp post-harvest withering III (3rd month); Pollen-pollen from disclosed flowers at more than 50% open flowers; Rachis-FS-rachis fruit set; Rachis-PFS-rachis post-fruit set; Rachis-V-rachis at véraison; Rachis-MR-rachis mid-ripening; Rachis-R-rachis ripening; Seed-V-seed veraison; Seed-MR-seed mid-ripening; Seed-FS-seed fruit set; Seed-PFS-seed post-fruit set; Seedling-seedling pool of 3 developmental stages; BerrySkin-PFS-berry skin post-fruit set; BerrySkin-V-berry skin at veraison; BerrySkin-MR-berry skin mid-ripening; BerrySkin-R-berry skin ripening; BerrySkin-PHWI-berry skin post-harvest withering I (1st month); BerrySkin-PHWII-berry skin post-harvest withering II (2nd month); BerrySkin-PHWIII-berry skin post-harvest withering III (3rd month); Stem-G-green stem; Stem-W-woody stem; Tendril-Y-young tendril (pool of tendrils from shoot of 7 leaves); Tendril-WD-well-developed tendril (pool of tendrils from shoot of 12 leaves); Tendril-FS-mature tendril (pool of tendrils at fruit set).
Figure 4. Tissue-specific expression patterns of VvPLs. The color scale on the right represents the RPKM normalized log2+1. Blue indicates low expression levels, yellow indicates moderate expression levels, and red indicates high expression levels. Transcriptome data were from the NCBI database (GSE36128) and represent multiple studies. The tissue samples were as follows: Stamen-pool of stamens from 10% and 50% open flowers; BerryPericarp-FS-berry pericarp at fruit set; BerryPericarp-PFS-berry pericarp post-fruit set; BerryPericarp-V-berry pericarp véraison; BerryPericarp-MR-berry pericarp mid-ripening; BerryPericarp-R-berry pericarp ripening; Bud-S-bud swell; Bud-B-bud burst; Bud-AB-bud after-burst; Bud-L-latent bud; Bud-W-winter bud; BerryFlesh-PFS-berry flesh post fruit set; BerryFlesh-V-berry flesh veraison; BerryFlesh-MR, berry flesh mid-ripening; BerryFlesh-R, berry flesh ripening; BerryFlesh-PHWI-berry flesh post-harvest withering I (1st month); BerryFlesh-PHWII-berry flesh post-harvest withering II (2nd month); Berry Flesh PHWIII-berry flesh post-harvest withering III (3rd month); Inflorescence-Y-young inflorescence; Inflorescence-WD-well-developed inflorescence; Flower-FB-flowering begins; Flower-F-flower at flowering; Root-root in vitro cultivation; Leaf-Y-young leaf; Leaf-FS-mature leaf; Leaf-S-senescing leaf; Carpel-pool of carpels from 10% and 50% open flowers; Petal-pool of petals from 10% and 50% open flowers; BerryPericarp-PHWI-berry pericarp post-harvest withering I (1st month); BerryPericarp-PHWII-berry pericarp post-harvest withering II (2nd month); BerryPericarp-PHWIII-berry pericarp post-harvest withering III (3rd month); Pollen-pollen from disclosed flowers at more than 50% open flowers; Rachis-FS-rachis fruit set; Rachis-PFS-rachis post-fruit set; Rachis-V-rachis at véraison; Rachis-MR-rachis mid-ripening; Rachis-R-rachis ripening; Seed-V-seed veraison; Seed-MR-seed mid-ripening; Seed-FS-seed fruit set; Seed-PFS-seed post-fruit set; Seedling-seedling pool of 3 developmental stages; BerrySkin-PFS-berry skin post-fruit set; BerrySkin-V-berry skin at veraison; BerrySkin-MR-berry skin mid-ripening; BerrySkin-R-berry skin ripening; BerrySkin-PHWI-berry skin post-harvest withering I (1st month); BerrySkin-PHWII-berry skin post-harvest withering II (2nd month); BerrySkin-PHWIII-berry skin post-harvest withering III (3rd month); Stem-G-green stem; Stem-W-woody stem; Tendril-Y-young tendril (pool of tendrils from shoot of 7 leaves); Tendril-WD-well-developed tendril (pool of tendrils from shoot of 12 leaves); Tendril-FS-mature tendril (pool of tendrils at fruit set).
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Figure 5. Expression patterns of VvPL genes at different fruit developmental stages of ‘Hanxiangmi’. (A) Expression heatmap of VvPL genes from the transcriptomes of grape berries at different developmental stages. The color scale on the right represents the RPKM normalized log2. Blue indicates low expression levels, yellow indicates moderate expression levels, and red indicates high levels. (B) Relative expression levels of VvPL genes verified by RT-qPCR in developing fruit at 2, 4, 6, 8, and 10 weeks after full bloom (2 W, 4 W, 6 W, 8 W, and 10 W). Data were shown as the means ± standard deviation. * represent significant differences at p < 0.05.
Figure 5. Expression patterns of VvPL genes at different fruit developmental stages of ‘Hanxiangmi’. (A) Expression heatmap of VvPL genes from the transcriptomes of grape berries at different developmental stages. The color scale on the right represents the RPKM normalized log2. Blue indicates low expression levels, yellow indicates moderate expression levels, and red indicates high levels. (B) Relative expression levels of VvPL genes verified by RT-qPCR in developing fruit at 2, 4, 6, 8, and 10 weeks after full bloom (2 W, 4 W, 6 W, 8 W, and 10 W). Data were shown as the means ± standard deviation. * represent significant differences at p < 0.05.
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Figure 6. Influence of overexpression of VvPL11 on tomato fruit firmness and ethylene production. (A) Phenotypic changes of WT and VvPL11-OE (lines OE-1, OE-2, and OE-3) during fruit developing. (B) RT-qPCR analysis of VvPL11 in transgenic plants and wild type ‘Micro-Tom’ tomato. (C) Fruit firmness. (D) Ethylene production. Data were shown as the means ± standard deviation. * represent significant differences at p < 0.05.
Figure 6. Influence of overexpression of VvPL11 on tomato fruit firmness and ethylene production. (A) Phenotypic changes of WT and VvPL11-OE (lines OE-1, OE-2, and OE-3) during fruit developing. (B) RT-qPCR analysis of VvPL11 in transgenic plants and wild type ‘Micro-Tom’ tomato. (C) Fruit firmness. (D) Ethylene production. Data were shown as the means ± standard deviation. * represent significant differences at p < 0.05.
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Figure 7. Overexpression of VvPL11 affected pectin degradation and the expression levels of pectin-related genes in tomato. (A) Propectin; (B) WSP; (C) SlPG2; (D) SlPME1; (E) SlEXP; and (F) SlPL11. (* p < 0.05, Student’s t-test). Data were shown as the means ± standard deviation. * represent significant differences at p < 0.05.
Figure 7. Overexpression of VvPL11 affected pectin degradation and the expression levels of pectin-related genes in tomato. (A) Propectin; (B) WSP; (C) SlPG2; (D) SlPME1; (E) SlEXP; and (F) SlPL11. (* p < 0.05, Student’s t-test). Data were shown as the means ± standard deviation. * represent significant differences at p < 0.05.
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Table
Table 1. The pectin lyase (VvPL) family in grape.
Table 1. The pectin lyase (VvPL) family in grape.
Protein NameNumber of Amino AcidsMolecular Weight (Da)Theoretical pISubcellular Location
VvPL144449,249.646.65Cell membrane. Chloroplast. Nucleus.
VvPL244349,927.89.44Cell wall. Chloroplast.
VvPL344349,930.339.66Chloroplast. Mitochondrion.
VvPL414015,562.169.36Chloroplast.
VvPL538142,374.559.06Cell membrane. Cell wall. Chloroplast.
VvPL654357,104.765.65Cell membrane. Cell wall.
VvPL749653,897.765.75Cell membrane. Cell wall. Chloroplast. Nucleus.
VvPL840344,441.016.88Cell membrane. Cell wall. Chloroplast.
VvPL937041,484.459.08Cell membrane. Cell wall. Chloroplast. Cytoplasm. Golgi apparatus. Vacuole.
VvPL1032036,103.929Cell wall.
VvPL1142947,876.528.48Cell wall. Chloroplast.
VvPL1240344,091.476.48Cell membrane. Cell wall. Chloroplast. Nucleus.
VvPL1340044,196.724.92Cell wall.
VvPL1446450,947.377.64Chloroplast. Nucleus.
VvPL1544549,642.077.99Chloroplast.
VvPL1637341,455.868.16Cell wall. Chloroplast. Cytoplasm. Golgi apparatus. Mitochondrion. Nucleus.
VvPL1748953,838.426Cell membrane. Cell wall. Chloroplast.
VvPL1833137,066.866.01Cell wall.
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Li, W.; He, C.; Wei, H.; Qian, J.; Xie, J.; Li, Z.; Zheng, X.; Tan, B.; Li, J.; Cheng, J.; et al. VvPL11 Is a Key Member of the Pectin Lyase Gene Family Involved in Grape Softening. Horticulturae 2023, 9, 182. https://doi.org/10.3390/horticulturae9020182

AMA Style

Li W, He C, Wei H, Qian J, Xie J, Li Z, Zheng X, Tan B, Li J, Cheng J, et al. VvPL11 Is a Key Member of the Pectin Lyase Gene Family Involved in Grape Softening. Horticulturae. 2023; 9(2):182. https://doi.org/10.3390/horticulturae9020182

Chicago/Turabian Style

Li, Wenxin, Chang He, Hongli Wei, Jiakang Qian, Jiannan Xie, Zhiqian Li, Xianbo Zheng, Bin Tan, Jidong Li, Jun Cheng, and et al. 2023. "VvPL11 Is a Key Member of the Pectin Lyase Gene Family Involved in Grape Softening" Horticulturae 9, no. 2: 182. https://doi.org/10.3390/horticulturae9020182

APA Style

Li, W., He, C., Wei, H., Qian, J., Xie, J., Li, Z., Zheng, X., Tan, B., Li, J., Cheng, J., Wang, W., Ye, X., & Feng, J. (2023). VvPL11 Is a Key Member of the Pectin Lyase Gene Family Involved in Grape Softening. Horticulturae, 9(2), 182. https://doi.org/10.3390/horticulturae9020182

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