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Genome-wide identification of the grapevine β-1,3-glucanase gene (VviBG) family and expression analysis under different stresses

BMC Plant Biology volume 24, Article number: 911 (2024) Cite this article

Abstract

Background

The β-1,3-glucanase gene is widely involved in plant development and stress defense. However, an identification and expression analysis of the grape β-1,3-glucanase gene (VviBG) family had not been conducted prior to this study.

Results

Here, 42 VviBGs were identified in grapevine, all of which contain a GH-17 domain and a variable C-terminal domain. VviBGs were divided into three clades α, β and γ, and six subgroups A–F, with relatively conserved motifs/domains and intron/exon structures within each subgroup. The VviBG gene family contained four tandem repeat gene clusters. There were intra-species synteny relationships between two pairs of VviBGs and inter-species synteny relationships between 20 pairs of VviBGs and AtBGs. The VviBG promoter contained many cis-acting elements related to stress and hormone responses. Tissue-specific analysis showed that VviBGs exhibited distinct spatial and temporal expression patterns. Transcriptome analysis indicated that many VviBGs were induced by wounds, UV, downy mildew, cold, salt and drought, especially eight VviBGs in subgroup A of the γ clade. RT-qPCR analysis showed that these eight VviBGs were induced under abiotic stress (except for VviBG41 under cold stress), and most of them were induced at higher expression levels by PEG6000 and NaCl than under cold treatment.

Conclusions

The chromosome localization, synteny and phylogenetic analysis of the VviBG members were first conducted. The cis-acting elements, transcriptome data and RT-qPCR analysis showed that VviBG genes play a crucial role in grape growth and stress (hormone, biotic and abiotic) responses. Our study laid a foundation for understanding their functions in grape resistance to different stresses.

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Background

Callose, or β-1,3-glucan, is a 1,3-β-D-linked glucose homopolymer, which is a polysaccharide found only in embryophytes. The regulation of plasmodesmata (PD) conductivity by callose is a dynamic process, and the accumulation of callose in the neck of PD is strictly controlled by the antagonistic effects of two enzymes, namely callose synthase (CalS) and β-1,3-glucanase (BG), which respectively confer synthesis and degradation of β-1,3-glucan [1].

BG acts as a hydrolase, catalyzing the intramolecular cleavage of 1,3-β-D-glucosidic bonds into individual β-1,3-glucan units. To date, a complex and diverse BG gene family has been found in tobacco [2], soybean [3], Arabidopsis [4], rice [5] and cotton [6]. Plant BGs are classified into three structural classes: class I vacuolar proteins [7], class II and class III acidic secreted proteins [8], and unique intercellular “ersatz” BGs contained within class I [9]. In Arabidopsis, there are 50 AtBGs, all of which contain an N-terminal signal peptide and a glycosyl hydrolase family 17 (GH-17) domain, with some also containing a C-terminal domain X8 (also known as CBM43) [4]. A recent study identified 67 (Gossypium. raimondii), 68 (G. arboretum), 130 (G. hirsutum acc. TM-1) and 158 (G. barbadense acc. 3–79) β-1,3-glucanase genes (GLUs) in four cotton species, and found that GLU42 and GLU43 positively regulate cotton resistance to Verticillium dahliae [6].

BGs are commonly referred to as pathogenesis-related (PR) proteins, primarily acting by directly degrading pathogen cell walls [10]. Pyrus pyrifolia gene PpGlu enhances tobacco resistance to several pathogenic fungi [11]. Ah-Glu gene enhances peanut resistance to fungal pathogen Cercospora personifita [12]. TcLr19Glu participates in the defense response of wheat against rust pathogens [13]. Wheat β-1,3-glucanase exerts inhibitory effects on fungi associated with wheat grains [14]. PnGlu1 gene from Panax notoginseng confers resistance in tobacco to Fusarium solani [15]. Gns6 is an important effector in the defense response of rice against pathogenic fungi [16]. Plant growth promoting bacteria induce the formation of a complex between the β-1,3-glucanase gene and GTP in host plants, resulting in resistance to rice blast fungus [17]. β-1,3-GLU2 is involved in the main defense response of mango against C. gloeosporioides, and SNP 21,881,933 enhances the activity of β-1,3-glucanase [18]. SlBG10 regulates tomato pollen, fruit and seed development and disease resistance by regulating callose deposition [19]. FaERF2 activates FaBG-1 and FaBG-2 genes to enhance strawberry resistance to Botrytis cinerea [20]. Co-overexpression of chitinase and β-1,3-glucanase enhances wheat resistance to Fusarium [21].

Additionally, they exhibit important roles in regulating plant development and abiotic stress. For instance, BGs may play a role in banana fruit softening and be induced by hormones and stress [22]. Arabidopsis PdBG1 and PdBG2 are involved in regulating the number and distribution of lateral roots [23], while the Drosera rotundifolia BG gene is expressed in vegetative organs and function in the development of transgenic tobacco [24]. The destruction of AtBG14 leads to increased deposition of callose in developing seeds and reduces seed longevity and dormancy [25]. GhGLU18 promotes fiber cell elongation and secondary cell wall deposition in cotton [26]. BGs reduce solute influx into membrane vesicles during freezing, thereby reducing osmotic stress and vesicle rupture during thawing [27]. Cold and gibberellin can excessively induce BG to reopen signal channels and break the dormancy of poplar and Paeonia suffruticosa [28, 29]. Meanwhile, sugarcane ScGluD2 is involved in plant defense against smut pathogen attack as well as responses to salt and heavy metal stress [30]. In addition to BG genes, promoter activity is also activated by stress or hormones. For example, the promoter of Brassica juncea BjPR2 is induced by fungal infection, plant hormones and wounding [31], while the promoter of the sesame BG gene is induced by ABA and drought stress [32].

Grapes, as one of the world’s widely cultivated economic fruit crops, are used for various purposes, including as a fresh and processed food. Cultivated grapevines usually exhibit low resistance to biotic and abiotic stress. BG genes have been shown to be extensively involved in plant responses to stress. Recently, VviBG genes in grapevines have been reported as cryoprotectants [33] and to play a role in resistance against different pathogens [34, 35]; however, a study of the identification and expression of VviBG gene family members in grapevines has not yet been reported. In this study, the VviBG family members were first identified, before undergoing chromosome localization, synteny analysis and phylogenetic analysis. Additionally, tissue specificity and expression levels of VviBGs under different stresses were analyzed using transcriptome data. Finally, the expression patterns of VviBGs under abiotic stress were investigated via RT-qPCR analysis. The identification and expression analysis of the VviBG family in our study laid a theoretical foundation for understanding their functions in grape resistance to different stresses.

Methods

Plant materials and treatments

The self-rooted cuttings of ‘Thompson Seedless’ were grown in a greenhouse (Henan Institute of Science and Technology) at 25–30℃ and 60–75% relative humidity. Two-month-old grape seedlings were selected for abiotic stress treatment. Grape seedlings were placed in an incubator at 4°C for cold treatment, irrigated with 10% PEG6000 for drought treatment, and irrigated with 150 mM NaCl for salt treatment. Grape seedlings grown in a controlled-environment chamber at 22℃ were used as controls. Samples were collected 0 h, 3 h, 6 h, 24 h and 48 h after treatment, with three biological replicates for each treatment. All leaf samples were immediately stored at -80°C for RNA extraction.

Identification of VviBG genes in grapevine

The amino acid sequence of the AtBG gene AT4G14080 was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/), it contains two conserved domains GH17 and CBM43 of the BG gene family and used as query to search for homologous proteins in the grape genome (https://plants.ensembl.org/index.html) using the BLAST program. Candidate proteins were further subjected to multiple sequence alignment to remove duplicates and incomplete protein sequences. Finally, the identification of conserved domains was performed using NCBI CDD tools (https://www.ncbi.nlm.nih.gov/cdd) and the InterPro database (https://www.ebi.ac.uk/interpro/) to identify the VviBG gene family members in grapevine, which were named based on their chromosomal positions. The Protein Parameter Calc (ProtParam-based) function of TBtools [36] was used to analyze various parameters of the VviBG proteins. Glycosyl phosphatidyl isohydrin (GPI) anchor prediction was performed using PredGPI (https://busca.biocomp.unibo.it/predgp).

Chromosome localization and synteny analysis

Chromosome localization of VviBGs was visualized using Gene Location Visualize in TBtools. The homology and synteny of VviBGs were analyzed with default parameters (CPU for BlastP: 2, E-value:1e-10, Num of BlastHits: 5) using One Step MCScanX in TBtools and visualized with Advanced Circos in TBtools.

Bioinformatics analysis of VviBGs

The genomic, CDS and protein sequences of VviBGs were obtained from the grape genome database (https://plants.ensembl.org/index.html). Multiple sequence alignment of VviBG proteins was performed using ClustalX2.1. The phylogenetic tree was constructed with MEGA 7.0 using the neighbor-joining method and 1000 bootstrap replicates. The exon/intron analysis of VviBGs was conducted using the GSDS 2.0 (http://gsds.gao-lab.org/index.php). The motif analysis of VviBGs was performed in MEME (http://meme-suite.org/), with parameters set as follows: default for advanced options and a maximum of 12 motifs. The conserved domain identification of VviBGs was carried out using the CD-search of NCBI (https://www.ncbi.nlm.nih.gov/cdd).

Analysis of cis-elements of VviBG promoters

The upstream 2000 bp promoter sequences of VviBGs were obtained using TBtools, and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the cis-elements of the VviBG promoters. Visualize and plotting of the types and numbers of cis-elements were then performed using Basic Biosequence View and Heatmap in TBtools, respectively.

Transcriptomic data analysis of VviBGs

Publicly available transcriptomic data (GSE36128) of 54 grape (‘Corvina’) samples comprising different tissues and developmental stages were utilized to analyze the tissue-specific expression patterns of VviBGs. Detailed information about grape different tissues and developmental stages can be found in the previous study [37]. Publicly available transcriptomic data (GSE37743) were used to analyze the expression patterns of VviBGs after 0 h, 24 h and 48 h of wounds, UV and pathogenic bacteria treatment in ‘Pinot Noir’ grape leaves. The information about the stress condition can be found in previous study [38]. Furthermore, our transcriptomic data (GSE276430) were analyzed to investigate the expression patterns of VviBGs at 0 h and 48 h after treatment with cold (4°C), drought (PEG6000) and salt (NaCl). Finally, the heatmaps of gene expression were generated using TBtools.

RT-qPCR analysis

Total RNA extraction of grape leaves and cDNA synthesis were performed using the EZNA Plant RNA Kit and M5 Super plus qPCR RT kit with gDNA remover (Agilent, China) respectively. The cDNA was diluted 5-fold at a concentration between 150 and 200 ng/uL and used for RT-qPCR reaction. RT-qPCR reactions were conducted using the SYBR qPCR Mixture (GenStar, China) and CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). Each reaction was performed with three biological replicates. VviGAPDH (GU585870) and VviACTIN (AY680701) were attempted as reference genes, and finally VviACTIN was used as the reference gene. Relative expression levels were calculated using the 2−ΔΔCT method [39]. The primers used for RT-qPCR are listed in Table S1.

Results

Identification and characterization of the VviBG gene family

Using the Arabidopsis AT4G14080 gene for BLAST, 73 homologous proteins were obtained in the grape genome, with 53 remaining after removing duplicates and short fragments. Finally, 42 members of the VviBGs in grapevine were identified through conserved domain analysis and named VviBG1–VviBG42 based on their chromosomal positions (Table S2). In Table S2, it was evident that the CDS length of VviBGs ranged from 837 (VviBG20) to 1584 bp (VviBG33), while the length of amino acids ranged from 290 to 527 aa. The molecular weights ranged from 32.24 to 57.67 kDa, and the isoelectric points ranged from 4.70 (VviBG4) to 9.39 (VviBG19). Among them, 12 VviBGs were unstable proteins with an instability index greater than 40. It was predicted that 14 VviBGs contained GPI anchor attachment.

Chromosomal localization and synteny analysis of VviBGs

Tandem duplication and whole genome-duplication have been found to be important for the expansion of gene families [40]. In order to further verify the duplication events of the VviBGs during evolution, our study investigated the chromosomal localization and synteny relationships of the VviBGs. As shown in Fig. 1, VviBGs were unevenly distributed on 19 chromosomes, among which chromosomes 6, 8 and 11 contained more genes (6, 7 and 5, respectively). Additionally, four tandem repeat gene clusters were found within the VviBGs, with cluster sizes ranging from 2 to 3 genes. VviBG11 and VviBG12 formed a tandem repeat gene cluster on chromosome 6, while VviBG15 and VviBG16 as well as VviBG17, VviBG18 and VviBG19 formed tandem repeat gene clusters on chromosome 8. VviBG41 and VviBG42 were tandem repeat genes located on chromosome Un (Fig. 1, Table S3).

Synteny analysis revealed two whole-genome duplication regions in the grape genome: VviBG5/VviBG39 and VviBG32/VviBG40 (Fig. 2a and Table S4). To further infer the evolutionary relationships between BG family members, we constructed synteny analysis of BG gene families in grape and Arabidopsis (Fig. 2b and Table S4). A total of 20 VviBG genes in grape showed synteny with Arabidopsis, and some VviBGs showed a synteny relationship with two AtBGs, such as VviBG5, VviBG21, VviBG26, VviBG29 and VviBG40 (Table S4).

Fig. 1
figure 1

Chromosomal localization analysis of VviBGs in grapevine. VviBGs are sequentially numbered according to their positions on chromosomes: VviBG1–VviBG42. Tandem repeat gene clusters of VviBGs are marked with different colors. Chromosomal localization of VviBGs was visualized using TBtools

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Fig. 2
figure 2

Synteny analysis of VviBGs within the grapevine and between grapevine and Arabidopsis. (a) Synteny analysis of VviBGs in grapevine. Gene pairs are connected by a red line. (b) Synteny analysis of VviBGs between grapevine and Arabidopsis. The red lines highlight the syntenic VviBG gene pairs. The chart was plotted with TBtools

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Multiple sequence alignment and phylogenetic analysis of VviBGs

Multiple sequence alignment reveals conserved and functional associations among gene family members. Therefore, in our study, the multiple sequence alignment analysis of VviBGs was conducted (Figure S1). The results showed that all VviBG proteins contained an N-terminal signal peptide (except VviBG20) and a GH-17 domain, but the C-terminal sequences were not conserved. In Arabidopsis, based on the presence or absence of C-terminal structural features, AtBGs were divided into five types [4], while VviBGs in grape were divided into four types, lacking type III (Figure S2). There were 12 members in type I, containing the CBM43 domain and C-terminal hydrophobic sequence; 9 members in type II, containing the CBM43 domain; 6 members in type IV, containing C-terminal hydrophobic sequence; and 15 members in type V, lacking the C-terminal structural features.

To study the evolutionary relationship between VviBGs and AtBGs, phylogenetic analysis was performed (Fig. 3). The results showed that 42 VviBGs and 50 AtBGs were classified into three clades (α, ß and γ), consistent with previous studies in Arabidopsis [4]. Among them, the α clade contained the largest number of VviBGs, with 20 members; followed by the γ clade, with 15 members; and finally, the ß clade, which contained 7 VviBGs. To better understand the evolutionary relationship of VviBGs in grape, phylogenetic analysis of VviBGs was conducted (Fig. 4a). The α, ß and γ clades were divided into 6 subgroups (A–F) according to the cotton [6].

Fig. 3
figure 3

Phylogenetic analysis of grapevine and Arabidopsis BG genes. The phylogenetic tree was constructed using the neighbor-joining method in MEGA7.0, with a bootstrap value of 1000, and was visualized using Evolview

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Analysis of motif/domain and gene structure of VviBGs

The motif analysis showed that the VviBGs exhibited strong conservation in the distribution pattern and quantity of motifs. VviBGs of the A, D and F subgroups contained 10 motifs, while VviBGs of the B, C and E subgroups contained 12 motifs (Fig. 4b). The conserved domain analysis indicated that 15 VviBGs of type V and 6 VviBGs of type IV containing the GH-17 domain were classified into subgroups A and D/F, respectively. 12 VviBGs of type I containing both GH-17 and CBM43 domains were classified into subgroups B (7 VviBGs) and C (5 VviBGs), while 9 VviBGs of group 2 containing GH-17 and CBM43 domains were classified into subgroups C (3 VviBGs) and E (6 VviBGs) (Fig. 4c). Gene structure analysis revealed that VviBGs of subgroups A and B both contained one intron, while VviBG genes of subgroups C, D and E contained varying numbers of introns ranging from 1 to 5. The F subgroup only contained VviBG35, which had no introns.

Fig. 4
figure 4

Phylogenetic, motif, conserved domain and gene structure analysis of the VviBGs in grapevine. (a) Phylogenetic analysis of VviBGs. (b) Motif analysis of VviBGs. (c). Conserved domain and gene structure analysis of VviBGs. Visualization was performed using TBtools

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Analysis of cis-acting elements in the promoters of VviBGs

In order to further analyze the functions of VviBGs in plant growth, development and stress response, the cis-acting elements of the VviBG promoters were analyzed (Fig. 5). In addition to the core promoter elements, a total of 33 types of elements were identified, categorized into four functional classes: light-responsive elements, stress-responsive elements (wounds, low temperature, stress, anaerobic and oxidative), hormone-responsive elements (gibberellin, auxin, abscisic acid, methyl jasmonate, salicylic acid and ethylene) and transcription factor binding elements (DREB, WRKY, MYB and MYC).

Then further analysis was conducted on the number of cis-acting elements contained in the promoter of VviBGs (Fig. 5c, Table S5). The results showed that the light-responsive element predominantly consisted of Box 4, with VviBG2, VviBG9, VviBG20 and VviBG42 containing up to eight. The main responsive elements included stress-responsive elements (STRE) and antioxidant-responsive elements (ARE), with each VviBG promoter in the ß clade containing 3–7 STRE elements, and each VviBG promoter in subgroup C of the α clade containing 1–6 ARE elements. The hormone-responsive elements mainly included ERE and ABRE, with almost all VviBG promoters containing ERE and ABRE, and VviBG2 and VviBG28 having the most ERE elements, with up to seven. The transcription factor binding elements mainly included MYC, present in almost all VviBG promoters, with VviBG16, VviBG17, VviBG29, VviBG35 and VviBG39 having the most (up to seven).

Fig. 5
figure 5

Analysis of the types and numbers of cis-acting elements in the promoters of VviBGs. (a) Phylogenetic analysis of VviBGs. (b) Schematic diagram of cis-acting elements in the VviBG promoters. (c) Heatmap analysis of the numbers of cis-acting elements in the VviBG promoters. Visualization was performed using TBtools

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RNA-seq analysis of tissue-specific expression patterns of VviBGs

To investigate the role of VviBGs in different tissues and developmental stages of grapevine, we analyzed the tissue-specific expression patterns of VviBGs in 54 grape (‘Corvina’) samples comprising different tissues and developmental stages using transcriptome data (GSE36128) (Fig. 6, Table S6). The results indicated that with the maturation of various tissues of grape, the expression of 5 VviBGs (VviBG6, VviBG10, VviBG11, VviBG4 and VviBG40) and 15 VviBGs (VviBG41, VviBG19, VviBG16, VviBG17, VviBG33, VviBG1, VviBG36, VviBG29, VviBG32, VviBG3, VviBG13, VviBG31, VviBG38, VviBG5 and VviBG39) showed an increasing and decreasing trend, respectively. Genes such as VviBG40, VviBG21, VviBG34, VviBG5, VviBG3, VviBG32, VviBG23 and VviBG39 were highly expressed in almost all tissues, while VviBG9 and VviBG12 showed very low expression levels in almost all tissues. Some VviBGs exhibited similar expression patterns in different tissues, such as VviBG7 and VviBG8, VviBG2 and VviBG14, VviBG28 and VviBG35, VviBG10 and VviBG11, VviBG19 and VviBG41, VviBG16 and VviBG17, VviBG20 and VviBG42. Some genes also exhibited tissue-specific expression, such as VviBG2 and VviBG14, which had relatively high expression levels in flower organs (stamen, pollen and flower), VviBG12, which was mainly highly expressed in tendril WD, and VviBG10, which was mostly expressed in roots.

Fig. 6
figure 6

Expression patterns of VviBGs in 54 different grapevine tissue samples. The transcriptome data were obtained from the grapevine expression database GSE37743. Log2 (RNA-normalized value + 1) was used to indicate the gene expression values. Heatmap visualization was performed using TBtools

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RNA-seq analysis of expression patterns of VviBGs under wounds, UV and downy mildew stress

To determine the expression of VviBGs under different stresses, the RNA-seq data (GSE37743) of ‘Pinot Noir’ leaves under wounds, UV and downy mildew stress were analyzed (Fig. 7, Table S7). The results revealed that 35 VviBGs were significantly induced or inhibited under at least one of the stresses. Ten VviBGs (VviBG4, VviBG6, VviBG10, VviBG11, VviBG16, VviBG17, VviBG18, VviBG19, VviBG40 and VviBG41) and two VviBGs (VviBG15 and VviBG36) were significantly induced and inhibited under all three stresses at 24 h and 48 h. Some VviBG genes were induced by two stresses, namely VviBG1, VviBG3, VviBG23, VviBG33 and VviBG38 were significantly induced by wounds and downy mildew; VviBG22 and VviBG26 were significantly induced by wounds and UV; VviBG2, VviBG29 and VviBG37 were significantly induced by UV and downy mildew. On the other hand, some genes were inhibited by one or two stress, such as VviBG20 and VviBG42 were significantly inhibited by wounds and downy mildew. VviBG31 was significantly inhibited by wounds, VviBG3 and VviBG27 were significantly inhibited by downy mildew, and VviBG5, VviBG8, VviBG13, VviBG30, VviBG32, VviBG34 and VviBG39 were significantly inhibited by UV.

Fig. 7
figure 7

Analysis of expression patterns of VviBGs under wounds, UV and downy mildew stress. The transcriptome data were obtained from the grapevine expression database GSE37743. Log2 (RNA-normalized value + 1) was used to indicate the gene expression values. Heatmap visualization was performed using TBtools

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RNA-seq analysis of expression of VviBGs under abiotic stress

Previously, we performed RNA-seq analysis on ‘Thompson Seedless’ leaves treated with three abiotic stresses (cold, PEG6000 and NaCl) (GSE276430). Then, the expression of VviBGs under abiotic stresses was analyzed utilizing the RNA-seq data (Fig. 8, Table S7). It was found that after 48 h of cold treatment, the expression levels of 10 VviBGs (VviBG4, VviBG6, VviBG10, VviBG11, VviBG19, VviBG21, VviBG22, VviBG26, VviBG34 and VviBG40) and 3 VviBGs (VviBG32, VviBG33 and VviBG37) increased and decreased, respectively. Among them, VviBG4 exhibited the highest induced fold change, although it was almost not expressed in the control. We also found that the expression levels of 10 VviBGs (VviBG6, VviBG10, VviBG11, VviBG16, VviBG17, VviBG18, VviBG19, VviBG26, VviBG34 and VviBG41) and 7 VviBGs (VviBG8, VviBG13, VviBG25, VviBG29, VviBG32, VviBG33 and VviBG36) increased and decreased respectively after PEG6000 treatment for 48 h, and VviBG11 was induced to express at the highest fold. Finally, it was shown that after 48 h of NaCl treatment, the expression levels of 6 VviBGs (VviBG6, VviBG10, VviBG11, VviBG17, VviBG19 and VviBG41) and 2 VviBGs (VviBG13 and VviBG32) increased and decreased, respectively, with VviBG10 exhibiting the highest induced fold change. Meanwhile, we observed that VviBG6, VviBG10, VviBG11 and VviBG19 were induced by all three abiotic stresses, VviBG26 and VviBG34 were induced by cold and PEG6000, and VviBG17 and VviBG41 were induced by PEG6000 and NaCl.

Fig. 8
figure 8

Expression patterns of VviBGs under cold (a), PEG6000 (b) and NaCl (c) treatments. Log2 (RNA-normalized value + 1) was used to indicate the gene expression values. Heat map visualization was performed using TBtools

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RT-qPCR analysis of expression patterns of VviBGs under abiotic stress

Based on the transcriptome data analysis of abiotic stress, eight genes (VviBG6, VviBG10, VviBG11, VviBG16, VviBG17, VviBG18, VviBG19 and VviBG41) were selected from subgroup A of the γ clade for RT-qPCR analysis (Fig. 9). The results showed that the eight genes were induced by all three abiotic stresses, except for VviBG41 under 4°C. Most VviBGs were not significantly induced at 3 h of abiotic stress and were even suppressed; however, with prolonged treatment time, VviBG6, VviBG10, VviBG11 and VviBG19 showed the highest induction after 48 h of the three stresses. Furthermore, most genes among the eight VviBGs showed a higher induction by PEG6000 and NaCl compared to 4°C, such as VviBG6, VviBG10, VviBG11, VviBG16, VviBG19 and VviBG41.

Fig. 9
figure 9

RT-qPCR analysis of VviBGs expression under 4°C, PEG6000 and salt treatments. Values represent the average of three biological replicates ± SD. Significance differences are indicated by asterisks (*P < 0.05 or **P < 0.01)

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Discussion

BGs are localized in various organs at different developmental and reproductive stages of plants, playing crucial roles in cell division, intercellular transport, flower formation, seed maturation and defense against biotic and abiotic stresses [41]. Studies have shown that overexpression of flax BGs leads to accumulation of pectin and phenolic substances, resulting in higher antioxidant capacity [42]. We speculated that VviBGs may also enhance grape antioxidant capacity in this way and thereby increase stress resistance. Our study identified 42 VviBGs in grape, twelve VviBGs of type I and six VviBGs of type IV contain C-terminal hydrophobic sequences encoding transient transmembrane domains, among which 14 genes contain GPI anchor attachments (Table S2), indicating that they may be localized to vacuoles [43] or the plasma membrane/cell wall [44, 45].

Four pairs of VviBG tandem repeats were detected in the grape genome (Fig. 1), suggesting that they were involved in the expansion of the VviBG gene family. Synteny analysis (Fig. 2a) revealed that proteins corresponding to VviBG5 and VviBG39 or VviBG32 and VviBG40 may have similar functions. The existence of one-to-many synteny relationships between VviBGs and AtBGs (Fig. 2b) suggested that VviBGs may have multiple orthologous genes in Arabidopsis, while also indicating a reduction in the number of VviBGs during evolution. VviBGs were divided into four types, lacking type III (Figure S2), indicating that VviBGs may have lost genes with two CBM43 domains during grapevine evolution.

Different types of cis-acting elements on the promoter reveal the potential functions and regulatory differences of genes. We found that the VviBG promoters contained many elements related to stress and hormone responses (Fig. 5). STRE responds to various stress conditions including heat, high osmotic pressure and ethanol [46]. ß clade VviBG promoters contained high amounts of STRE, suggesting that these genes may confer grape stress resistance by responding to external stresses. On the other hand, ARE belongs to antioxidant response elements [47], the α clade VviBG promoters contained many ARE elements, indicating that they may confer grape stress resistance through oxidative stress pathways. ERE and ABRE denote ethylene and abscisic acid response elements, respectively, and almost all VviBG promoters were found to contain these two hormones, suggesting that they play a major role in regulating VviBGs in grape growth, development and stress response. The VviBG promoters also contained many transcription factor binding elements, in the future, the upstream transcription factors of VviBGs can be identified to explore the regulatory mechanism of VviBGs. The promoter activity of BG in other species is also induced by stress and hormones, such as the promoter of BjPR2 being induced by fungi, hormones and wounds [31], or the BG gene promoter of sesame being induced by ABA and drought [32]. This indicates that the regulatory mechanism of BG promoters in plant growth and stress response is relatively conservative.

In our study, 5 VviBGs and 15 VviBGs showed a trend of increasing and decreasing with the maturation of various tissues, respectively, indicating that they may play opposite roles at different stages of grape. Gene expression analysis suggests that VviBG18 may play a key role in flower organ development, berry maturation and post-harvest processes; VviBG2 and VviBG14 may mainly participate in flower and male organ development; VviBG12 and VviBG10 may play a role in the tendrils lignification and root development of grape, respectively. We also found that during the maturation process of various tissues, the induced VviBGs were mainly concentrated in the γ clade, while the inhibited VviBGs were mainly concentrated in the α clade, suggesting that VviBGs from the same clades may play similar roles in grape development. VviBG7 and VviBG8, VviBG2 and VviBG14, VviBG28 and VviBG35 showed distant phylogenetic relationships in the phylogenetic analysis, but exhibited similar expression profiles, which revealed that VviBGs with distant relationships may play similar roles in grape growth and development.

The transcriptome analysis (Fig. 7, Table S7) indicated that VviBG6, VviBG10, VviBG11 and VviBG18 may play a crucial role in grape response to wounds, UV and pathogens, especially VviBG10. The transcriptome analysis (Fig. 8, Table S8) also revealed that VviBG6, VviBG10, VviBG11 and VviBG19 may be involved in the grape response to abiotic stress (4℃, PEG6000 and salt), especially drought stress. AtBG gene At4g16260 is induced by pathogens and may play a role under drought and salt stress, as the drought and salt stress positive regulatory factor AtbHLH112 binds to the At4g16260 promoter [48]. This shows that the results of our study are consistent with Arabidopsis, as we found that VviBG6 and At4g16260 are orthologous genes, and may have similar functions.

To date, there have been many studies on the disease resistance of BGs; however there are few studies on their function and mechanism under abiotic stress. RT-qPCR analysis (Fig. 9) showed that the expression of 8 VviBGs of subgroup A in the γ clade mainly increased in the later stages of 4℃, drought and salt treatments, especially at 48 h, indicating that the VviBGs may not take effect until some time after treatments. VviBG6, VviBG11 and VviBG19 were highly expressed under drought and salt stress, suggesting they may play a significant role under these two stresses. VviBG18 was significantly upregulated under salt stress compared to the other two stresses, indicating that it may mainly play a role in the response to salt stress. VviBG17 was induced to have the highest expression fold at 4℃, so it may be involved in the response to cold stress. According to the induced expression fold of VviBG genes, we also found that most genes may play a greater role in the response to drought and salt stress than cold stress.

Conclusion

Our study identified 42 VviBGs in grapevine, all containing a GH-17 domain and C-terminal non conserved sequence features. We divided these into three clades α, ß and γ, and six subgroups. There were four tandem repeat gene clusters in the VviBG gene family, two pairs of synteny relationships in grapevine and twenty pairs of synteny relationships between grape and Arabidopsis. The VviBG promoters contained many cis-acting elements related to stress and hormone responses. VviBGs exhibited distinct spatial and temporal organization-specific expression patterns. Most VviBGs were induced by wounds, UV, pathogens, cold, salt, and drought, especially the 8 VviBGs in subgroup A. Most of the 8 VviBGs were induced by PEG6000 and NaCl with expression multiple times higher than those induced by cold. The comprehensive analyses of the VviBG gene family in grapevine will help to further elucidate the role of VviBGs in the grape responses to biotic and abiotic stress.

Data availability

The data presented in the study are deposited in the NCBI repository, GEO accession: GSE276430.

Abbreviations

ARE:

Antioxidant-responsive elements

BG:

β-1,3-glucanase

CalS:

Callose synthase

GH-17:

Glycosyl hydrolase family 17

GPI:

Glycosyl phosphatidyl isohydrin

PD:

Plasmodesmata

PR:

Pathogenesis-related

STRE:

Stress-responsive elements

References

  1. Chen X-Y, Kim J-Y. Callose synthesis in higher plants. Plant Signal Behav. 2009;4(6):489–92.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Linthorst HJ, Melchers SL, Mayer A, et al. Analysis of gene families encoding acidic and basic beta-1,3-glucanases of tobacco. Proc Natl Acad Sci U S A. 1990;87(22):8756–60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Jin W, Horner HT, Palmer RG, et al. Analysis and mapping of gene families encoding beta-1,3-glucanases of soybean. Genetics. 1999;153(1):445–52.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Doxey AC, Yaish MWF, Moffatt BA, et al. Functional divergence in the Arabidopsis β-1,3-Glucanase gene family inferred by phylogenetic reconstruction of expression states. Mol Biol Evol. 2007;24(4):1045–55.

    Article  PubMed  CAS  Google Scholar 

  5. Wan L, Zha W, Cheng X, et al. A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development. Planta. 2011;233(2):309–23.

    Article  PubMed  CAS  Google Scholar 

  6. Xu X, Feng Y, Fang S, et al. Genome-wide characterization of the β-1,3-glucanase gene family in Gossypium by comparative analysis. Sci Rep. 2016;6(1):29044.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Shinshi H, Wenzler H, Neuhaus JM, et al. Evidence for N- and C-terminal processing of a plant defense-related enzyme: primary structure of tobacco prepro-beta-1,3-glucanase. Proc Natl Acad Sci U S A. 1988;85(15):5541–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Payne G, Ward E, Gaffney T, et al. Evidence for a third structural class of beta-1,3-glucanase in tobacco. Plant Mol Biol. 1990;15(6):797–808.

    Article  PubMed  CAS  Google Scholar 

  9. Beffa RS, Neuhaus JM, Meins F Jr. Physiological compensation in antisense transformants: specific induction of an ersatz glucan endo-1,3-beta-glucosidase in plants infected with necrotizing viruses. Proc Natl Acad Sci U S A. 1993;90(19):8792–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Liu YF, Liu LJ, Yang S, et al. Molecular characterization and functional analysis of a pathogenesis-related β-1,3-glucanase gene in spruce. Eur J Plant Pathol. 2022;164(2):177–92.

    Article  CAS  Google Scholar 

  11. Liu D, He X, Li W, et al. A β-1,3-glucanase gene expressed in fruit of Pyrus pyrifolia enhances resistance to several pathogenic fungi in transgenic tobacco. Eur J Plant Pathol. 2013;135(2):265–77.

    Article  CAS  Google Scholar 

  12. Qiao LX, Ding X, Wang HC, et al. Characterization of the β-1,3-glucanase gene in peanut (Arachis hypogaea L.) by cloning and genetic transformation. Genet Mol Res. 2014;13(1):1893–904.

    Article  PubMed  CAS  Google Scholar 

  13. Gao L, Wang S, Zhang Y, et al. Identification and characterization of a β-1,3-glucanase gene, TcLr19Glu, involved in wheat resistance against Puccinia Triticina. J Plant Biochem Biot. 2016;25(3):319–26.

    Article  CAS  Google Scholar 

  14. Zhang S, Zhang W, Zhai H, et al. Expression of a wheat β-1,3-glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel. Protein Expres Purif. 2019;154:134–9.

    Article  CAS  Google Scholar 

  15. Taif S, Zhao Q, Pu L, et al. A β-1,3‐glucanase gene from Panax notoginseng confers resistance in tobacco to Fusarium solani. Ind Crop Prod. 2020;143:111947.

    Article  CAS  Google Scholar 

  16. Wang Y, Liu M, Wang X, et al. A novel β-1,3-glucanase Gns6 from rice possesses antifungal activity against Magnaporthe oryzae. J Plant Physiol. 2021;265:153493.

    Article  PubMed  CAS  Google Scholar 

  17. Jha Y, Dehury B, Kumar SPJ, et al. Delineation of molecular interactions of plant growth promoting bacteria induced β-1,3-glucanases and guanosine triphosphate ligand for antifungal response in rice: a molecular dynamics approach. Mol Biol Rep. 2022;49(4):2579–89.

    Article  PubMed  CAS  Google Scholar 

  18. Felipe JEL, Lachica JAP, Dela Cueva FM, et al. Validation and molecular analysis of β-1,3-GLU2 SNP marker associated with resistance to Colletotrichum gloeosporioides in mango (Mangifera indica L). Physiol Mol Plant P. 2022;118:101804.

    Article  CAS  Google Scholar 

  19. Pei Y, Xue Q, Zhang Z, et al. β-1,3-GLUCANASE10 regulates tomato development and disease resistance by modulating callose deposition. Plant Physiol. 2023;192(4):2785–802.

    Article  PubMed  CAS  Google Scholar 

  20. Peng Y, Liang M, Zhang X, et al. FaERF2 activates two β-1,3-glucanase genes to enhance strawberry resistance to Botrytis Cinerea. Plant Sci. 2024;347:112179.

    Article  PubMed  CAS  Google Scholar 

  21. Mohammadizadeh-Heydari N, Tohidfar M, Zanjani M. Co-overexpression of chitinase and β-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium. BMC Biotechnol. 2024;24(1):35.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Choudhury SR, Roy S, Singh SK, et al. Molecular characterization and differential expression of β-1,3-glucanase during ripening in banana fruit in response to ethylene, auxin, ABA, wounding, cold and light-dark cycles. Plant Cell Rep. 2010;29(8):813–28.

    Article  Google Scholar 

  23. Benitez-Alfonso Y, Faulkner C, Pendle A, et al. Symplastic intercellular connectivity regulates lateral root patterning. Dev Cell. 2013;26(2):136–47.

    Article  PubMed  CAS  Google Scholar 

  24. Michalko J, Renner T, Mészáros P, et al. Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V β-1,3-glucanase. Planta. 2017;245(1):77–91.

    Article  PubMed  CAS  Google Scholar 

  25. Wang C, Lyu Y, Zhang Q, et al. Disruption of BG14 results in enhanced callose deposition in developing seeds and decreases seed longevity and seed dormancy in Arabidopsis. Plant J. 2023;113(5):1080–94.

    Article  PubMed  CAS  Google Scholar 

  26. Fang S, Shang X, He Q, et al. A cell wall-localized β-1,3-glucanase promotes fiber cell elongation and secondary cell wall deposition. Plant Physiol. 2023;194(1):106–23.

    Article  PubMed  Google Scholar 

  27. Hincha DK, Meins F Jr, Schmitt JM. β-1,3-Glucanase is cryoprotective in vitro and is accumulated in leaves during cold acclimation. Plant Physiol. 1997;114(3):1077–83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Rinne PLH, Welling A, Vahala J, et al. Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-β-Glucanases to reopen signal conduits and release dormancy in populus. Plant Cell. 2011;23(1):130–46.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Gao X, Yuan Y, Liu Z, et al. Chilling and gibberellin acids hyperinduce β-1,3-glucanases to reopen transport corridor and break endodormancy in tree peony (Paeonia suffruticosa). Plant Physiol Biochem. 2021;167:771–84.

    Article  PubMed  CAS  Google Scholar 

  30. Su Y, Wang Z, Liu F, et al. Isolation and characterization of ScGluD2, a new sugarcane beta-1,3-Glucanase D family gene induced by Sporisorium scitamineum, ABA, H2O2, NaCl, and CdCl2 stresses. Front. Plant Sci. 2016;7:1348.

    Google Scholar 

  31. Ali S, Chandrashekar N, Rawat S, et al. Isolation and molecular characterization of pathogenesis related PR2 gene and its promoter from Brassica juncea. Biol Plant. 2017;61(4):763–73.

    Article  CAS  Google Scholar 

  32. Parveen S, Mazumder M, Bhattacharya A, et al. Identification of anther-specific genes from sesame and functional assessment of the upstream region of a tapetum-specific β-1,3-glucanase gene. Plant Mol Biol Rep. 2018;36(2):149–61.

    Article  CAS  Google Scholar 

  33. Romero I, Fernandez-Caballero C, Goñi O, et al. Functionality of a class I beta-1,3-glucanase from skin of table grapes berries. Plant Sci. 2008;174(6):641–8.

    Article  CAS  Google Scholar 

  34. Mestre P, Arista G, Piron MC, et al. Identification of a Vitis vinifera endo-β-1,3-glucanase with antimicrobial activity against Plasmopara Viticola. Mol Plant Pathol. 2017;18(5):708–19.

    Article  PubMed  CAS  Google Scholar 

  35. Soon-Young A, Kim SA, Yun H. Differential expression of β -1,3-Glucanase transcripts induced by pathogens in the leaves of Vitis Flexuosa. Plant Breed Biotechnol. 2014;2(2):176–83.

    Article  Google Scholar 

  36. Chen C, Wu Y, Li J, et al. TBtools-II: a one for all, all for one bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733–42.

    Article  PubMed  CAS  Google Scholar 

  37. Fasoli M, Dal Santo S, Zenoni S, et al. The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program. Plant Cell. 2012;24(9):3489–505.

    Article  PubMed  CAS  Google Scholar 

  38. Vannozzi A, Dry IB, Fasoli M, et al. Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses. BMC Plant Biol. 2012;12(1):130.

    Article  PubMed  CAS  Google Scholar 

  39. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods. 2001;25(4):402–8.

    Article  PubMed  CAS  Google Scholar 

  40. Kong H, Landherr LL, Frohlich MW, et al. Patterns of gene duplication in the plant SKP1 gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth. Plant J. 2007;50(5):873–85.

    Article  PubMed  CAS  Google Scholar 

  41. Balasubramanian V, Vashisht D, Cletus J, et al. Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi. Biotechnol Lett. 2012;34(11):1983–90.

    Article  PubMed  CAS  Google Scholar 

  42. Wojtasik W, Kulma A, Dyminska L, et al. Fibres from flax overproducing β-1,3-glucanase show increased accumulation of pectin and phenolics and thus higher antioxidant capacity. BMC Biotechnol. 2013;13:10.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Sticher L, Hinz U, Meyer AD, et al. Intracellular transport and processing of a tobacco vacuolar β-1,3-glucanase. Planta. 1992;188(4):559–65.

    Article  PubMed  CAS  Google Scholar 

  44. Henrissat B, Davies GJ. Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol. 2000;124(4):1515–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Borner GHH, Sherrier DJ, Stevens TJ, et al. Prediction of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A genomic analysis. Plant Physiol. 2002;129(2):486–99.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Schmitt AP, McEntee K. Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 1996;93(12): 5777-82.

  47. Raghunath A, Sundarraj K, Nagarajan R, et al. Antioxidant response elements: discovery, classes, regulation and potential applications. Redox Biol. 2018;17:297–314.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Liu Y, Ji X, Nie X, et al. Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytol. 2015;207(3):692–709.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the Key Research and Development Program of Henan Province, China (241111113200), the R&D and Promotion Program (Science and Technology Research Project) of Henan Province, China (242102110319) and the Project 2 of the High Level Talent Introduction Program (103020224001/065).

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Authors and Affiliations

  1. College of Horticulture and Landscape Architecture, Henan Institute of Science and Technology, Xinxiang, 453003, China

    Ling Wang, Ruilong Li, Kaiwei Li, Ruijin Zhou, Guilong Lu, Peng Li & Guirong Li

  2. Henan Province Engineering Research Centers of Horticultural Plant Resource Utilization and Germplasm Enhancement, Xinxiang, 453003, China

    Ling Wang, Ruilong Li, Kaiwei Li, Ruijin Zhou, Guilong Lu, Peng Li & Guirong Li

  3. State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China

    Ziyang Qu

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Contributions

LW and GL conceived and designed the experiments. LW, RL and ZQ performed the experiments. LW, KL, GL and PL analyzed the data and manuscript modification. RZ and GL contributed reagents/materials/analysis tools. LW contributed to the writing of the manuscript.

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Wang, L., Li, R., Li, K. et al. Genome-wide identification of the grapevine β-1,3-glucanase gene (VviBG) family and expression analysis under different stresses. BMC Plant Biol 24, 911 (2024). https://doi.org/10.1186/s12870-024-05597-1

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