VvLRXs gene identification
To identify potential LRXs in the grapevine genome (PN_T2T) [34], Arabidopsis LRX protein sequences were used as a reference, and a BLASTP search was conducted. This initial search yielded approximately 50 candidate proteins. The presence of conserved LRR and extensin domains was confirmed using SMART and NCBI–CDD tools. Ultimately, 14 VvLRXs were identified (Fig. S1), a number higher than those found in Arabidopsis, rice, maize, and tomato (Table S2). These 14 grapevine LRX genes were designated VvLRX1–VvLRX14 based on their chromosomal locations.
The molecular weights of the 14 VvLRXs varied from 29.84 kDa (VvLRX13) to 152.84 kDa (VvLRX11), and their amino acid lengths varied from 275 amino acids (VvLRX13) to 1388 amino acids (VvLRX11). Additionally, the isoelectric points of these proteins varied from 5.47 (VvLRX4) to 9.90 (VvLRX12) (Table 1).
Table 1
Characteristics of LRXs in Grapevine (Vitis vinifera L.)
Gene name | Gene ID | Chromosome location | Exon | Size (amino acids) | MW (kDa) | pI |
VvLRX1 | Vitis03g00581 | PN3:4606131–4608944 | 1 | 937 | 97.67 | 9.19 |
VvLRX2 | Vitis03g00703 | PN3:5861853–5863384 | 1 | 445 | 49.14 | 8.18 |
VvLRX3 | Vitis03g00704 | PN3:5875089–5876942 | 1 | 507 | 55.46 | 8.15 |
VvLRX4 | Vitis05g00653 | PN5:5770500–5771879 | 1 | 394 | 43.21 | 5.47 |
VvLRX5 | Vitis09g01122 | PN9:14210055–14212754 | 1 | 740 | 79.10 | 8.78 |
VvLRX6 | Vitis09g01427 | PN9:20795504–20799633 | 2 | 1120 | 123.03 | 5.87 |
VvLRX7 | Vitis11g00433 | PN11:3440956–3444229 | 1 | 752 | 80.58 | 5.81 |
VvLRX8 | Vitis11g00893 | PN11:8917201–8920586 | 2 | 989 | 108.47 | 6.66 |
VvLRX9 | Vitis11g00900 | PN11:9103494–9107050 | 2 | 976 | 107.03 | 6.41 |
VvLRX10 | Vitis12g00363 | PN12:4188271–4189500 | 1 | 409 | 44.57 | 6.56 |
VvLRX11 | Vitis12g01667 | PN12:21278398–21287152 | 4 | 1388 | 152.84 | 6.77 |
VvLRX12 | Vitis18g00827 | PN18:8373625–8375243 | 1 | 426 | 47.30 | 9.90 |
VvLRX13 | Vitis18g00828 | PN18:8379036–8380632 | 1 | 275 | 29.84 | 6.28 |
VvLRX14 | Vitis18g00962 | PN18:9611744–9614217 | 1 | 798 | 83.40 | 9.20 |
Phylogenetic and synteny analyses of VvLRXs
To examine the evolutionary and phylogenetic relationships among 11 AtLRX and 14 VvLRX proteins, we constructed an unrooted phylogenetic tree (Fig. 1a). According to the analysis results, the LRX proteins were classified into four primary groups (I–IV). Groups I and II each contained three proteins, and groups III and IV each had four proteins. This classification differs from that of most species, where the LRX proteins typically fall into two groups.
VvLRXs are distributed across six grapevine chromosomes, although not uniformly (Fig. 1b). Specifically, one gene was located on chromosome 5; two genes each on chromosomes 9 and 12; and three genes each on chromosomes 3, 11, and 18. Notably, more genes were positioned in the middle regions of the chromosomes than at the proximal or distal ends. To understand the evolutionary mechanisms of VvLRXs, duplication events were analyzed. Based on sequence homology, six VvLRXs (42.86%) formed three tandem duplication pairs, whereas another six VvLRXs (42.86%) formed three segmental duplication pairs (Fig. 1b, Table S3).
Conserved motif and gene structure analyses of VvLRXs
The conserved motifs and gene structure of VvLRXs were examined using GSDS 2.0 and MEME, respectively. The relationship between evolution, conserved motifs, and gene structure was analyzed by constructing a NJ phylogenetic tree (Fig. 2a), consistent with the findings in Fig. 1a. Conserved protein motifs must be identified for understanding evolutionary processes. Three conserved motifs were identified within the VvLRXs, with lengths of 21, 15, and 21 amino acids, respectively (Fig. 2b and Fig. S2). Members within the same group had similar motif locations and distributions. The positions and distributions of motifs 1 and 2 were similar across groups I–III. However, motif 3 was significantly less prevalent in group III compared with groups I and II. Group IV sequences completely lacked motif 3, whereas motif 2 was significantly more abundant in group IV than in groups I–III.
Additionally, exon and intron distribution is a critical aspect of the gene structure. Therefore, the structural characteristics of the 14 VvLRXs were analyzed, including the location, number, and length of their exons and introns (Fig. 2c). Members within the same subfamily exhibited a similar gene structure, reinforcing the results of the grapevine LRX classification. Groups I–III each had one exon. By contrast, group IV had 2–4 exons. Moreover, the length of all group IV was clearly greater than that of groups I–III. All members of groups I and III had both 3′-UTR and 5′-UTR regions, whereas only a few members of groups II and IV had both UTRs. Gene structure variations inevitably lead to functional differences among the genes.
Cis‑regulatory element analysis of VvLRXs
To predict the transcriptional properties and functions of VvLRXs, cis-regulatory elements in the promoter sequences of these genes were analyzed using PlantCARE. Five hormone-related elements, including auxin-, MeJA-, gibberellin-, salicylic acid-, and abscisic acid-responsive elements, were identified. Additionally, five putative cis-elements associated with stress responses and seven cis-elements associated with plant growth and development were detected (Fig. 3a). The most common cis-acting elements in groups I–IV were anaerobic induction elements, MeJA-responsive elements, abscisic acid-responsive elements, and salicylic acid-responsive elements, respectively. Furthermore, the number of cis-acting elements related to plant growth and development, stress response, and hormones also differed among the different groups. group IV had the most number of plant growth- and development-cis-elements, group I had the most number of stress response-related cis-elements, and group III had the most number of hormone-related cis-elements (Fig. 3b). VvLRXs have various types and number of cis-acting elements, indicating that they have different biological functions.
Tissue-specific expression profile of VvLRXs
For a more comprehensive understanding of the potential functions of VvLRXs, the expression levels of VvLRXs across 21 organs or tissues at various grapevine developmental stages were examined by referring to the BAR database (Fig. 4). In general, VvLRXs exhibit constitutive expression across almost all examined tissues. The VvLRX expression profiles varied among different groups in various tissues and organs. VvLRXs in groups I and II exhibited broader expression across various tissues and organs than those in group III. Notably, VvLRX5 and VvLRX7 in group I demonstrated the highest expression levels across multiple tissues and organs. Additionally, even same group members differed in their expression profiles. For example, among the four genes in group IV (VvLRX6, VvLRX8, VvLRX9, and VvLRX11), VvLRX11 exhibited significantly higher expression across different tissues and organs than the other three genes. In summary, our findings suggest that VvLRXs is a comprehensive regulator of grapevine growth and response to environmental stresses.
Expression profiles of VvLRXs under salt stress
Building on the aforementioned findings, numerous hormone- and stress-related cis-acting elements were identified in the VvLRX promoters. Additionally, a study demonstrated that LRX3/4/5 are vital for the salt tolerance of Arabidopsis [27]. To explore the response of VvLRXs to salt stress, the expression levels of these genes was assessed in grape rootstock leaves at 3, 6, 12, 24, and 48 h after the salt treatment. VvLRX1/4/5/7/8/10/11/14 expression significantly increased over time, with VvLRX7 exhibiting the most pronounced upregulation (Fig. 5). Salt treatment also induced VvLRX2/3/13 expression, although the changes were not statistically significant. Interestingly, VvLRX12 expression first increased and then decreased, whereas VvLRX6 expression first decreased and then increased.
Expression profiles of VvLRXs in different grape rootstocks under salt stress
Salt tolerance varies widely among grape rootstocks. On evaluating the physiological and morphological characteristics, and antioxidant enzyme activities of 10 common grape rootstocks under salt stress, studies have been able to identify the intolerant genotypes Beta, 101 − 14, and 5BB, and the tolerant genotypes 1103P, 520A, and 3309C [35]. VvLRX1/4/5/7/8/10/11/14 expression was significantly upregulated in 5BB under salt stress (Fig. 5). The expression of these eight VvLRXs in the leaves of different grape rootstocks after the salt treatment was investigated. The result showed that only VvLRX7 expression was positively correlated with the salt tolerance of the different grape rootstocks (Fig. 6).
VvLRX7 overexpression enhanced salt tolerance in Arabidopsis
As previously noted, VvLRX7 was the most significantly upregulated VvLRX gene in grape rootstock leaves at 3, 6, 12, 24, and 48 h after salt treatment. Additionally, among VvLRX genes, the expression level of only VvLRX7 was positively correlated with the salt tolerance of different grapevine rootstock genotypes. VvLRX7 also shares high homology with AtLRX3/4/5, which are essential for salt tolerance in Arabidopsis. VvLRX7 may be a key regulator in the salt stress response of plants. To investigate the role of VvLRX7 in the salt stress response, 12 VvLRX7 overexpression (VvLRX7-OE) Arabidopsis lines were generated using an Agrobacterium-mediated method. qPCR confirmed that the VvLRX7 mRNA expression level in the leaves of these transgenic lines increased (Fig. 6a). The lines with the highest VvLRX7 expression (#2, #7, and #11) were selected for further experiments. To determine whether VvLRX7 overexpression affected salt tolerance in Arabidopsis, #2, #7, and #11 were grown in the presence of 150 mM NaCl. All VvLRX7-OE lines exhibited significantly higher germination and survival rates than WT lines (Fig. 6b, c, d), which suggest that VvLRX7 promotes plant germination and survival under salt stress.