Genome-wide Identification and Characterization of the LRX Gene Family in Grapevine (Vitis vinifera L.) and Functional Characterization of VvLRX7 in Plant Salt Response

Genome-wide Identification and Characterization of the LRX Gene Family in Grapevine (Vitis vinifera L.) and Functional Characterization of VvLRX7 in Plant Salt Response | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide Identification and Characterization of the LRX Gene Family in Grapevine (Vitis vinifera L.) and Functional Characterization of VvLRX7 in Plant Salt Response Kai Liu, Xiujie Li, Chaoping Wang, Yan Han, Ziguo Zhu, Bo Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4776721/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2024 Read the published version in BMC Genomics → Version 1 posted 4 You are reading this latest preprint version Abstract Background Leucine-rich repeat (LRR) extensins (LRXs), cell wall-localized chimeric extensin proteins, are essential for the development of plants and in their stress resistance. Despite their significance, an extensive genome-wide analysis of the LRX gene family in grapevine ( Vitis vinifera L.) is lacking. Results We here detected 14 grapevine LRX genes and classified them into four groups through phylogenetic analysis. Their physiological and biochemical properties and gene/protein structures were also analyzed. According to synteny analysis, the expansion of the grapevine LRX gene family has been appreciably affected by tandem and segmental duplications. On investigating tissue-specific expression profiles and cis-regulatory elements, VvLRXs were found to be the comprehensive regulators of grapevine growth and response to environmental stresses. Salt stress treatments induced the expression of several VvLRX s, and VvLRX7 was the most significantly upregulated. Furthermore, VvLRX7 expression was positively correlated with the salt tolerance of grape rootstocks. VvLRX7 overexpression in Arabidopsis markedly enhanced its salt tolerance. Conclusion This study has provided a general understanding of the characteristics and evolution of the LRX gene family in grapevine. And it has been demonstrated that VvLRX7 may be key regulator in plant salt response. This provides a basis for future studies of the function of grapevine LRXs and the improvement of salt stress tolerance in grapevine. Genome-wide analysis Leucine-rich repeat extensins Grapevine Gene expression Gene function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Cell wall is a unique and complex structure present in plant cells but absent in animal cells. The cell wall shapes each cell and shields them against biological and abiotic stresses [ 1 , 2 ]. This dynamic structure is primarily composed of polysaccharides such as pectin, hemicellulose, and cellulose, which are augmented by a small contingent of proteins [ 3 ]. Although these proteins are present in a minor proportion, they are crucial for regulating cellular communication, embryogenesis, and responses to injury [ 4 , 5 ]. Extensins (EXTs), exhibit Ser-Pro2–n repeats, are the initial structural proteins of the cell wall [ 6 , 7 ]. They mainly facilitate cell wall loosening and expansion, thereby representing the sole class of cell wall proteins that induce in vitro cell enlargement and overall cell wall growth [ 8 , 9 ]. Leucine-rich repeat (LRR) extensins (LRXs), a special group within EXTs, have highly conserved LRR and EXT domains [ 10 ]. The LRR domain facilitates a spectrum of protein–protein interactions, extending from small peptides to large proteins, and the EXT domain likely participates in the positioning and stabilization of these cell wall proteins [ 11 , 12 ]. In Arabidopsis , 11 LRXs were cataloged into two distinct clades based on their expression patterns: class I is found in vegetative tissues (LRX1–LRX7) and class II in pollen (LRX8–LRX11) [ 10 , 13 ]. Class I LRXs play pivotal roles in cell wall construction in root hairs [ 14 , 15 ], and class II LRXs are integral to orchestrating the maintenance of cell wall integrity during pollen tube growth [ 16 ]. Grapevine ( Vitis vinifera L.) is among the oldest fruit trees that is widely cultivated globally [ 17 ]. Grapevine fruits can be eaten fresh and used for producing wine, juice, and dried fruit and are thus of substantial economic importance [ 18 ]. Approximately 7% of the Earth's land surface, or approximately 1 billion hectares, currently encounters the problem of salinization, and this affected area is continuously expanding. Soil salinization has become among the greatest environmental challenges of agricultural production [ 19 – 22 ]. Salt is the key source of abiotic stress affecting fruit trees such as grapevine. Salt stress can lead to nutritional imbalance, ionic toxicity, osmotic stress, and oxidative damage, which thus restricts the growth, yield, and fruit quality of fruit trees [ 19 , 21 , 23 , 24 ]. Improving the salt tolerance of grape varieties and rootstocks through selective breeding and use of grape resources with an inherent salt tolerance ability is a critical strategy for expanding cultivation in medium and low saline-alkali soils and improving grape yield and quality in such challenging conditions. However, molecular mechanisms underlying salt tolerance in grapevine remain poorly understood. The extended duration of breeding cycles has also hampered the breeding of high-salt-tolerant varieties and rootstocks. Recent in-depth studies have highlighted the substantial contributions of LRXs to plant growth and development. Of the LRXs expressed in vegetative tissues, LRX1 / 2 are crucial for root hair formation. Root hairs in the lrx1 single mutant were malformed, whereas those in the lrx2 single mutant maintained a normal appearance [ 14 ]. Notably, root hairs of the lrx1lrx2 double mutant have more severe abnormalities than those of the lrx1 single mutant alone, which suggests a synergistic effect of LRX1/2 on root hair morphology [ 15 ]. LRX3 / 4 / 5 single mutants typically show no notable phenotypic changes. By contrast, the lrx3lrx4lrx5 triple mutant exhibits stunted growth, compromised cell and vacuole formation, and increased anthocyanin accumulation [ 25 – 27 ]. Furthermore, LRXs specifically expressed in pollen ( LRX8 – 11 ) are necessary for regulating pollen germination and tube elongation. The quadruple mutants lrx8 – lrx11 exhibit reduced male fertility, decreased seed formation, and inhibition of pollen tube development, thereby highlighting the critical roles of these proteins in reproduction [ 28 , 29 ]. LRXs not only plays an important role in regulating plant growth and development, but also participates in stress response, especially salt stress response. In Arabidopsis , the RAPID ALKALINIZATION FACTOR (RALF) peptide directly binds to FERONIAs (FERs) and LRXs [ 30 ]. Under salt stress, the mature RALF22 peptide is liberated because of S1P protease, which subsequently initiates FER internalization through endosomal mechanisms. Comparable phenotypic manifestations, including growth inhibition and increased salt stress sensitivity, are observed in the lrx345 triple mutant, fer mutant, and RALF22 /23 overexpression plants. These findings support the hypothesis that the interaction among LRX, RALF, and FER forms a crucial signaling pathway that translates cell wall signals into mechanisms coordinating plant growth and salt stress responses. This thus underscores their pivotal roles in plant adaptability and stress management [ 27 ]. Based on previous study results, we propose that LRXs in grapevine are pivotal in modulating salt stress response. The LRX gene family in species such as Arabidopsis thaliana [ 31 ], tomato ( Lycopersicon esculentum ) [ 32 ], maize ( Zea mays ) [ 33 ], and rice ( Oryza sativa ) [ 31 ] has been extensively analyzed, but the functions of these genes within the grapevine genome remain largely unexplored. To enhance our understanding of the roles of LRX genes in grapevine, a genome-wide search was conducted to identify these genes. In total, 14 full-length LRX genes were successfully detected within the grapevine genome. Using comprehensive bioinformatics methods, we here analyzed the physicochemical attributes, gene/protein structures, chromosomal locations, evolutionary relationships, and tissue-specific expression profiles of these genes. Additionally, using qRT-PCR, the VvLRX expression profiles were assessed after salt treatment. The functionality of VvLRX7 was specifically explored through transgenic approaches. These insights advance our overall comprehension of the functions of the LRX gene family in grapevine, thereby highlighting the potential roles of this gene family in salt stress response mechanisms. Results 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. 1 a). 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. 1 b). 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. 1 b, 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. 2 a), consistent with the findings in Fig. 1 a. 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. 2 b 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. 2 c). 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. 3 a). 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. 3 b). 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 VvLRX s 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 VvLRX s 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. 6 a). 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. 6 b, c, d), which suggest that VvLRX7 promotes plant germination and survival under salt stress. Discussion LRX proteins, which are cell wall-localized chimeric extensins, are essential for modulating plant development and stress resistance [ 25 , 36 , 37 ]. Comprehensive studies have been conducted to systematically catalog the LRX gene family in several species, including A. thaliana [ 31 ], tomato ( L. esculentum ) [ 32 ], maize ( Z. mays ) [ 33 ], and rice ( Oryza sativa ) [ 31 ]. However, the LRX gene family in grapevine ( V. vinifera . L) had not been genome-wide analyzed before this study. Therefore, a genome-wide identification of the LRX gene family in grapevine was conducted, and 14 VvLRX genes were detected. Then, the physicochemical properties, gene/protein structures, chromosomal locations, evolutionary relationships, tissue-specific expression profiles, and expression responses under salt stress conditions of these genes were characterized. Moreover, the role of VvLRX7 in augmenting salt tolerance in plants was specifically investigated. The study findings provide crucial insights and a valuable foundation for the subsequent functional analyses of VvLRX genes. LRX genes of higher plants are broadly categorized into two distinct clades: one chiefly expressed in vegetative tissues and the other in pollen grains and tubes [ 10 ]. In the current study, phylogenetic analyses and sequence alignments revealed that VvLRXs segregate into four groups (Fig. 1 a), a classification deviating from the typical grouping observed in most species [ 38 ]. This difference underscores that species-specific characteristics influence the gene family organization [ 39 , 40 ]. The function of the grapevine LRX gene family might be different from that of the LRX gene family previously identified in other species. In grapevine, groups III and IV of VvLRXs exhibited more heterogeneous compositions than groups I and II (Fig. 1 b). This diversity suggests that groups III and IV was evolutionarily older, providing extended periods for gene duplication and structural rearrangement. Notably, the LRX gene family in grapevine has expanded to 14 VvLRXs , which is more than the numbers reported in Arabidopsis (11 AtLRX genes), rice (8 OsLRX genes), and maize (2 StLRX genes) (Table S2). Tandem and segmental duplications are vital for gene family expansion and the adaptive responses of plants to environmental fluctuations [ 41 , 42 ]. Approximately 50% VvLRXs are located within duplicated genomic blocks (Fig. 1 b, Table S3), which highlights the significance of these duplication processes in promoting the diversification of the VvLRX gene family in grapevine. Furthermore, our analysis indicates that the number of LRX genes and genome size across species were not positively correlated (Table S2). For instance, despite having a significantly larger genome (~ 2300 Mbp), Z. mays [ 43 ] has fewer LRX genes than A. thaliana [ 44 ] (genome size: ~135 Mbp). This contrasts with prokaryotes [ 45 ] and viruses [ 46 ], where the gene number and genome size are typically positively correlated. This correlation is absent in eukaryotes possibly because of the presence of a higher proportion of noncoding sequences in their genomes than in prokaryotic and viral genomes [ 47 ]. The structural features of various gene families offer insights into their evolutionary patterns [ 48 , 49 ]. By contrast, conserved motifs shed light on their specific protein functions [ 50 , 51 ]. In the current study, the motif arrangement and gene structure of VvLRXs changed with a change in group (Fig. 2 , Fig. S2). This suggested that the gene function of different VvLRX groups evolved over time to help organisms adapt to environmental changes. Furthermore, groups I–III VvLRXs are more similar in the gene structure and conserved domains than group IV VvLRXs (Fig. 2 , Fig. S2). Functional studies of LRX genes in Arabidopsis have indicated that proteins with similar gene structures and conserved domains tend to play analogous regulatory roles [ 31 ]. Therefore, groups I–III VvLRXs may have somewhat similar functions. Cis-acting elements, such as inducible elements, enhancers, and promoters, play crucial role in modulating plant growth, development, and stress responses by activating or inhibiting specific gene expression [ 52 , 53 ]. We here identified numerous cis-acting elements related to plant growth, development, stress responses, and hormones within VvLRX promoters (Fig. 3 ). The number and types of these elements varied among different VvLRXs , thereby contributing to the diverse regulatory roles of these genes in the growth, development, and environmental stress tolerance of grapes. Tissue-specific expression is a key factor for predicting the function of genes [ 54 , 55 ]. Comprehensive data on the tissue-specific expression of LRX genes in grapevine are currently lacking. We here found that VvLRXs were constitutively expressed in all tissues tested in grapevine (Fig. 4 ), suggesting that these genes have diverse functions in the growth and stress resistance of grapevine. Additionally, many genes within the same group (e.g., VvLRX10 / 14 ), and even gene pairs (e.g., VvLRX12 / 13 ), exhibited different tissue-specific expression patterns, which was attributable to subfunctionalization occurring between gene pairs [ 56 ]. LRX gene functions in model plants have been well characterized, with a specific subfamily having a specific function. LRX genes have broad regulatory roles in plant growth and development. Among the vegetatively expressed LRXs , LRX1/2 are essential for forming cell walls in root hairs [ 14 , 15 ]. LRX3 / 4 / 5 also regulate cell wall formation. The lrx3lrx4lrx5 triple mutant results in stunted growth, broader rosette leaves with defects in cell–cell adhesion in the epidermal layer, cell and vacuole growth defects, and increased anthocyanin accumulation [ 25 – 27 ]. The pollen-expressed LRXs ( LRX8 / 9 / 10 / 11 ) work synergistically to maintain cell wall integrity in the pollen tube and are critical for pollen germination [ 10 , 13 ]. LRX3 / 4 / 5 are known to be vital for plant salt tolerance, with the lrx345 triple mutant and fer mutant plants being hypersensitive to salt stress [ 27 , 30 ]. In our current study, VvLRX7 was found to share the highest homology with Arabidopsis LRX3 / 4 / 5 (Fig. 1 a), and its expression was strongly induced under salt stress (Fig. 5 ). Moreover, VvLRX7 was the only VvLRXs with an expression level positively correlated with the salt tolerance of different grape stocks (Fig. 6 ). These results indicate that VvLRX7 is crucial in the responses of grapevine to salt stress. We then cloned VvLRX7 and obtained 12 VvLRX7 -OE Arabidopsis lines. The lines with the highest VvLRX7 expression (#2, #7, and #11) were selected to verify salt tolerance (Fig. 7 a). VvLRX7 -overexpressing Arabidopsis lines exhibited increased tolerance to NaCl (Fig. 7 b, c, d)), indicating that VvLRX7 positively regulates in the plant’s response to salt stress. In summary, this study performed an extensive analysis of the LRX gene family in grapevine. qRT-PCR was performed to examine VvLRX expression after the salt treatment. We then investigated the specific function of the VvLRX7 gene by using transgenic technology. The findings offer valuable resources for future studies on the LRX gene family in grapevine and other plant species. Conclusion In current study, 14 full-length LRX genes were detected in grapevine. These were then classified into four distinct groups. According to the synteny analysis, both segmental and tandem duplications were essential for LRX gene family expansion in grapevine. Gene/protein structure examination indicated substantial divergence among the different VvLRX groups throughout evolution. According to the results of analysis of cis-regulatory elements and tissue-specific expression patterns, VvLRXs may play roles in various aspects of grapevine growth and resistance to environmental stress. Of note, several VvLRXs were upregulated in the leaves of grape rootstocks after salt treatment, with VvLRX7 exhibiting the most pronounced increase in expression. A positive correlation was noted between VvLRX7 expression and salt tolerance of grape rootstocks. Additionally, VvLRX7 overexpression in Arabidopsis significantly enhanced salt tolerance of the plant. These findings offer valuable insights into the LRX gene function in grapevine and the potential applications of these genes in improving stress tolerance. Materials and methods Grapevine LRX gene s i dentification The amino acid sequences of Arabidopsis LRXs were obtained from TAIR[†] and served as references for identifying grapevine LRX proteins by through BLAST [57] searches in Phytozome2[‡] and Winberige[ § ] databases. Sequences with an e-value of <1e ⁻30 were selected for further analysis. Using NCBI–CDD and SMART, the potential VvLRX sequences were analyzed for confirming the existence of LRR and extensin domains. The Expasy ProtParam tool[∗∗] was employed to predict the size, molecular weight, and isoelectric point of LRXs in grapevine. Phylogenetic and synteny analyses To create an unrooted phylogenetic tree based on the alignment of the complete amino acid sequences of LRX proteins from Arabidopsis and grapevine, the neighbor-joining (NJ) method was employed by using MEGA-7 software. The Clustal W program allowed sequence alignments, and bootstrap values were derived from 1000 replicates to ensure reliability [58]. Using MCScanX [59], gene duplication patterns were analyzed to conduct synteny analysis. Chromosome locations were identified by referring to the Winberige website, and chromosomal positions and synteny relationships were visualized using TBtools [60]. Gene structure and conserved motif analysis The Gene Structure Display Server (GSDS)[††] was used for analyzing the exon–intron structures of the 14 VvLRX s [61]. The visualizations were produced using TBtools. Conserved motifs within the 14 VvLRXs were identified using MEME [62][‡‡], with motif widths of 6–200 amino acids. Promoter analysis Using TBtools, the promoter region, identified as 2-kb sequences upstream of individual VvLRXs , was extracted from the Winberige database. Cis-acting elements within these sequences were identified using PlantCARE[ § § ] and visualized using TBtools. Tissue-specific expression profile analysis Tissue-specific VvLRX expression data were obtained from the BAR database[∗∗∗]. These data included gene expression profiles across major grapevine organs, such as roots, stems, leaves, buds, flowers, fruits, seedlings, and pollen. The FPKM values for all genes were subjected to quality control. Normalized log2 expression values were converted, and a corresponding heatmap was generated using TBtools software. Plant material Sixteen-month-old potted grape rootstocks, namely 5BB ( V. berlandieri × V. riparia ), Beta ( V. riparia × V. labrusca ), 101-14 ( V. riparia × V. rupestris ), 1103P ( V. berlandieri × V. rupestris ), 520A ( V. berlandieri × V. riparia ), and 3309C ( V. riparia × V. rupestris ), were maintained under controlled greenhouse conditions at the Shandong Academy of Agricultural Sciences (Jinan, China; 36°42′N, 117°4′E). The greenhouse conditions were 8 h dark and 16 h light at 26°C under 60% relative humidity. The grape rootstocks were cultivated in a mixture of sand, peat, and soil in a 1:1:3 ratio (v/v/v). To induce salt stress for the experiment, the grape rootstocks were irrigated with 150 mM NaCl solution. For the gene expression analysis, the newly emerged and still shining leaves from the 5BB rootstock were sampled at 0, 3, 6, 12, 24, and 48 h after the treatment. Similarly, the newly emerged and still shining leaves from 520A, 1103P, 3309C, 101-14, and Beta rootstocks were sampled at 48 h after the treatment. All samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C for subsequent RNA extraction. qRT-PCR validation SnapGene software was used to design qRT-PCR primers (Table S1). The PrimeScript™ RT Master Mix (Perfect Real Time) from TaKaRa was used for cDNA synthesis. TB Green Premix DimerEraser (TaKaRa) was used as a fluorescence labeling agent. The internal reference gene selected was VvUBI [21]. qRT-PCR was conducted on a CFX96™ Real-Time System (Bio-Rad Laboratories). A 25-µL reaction mixture was prepared by mixing 2 µL template cDNA, 1 mM forward and reverse primers, and 12.5 µL of TB Green Premix DimerEraser. The PCR program included initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 30 s. The 2 −ΔΔCt method was used to calculate relative gene expression levels [63]. Statistical analyses were performed using Student's t-test in GraphPad Prism 9, and error bars represent standard deviations from three biological replicates. V ector and transgenesis c onstruction The overexpression vector was constructed by amplifying the 2259-bp coding sequence (CDS) of VvLRX7 from the cDNA of 5BB grape stock leaves. Gene cloning was confirmed through sequencing. Using the ABclonal MultiF Seamless Assembly Mix (ABclonal Technology), the CDS was inserted into the plant expression vector PCambia1300. The recombinant PCambia1300-VvLRX7 vector was subsequently transformed into Agrobacterium tumefaciens EHA105. Table S1 presents the list of amplification primers used. Transgenic A. thaliana plants (Col-0 ecotype) harboring the 35S::LRX7 expression cassette were generated through Agrobacterium tumefaciens -mediated transformation [64]. Declarations Acknowledgements Not applicable. Authors’ contributions B.L., X.J.L., K.L. conceived and planned the research. B.L. supervised the research. K.L., X.J.L., C.P.W., Y.H., Z.G.Z., and B.L. performed the experiments. K.L. conducted data analysis. K.L. wrote the manuscript. B.L. edited the manuscript. All authors have read and agreed to the final version of the manuscript. Funding This work was financially supported by the National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land (Grant No. GYJ2023004), Science and Technology Innovation Program of Shandong Academy of Agricultural Sciences (Grant No. CXGC2024D16), Major Agricultural Technology Collaborative Promotion Program of Shandong (Grant No. SDNYXTTG-2024-26), Guide Foundation of Shandong Academy of Grape (Grant No. SDAG2021B08). Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Shandong Academy of Grape, Shandong Academy of Agricultural Science, Jinan 250100, China 2 National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land, P. R. 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Footnotes † https://www.arabidopsis.org/browse/gene_family ‡ https://phytozome-next.jgi.doe.gov/blast-search § http://www.winberige.cc/ftp.html ∗∗ https://web.expasy.org/protparam/ †† http://gsds.cbi.pku.edu.cn/ ‡‡ https://meme-suite.org/meme/tools/meme §§ http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ∗∗∗ https://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4776721","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330175928,"identity":"bd816f06-ec94-425d-a4d3-9fbfb6fad951","order_by":0,"name":"Kai Liu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Liu","suffix":""},{"id":330175929,"identity":"d4ec9580-02bf-4c88-8a5a-4ed8c918abc4","order_by":1,"name":"Xiujie Li","email":"","orcid":"","institution":"Shandong Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Xiujie","middleName":"","lastName":"Li","suffix":""},{"id":330175930,"identity":"22d9c3b4-120b-4093-92fc-5e7eb5aa4aed","order_by":2,"name":"Chaoping Wang","email":"","orcid":"","institution":"Shandong Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Chaoping","middleName":"","lastName":"Wang","suffix":""},{"id":330175931,"identity":"268a5f9d-1d44-4889-ab49-cb3d59f77d38","order_by":3,"name":"Yan Han","email":"","orcid":"","institution":"Shandong Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Han","suffix":""},{"id":330175932,"identity":"98d49c8f-4850-49eb-bfc0-52900c7bdc72","order_by":4,"name":"Ziguo Zhu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Ziguo","middleName":"","lastName":"Zhu","suffix":""},{"id":330175933,"identity":"9f37556a-dfb8-4a4f-845e-5c8654c78334","order_by":5,"name":"Bo Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYFCCBAaGBwYHGPiZmQ8/IFJLMlAXUItkO1uaAQlaGA4wGJznUZAgSoN8e/7BDwkFd+SMD/MwGDDU2EQT1GJw5jGzRILBM2Ozw7wHHjAcS8ttIKhFIpkBqOVw4rbDfAkGjA2HCWuRn5HM/AOkZXMzj4EEUVoYbiSzgW3ZwEysFqBfzCxAfpE4DAzkBGL8It+e+PjGhz935Pj7Dx9+8KHGhgiHoYAE0pSPglEwCkbBKMAFAD7FQOS2dhtPAAAAAElFTkSuQmCC","orcid":"","institution":"Shandong Academy of Agricultural Science","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-07-21 12:26:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4776721/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4776721/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-024-11087-3","type":"published","date":"2024-11-29T15:58:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62557544,"identity":"61813a95-a301-4003-adde-ee7cea27963f","added_by":"auto","created_at":"2024-08-15 20:12:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":507883,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic and synteny analyses of \u003cem\u003eLRXs\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Phylogenetic relationship between \u003cem\u003eLRXs\u003c/em\u003efrom grapevine and \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003eLRXs\u003c/em\u003e are represented by a green triangle, whereas grapevine \u003cem\u003eLRXs\u003c/em\u003e are indicated by purple circles. \u003cstrong\u003eb\u003c/strong\u003e Duplication events and chromosomal distribution of \u003cem\u003eVvLRXs\u003c/em\u003e. Segmental duplicated genes are linked by red lines, and tandem duplicated genes are linked by blue lines.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/815acfc7bdfd3b802dece14c.png"},{"id":62557762,"identity":"5c13a1c5-95c0-4a2a-a009-65c4f5e5aae1","added_by":"auto","created_at":"2024-08-15 20:20:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":251591,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motif and structures of \u003cem\u003eVvLRXs\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Phylogenetic relationship of \u003cem\u003eVvLRXs\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e Conserved motifs of VvLRXs. Motifs 1, 2, and 3 are depicted by blue, red, and green squares, respectively. \u003cstrong\u003ec\u003c/strong\u003e Gene structure of \u003cem\u003eVvLRXs\u003c/em\u003e. UTRs and CDSs are represented by red and green squares, respectively, and black lines indicate introns.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/97e500d70524c3cb087b7562.png"},{"id":62557546,"identity":"44dd103c-c9c7-452c-bcce-2567c688ffc9","added_by":"auto","created_at":"2024-08-15 20:12:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":505476,"visible":true,"origin":"","legend":"\u003cp\u003eMajor cis-acting elements in \u003cem\u003eVvLRX\u003c/em\u003e promoters. \u003cstrong\u003ea\u003c/strong\u003e The location of various cis-element types in chromosomes. The different types of cis-elements are represented by different colored circles. \u003cstrong\u003eb\u003c/strong\u003e Heatmap of number of different cis-element types. Red boxes indicate a higher number, whereas blue boxes indicate a lower number of each cis-element type.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/d20045d5ce2c1b9bdae8dfb7.png"},{"id":62557551,"identity":"11d1e1b2-5041-40ce-9f55-c64fb8ae7e1b","added_by":"auto","created_at":"2024-08-15 20:12:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":385545,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression profile of \u003cem\u003eVvLRXs\u003c/em\u003e. The heatmap was drawn by referring to the in silico analysis results of tissue-specific expression data for \u003cem\u003eVvLRXs\u003c/em\u003e obtained from the BAR database. Normalized log2 transformed values were used with hierarchical clustering. The different \u003cem\u003eVvLRX\u003c/em\u003e expression levels are represented by a gradient of color from blue to red.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/f5a63bac007ffd9431f42ae7.png"},{"id":62557763,"identity":"30a53c59-3916-44e6-a23c-f27f3ebed698","added_by":"auto","created_at":"2024-08-15 20:20:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":421530,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of 14 \u003cem\u003eVvLRXs\u003c/em\u003e after the salt treatment based on qRT-PCR data. Error bars = SD. Identical lowercase letters indicate no significant differences at the P \u0026lt; 0.05 level, Duncan’s multiple range test. \u003cem\u003eVvLRXs\u003c/em\u003esignificantly upregulated in leaves under salt stress were marked with a red five-pointed star.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/418d0fd431d40dde5f21cdbf.png"},{"id":62557549,"identity":"6844e78a-7e5a-4884-b1bd-ce02f485fd19","added_by":"auto","created_at":"2024-08-15 20:12:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":290517,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of selected \u003cem\u003eVvLRX\u003c/em\u003es in different grape rootstocks after the salt treatment. Error bars = SD. Identical lowercase letters indicate no significant differences at the P \u0026lt; 0.05 level, Duncan’s multiple range test. \u003cem\u003eVvLRXs\u003c/em\u003ewhose expression levels were positively correlated with the salt tolerance of different grape rootstocks are marked with green five-pointed stars.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/2e1038902adde0bf2fbe38d6.png"},{"id":62557935,"identity":"cb83648b-8901-4f37-9fe3-5b6617cd0523","added_by":"auto","created_at":"2024-08-15 20:28:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":557541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVvLRX7\u003c/em\u003e overexpression improved salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e \u003cem\u003eVvLRX7\u003c/em\u003eexpression levels in \u003cem\u003eVvLRX7\u003c/em\u003e-OE lines and WT. \u003cstrong\u003eb\u003c/strong\u003e Phenotypes of \u003cem\u003eVvLRX7\u003c/em\u003e-OE lines and WT grown on MS and MS+NaCl (150 mM) media. Bar = 1 cm. \u003cstrong\u003ec\u003c/strong\u003e Germination rates of \u003cem\u003eVvLRX7\u003c/em\u003e-OE lines and WT seedlings on MS+NaCl (150 mM) medium. \u003cstrong\u003ed\u003c/strong\u003e Survival rates of \u003cem\u003eVvLRX7\u003c/em\u003e-OE lines and WT seedlings on MS+NaCl (150 mM) medium. Error bars = SD. (Student’s t-tests, *P \u0026lt; 0.05, **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/cdd1074b84614c522c580730.png"},{"id":70389936,"identity":"76c936ad-9ffb-4f78-a6a6-1cf266c179be","added_by":"auto","created_at":"2024-12-02 17:29:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3930756,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/e99dfdfd-091d-4a27-aaac-b23d946272e6.pdf"},{"id":62557547,"identity":"ee3804f1-a033-4e2d-8edf-478ce128db1a","added_by":"auto","created_at":"2024-08-15 20:12:18","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":322223,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4776721/v1/a7091cecc2c2efbc538210bf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide Identification and Characterization of the LRX Gene Family in Grapevine (Vitis vinifera L.) and Functional Characterization of VvLRX7 in Plant Salt Response","fulltext":[{"header":"Background","content":"\u003cp\u003eCell wall is a unique and complex structure present in plant cells but absent in animal cells. The cell wall shapes each cell and shields them against biological and abiotic stresses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This dynamic structure is primarily composed of polysaccharides such as pectin, hemicellulose, and cellulose, which are augmented by a small contingent of proteins [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although these proteins are present in a minor proportion, they are crucial for regulating cellular communication, embryogenesis, and responses to injury [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Extensins (EXTs), exhibit Ser-Pro2\u0026ndash;n repeats, are the initial structural proteins of the cell wall [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. They mainly facilitate cell wall loosening and expansion, thereby representing the sole class of cell wall proteins that induce \u003cem\u003ein vitro\u003c/em\u003e cell enlargement and overall cell wall growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Leucine-rich repeat (LRR) extensins (LRXs), a special group within EXTs, have highly conserved LRR and EXT domains [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The LRR domain facilitates a spectrum of protein\u0026ndash;protein interactions, extending from small peptides to large proteins, and the EXT domain likely participates in the positioning and stabilization of these cell wall proteins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, 11 LRXs were cataloged into two distinct clades based on their expression patterns: class I is found in vegetative tissues (LRX1\u0026ndash;LRX7) and class II in pollen (LRX8\u0026ndash;LRX11) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Class I LRXs play pivotal roles in cell wall construction in root hairs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and class II LRXs are integral to orchestrating the maintenance of cell wall integrity during pollen tube growth [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGrapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) is among the oldest fruit trees that is widely cultivated globally [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Grapevine fruits can be eaten fresh and used for producing wine, juice, and dried fruit and are thus of substantial economic importance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Approximately 7% of the Earth's land surface, or approximately 1\u0026nbsp;billion hectares, currently encounters the problem of salinization, and this affected area is continuously expanding. Soil salinization has become among the greatest environmental challenges of agricultural production [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Salt is the key source of abiotic stress affecting fruit trees such as grapevine. Salt stress can lead to nutritional imbalance, ionic toxicity, osmotic stress, and oxidative damage, which thus restricts the growth, yield, and fruit quality of fruit trees [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Improving the salt tolerance of grape varieties and rootstocks through selective breeding and use of grape resources with an inherent salt tolerance ability is a critical strategy for expanding cultivation in medium and low saline-alkali soils and improving grape yield and quality in such challenging conditions. However, molecular mechanisms underlying salt tolerance in grapevine remain poorly understood. The extended duration of breeding cycles has also hampered the breeding of high-salt-tolerant varieties and rootstocks.\u003c/p\u003e \u003cp\u003eRecent in-depth studies have highlighted the substantial contributions of LRXs to plant growth and development. Of the \u003cem\u003eLRXs\u003c/em\u003e expressed in vegetative tissues, \u003cem\u003eLRX1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e are crucial for root hair formation. Root hairs in the \u003cem\u003elrx1\u003c/em\u003e single mutant were malformed, whereas those in the \u003cem\u003elrx2\u003c/em\u003e single mutant maintained a normal appearance [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, root hairs of the \u003cem\u003elrx1lrx2\u003c/em\u003e double mutant have more severe abnormalities than those of the \u003cem\u003elrx1\u003c/em\u003e single mutant alone, which suggests a synergistic effect of \u003cem\u003eLRX1/2\u003c/em\u003e on root hair morphology [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eLRX3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e single mutants typically show no notable phenotypic changes. By contrast, the \u003cem\u003elrx3lrx4lrx5\u003c/em\u003e triple mutant exhibits stunted growth, compromised cell and vacuole formation, and increased anthocyanin accumulation [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, \u003cem\u003eLRXs\u003c/em\u003e specifically expressed in pollen (\u003cem\u003eLRX8\u003c/em\u003e\u0026ndash;\u003cem\u003e11\u003c/em\u003e) are necessary for regulating pollen germination and tube elongation. The quadruple mutants \u003cem\u003elrx8\u003c/em\u003e\u0026ndash;\u003cem\u003elrx11\u003c/em\u003e exhibit reduced male fertility, decreased seed formation, and inhibition of pollen tube development, thereby highlighting the critical roles of these proteins in reproduction [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. LRXs not only plays an important role in regulating plant growth and development, but also participates in stress response, especially salt stress response. In \u003cem\u003eArabidopsis\u003c/em\u003e, the RAPID ALKALINIZATION FACTOR (RALF) peptide directly binds to FERONIAs (FERs) and LRXs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Under salt stress, the mature RALF22 peptide is liberated because of S1P protease, which subsequently initiates FER internalization through endosomal mechanisms. Comparable phenotypic manifestations, including growth inhibition and increased salt stress sensitivity, are observed in the \u003cem\u003elrx345\u003c/em\u003e triple mutant, \u003cem\u003efer\u003c/em\u003e mutant, and \u003cem\u003eRALF22\u003c/em\u003e/23 overexpression plants. These findings support the hypothesis that the interaction among LRX, RALF, and FER forms a crucial signaling pathway that translates cell wall signals into mechanisms coordinating plant growth and salt stress responses. This thus underscores their pivotal roles in plant adaptability and stress management [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on previous study results, we propose that \u003cem\u003eLRXs\u003c/em\u003e in grapevine are pivotal in modulating salt stress response. The \u003cem\u003eLRX\u003c/em\u003e gene family in species such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], tomato (\u003cem\u003eLycopersicon esculentum\u003c/em\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], maize (\u003cem\u003eZea mays\u003c/em\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and rice (\u003cem\u003eOryza sativa\u003c/em\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] has been extensively analyzed, but the functions of these genes within the grapevine genome remain largely unexplored. To enhance our understanding of the roles of \u003cem\u003eLRX\u003c/em\u003e genes in grapevine, a genome-wide search was conducted to identify these genes. In total, 14 full-length \u003cem\u003eLRX\u003c/em\u003e genes were successfully detected within the grapevine genome. Using comprehensive bioinformatics methods, we here analyzed the physicochemical attributes, gene/protein structures, chromosomal locations, evolutionary relationships, and tissue-specific expression profiles of these genes. Additionally, using qRT-PCR, the \u003cem\u003eVvLRX\u003c/em\u003e expression profiles were assessed after salt treatment. The functionality of \u003cem\u003eVvLRX7\u003c/em\u003e was specifically explored through transgenic approaches. These insights advance our overall comprehension of the functions of the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine, thereby highlighting the potential roles of this gene family in salt stress response mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eVvLRXs\u003c/b\u003e \u003cb\u003egene identification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify potential LRXs in the grapevine genome (PN_T2T) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], \u003cem\u003eArabidopsis\u003c/em\u003e 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\u0026ndash;CDD tools. Ultimately, 14 VvLRXs were identified (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), a number higher than those found in \u003cem\u003eArabidopsis\u003c/em\u003e, rice, maize, and tomato (Table S2). These 14 grapevine \u003cem\u003eLRX\u003c/em\u003e genes were designated \u003cem\u003eVvLRX1\u0026ndash;VvLRX14\u003c/em\u003e based on their chromosomal locations.\u003c/p\u003e \u003cp\u003eThe 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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of LRXs in Grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003cp\u003e(amino acids)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW\u003c/p\u003e \u003cp\u003e(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis03g00581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN3:4606131\u0026ndash;4608944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis03g00703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN3:5861853\u0026ndash;5863384\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis03g00704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN3:5875089\u0026ndash;5876942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis05g00653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN5:5770500\u0026ndash;5771879\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis09g01122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN9:14210055\u0026ndash;14212754\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e79.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis09g01427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN9:20795504\u0026ndash;20799633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e123.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis11g00433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN11:3440956\u0026ndash;3444229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis11g00893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN11:8917201\u0026ndash;8920586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e108.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis11g00900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN11:9103494\u0026ndash;9107050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e107.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis12g00363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN12:4188271\u0026ndash;4189500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis12g01667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN12:21278398\u0026ndash;21287152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1388\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e152.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis18g00827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN18:8373625\u0026ndash;8375243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis18g00828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN18:8379036\u0026ndash;8380632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e29.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvLRX14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitis18g00962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePN18:9611744\u0026ndash;9614217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic and synteny analyses of\u003c/b\u003e \u003cb\u003eVvLRXs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the evolutionary and phylogenetic relationships among 11 AtLRX and 14 VvLRX proteins, we constructed an unrooted phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). According to the analysis results, the LRX proteins were classified into four primary groups (I\u0026ndash;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.\u003c/p\u003e \u003cp\u003e \u003cem\u003eVvLRXs\u003c/em\u003e are distributed across six grapevine chromosomes, although not uniformly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). 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 \u003cem\u003eVvLRXs\u003c/em\u003e, duplication events were analyzed. Based on sequence homology, six \u003cem\u003eVvLRXs\u003c/em\u003e (42.86%) formed three tandem duplication pairs, whereas another six \u003cem\u003eVvLRXs\u003c/em\u003e (42.86%) formed three segmental duplication pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Table S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConserved motif and gene structure analyses of\u003c/b\u003e \u003cb\u003eVvLRXs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), consistent with the findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb 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\u0026ndash;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\u0026ndash;III.\u003c/p\u003e \u003cp\u003eAdditionally, exon and intron distribution is a critical aspect of the gene structure. Therefore, the structural characteristics of the 14 \u003cem\u003eVvLRXs\u003c/em\u003e were analyzed, including the location, number, and length of their exons and introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Members within the same subfamily exhibited a similar gene structure, reinforcing the results of the grapevine LRX classification. Groups I\u0026ndash;III each had one exon. By contrast, group IV had 2\u0026ndash;4 exons. Moreover, the length of all group IV was clearly greater than that of groups I\u0026ndash;III. All members of groups I and III had both 3\u0026prime;-UTR and 5\u0026prime;-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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCis‑regulatory element analysis of\u003c/b\u003e \u003cb\u003eVvLRXs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo predict the transcriptional properties and functions of \u003cem\u003eVvLRXs\u003c/em\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The most common cis-acting elements in groups I\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). \u003cem\u003eVvLRXs\u003c/em\u003e have various types and number of cis-acting elements, indicating that they have different biological functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue-specific expression profile of\u003c/b\u003e \u003cb\u003eVvLRXs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor a more comprehensive understanding of the potential functions of \u003cem\u003eVvLRXs\u003c/em\u003e, the expression levels of \u003cem\u003eVvLRXs\u003c/em\u003e across 21 organs or tissues at various grapevine developmental stages were examined by referring to the BAR database (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In general, \u003cem\u003eVvLRXs\u003c/em\u003e exhibit constitutive expression across almost all examined tissues. The \u003cem\u003eVvLRX\u003c/em\u003e expression profiles varied among different groups in various tissues and organs. \u003cem\u003eVvLRXs\u003c/em\u003e in groups I and II exhibited broader expression across various tissues and organs than those in group III. Notably, \u003cem\u003eVvLRX5\u003c/em\u003e and \u003cem\u003eVvLRX7\u003c/em\u003e 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 (\u003cem\u003eVvLRX6\u003c/em\u003e, \u003cem\u003eVvLRX8\u003c/em\u003e, \u003cem\u003eVvLRX9\u003c/em\u003e, and \u003cem\u003eVvLRX11\u003c/em\u003e), \u003cem\u003eVvLRX11\u003c/em\u003e exhibited significantly higher expression across different tissues and organs than the other three genes. In summary, our findings suggest that \u003cem\u003eVvLRXs\u003c/em\u003e is a comprehensive regulator of grapevine growth and response to environmental stresses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiles of\u003c/b\u003e \u003cb\u003eVvLRXs\u003c/b\u003e \u003cb\u003eunder salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBuilding on the aforementioned findings, numerous hormone- and stress-related cis-acting elements were identified in the \u003cem\u003eVvLRX\u003c/em\u003e promoters. Additionally, a study demonstrated that \u003cem\u003eLRX3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e are vital for the salt tolerance of \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To explore the response of \u003cem\u003eVvLRX\u003c/em\u003es 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. \u003cem\u003eVvLRX1\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e/\u003cem\u003e8\u003c/em\u003e/\u003cem\u003e10\u003c/em\u003e/\u003cem\u003e11\u003c/em\u003e/\u003cem\u003e14\u003c/em\u003e expression significantly increased over time, with \u003cem\u003eVvLRX7\u003c/em\u003e exhibiting the most pronounced upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Salt treatment also induced \u003cem\u003eVvLRX2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e/\u003cem\u003e13\u003c/em\u003e expression, although the changes were not statistically significant. Interestingly, \u003cem\u003eVvLRX12\u003c/em\u003e expression first increased and then decreased, whereas \u003cem\u003eVvLRX6\u003c/em\u003e expression first decreased and then increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiles of\u003c/b\u003e \u003cb\u003eVvLRXs\u003c/b\u003e \u003cb\u003ein different grape rootstocks under salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSalt 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\u0026thinsp;\u0026minus;\u0026thinsp;14, and 5BB, and the tolerant genotypes 1103P, 520A, and 3309C [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. \u003cem\u003eVvLRX1\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e/\u003cem\u003e8\u003c/em\u003e/\u003cem\u003e10\u003c/em\u003e/\u003cem\u003e11\u003c/em\u003e/\u003cem\u003e14\u003c/em\u003e expression was significantly upregulated in 5BB under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The expression of these eight \u003cem\u003eVvLRX\u003c/em\u003es in the leaves of different grape rootstocks after the salt treatment was investigated. The result showed that only \u003cem\u003eVvLRX7\u003c/em\u003e expression was positively correlated with the salt tolerance of the different grape rootstocks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVvLRX7\u003c/b\u003e \u003cb\u003eoverexpression enhanced salt tolerance in\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs previously noted, \u003cem\u003eVvLRX7\u003c/em\u003e was the most significantly upregulated \u003cem\u003eVvLRX\u003c/em\u003e gene in grape rootstock leaves at 3, 6, 12, 24, and 48 h after salt treatment. Additionally, among \u003cem\u003eVvLRX\u003c/em\u003e genes, the expression level of only \u003cem\u003eVvLRX7\u003c/em\u003e was positively correlated with the salt tolerance of different grapevine rootstock genotypes. \u003cem\u003eVvLRX7\u003c/em\u003e also shares high homology with \u003cem\u003eAtLRX3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e, which are essential for salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eVvLRX7\u003c/em\u003e may be a key regulator in the salt stress response of plants. To investigate the role of \u003cem\u003eVvLRX7\u003c/em\u003e in the salt stress response, 12 \u003cem\u003eVvLRX7\u003c/em\u003e overexpression (\u003cem\u003eVvLRX7\u003c/em\u003e-OE) \u003cem\u003eArabidopsis\u003c/em\u003e lines were generated using an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated method. qPCR confirmed that the \u003cem\u003eVvLRX7\u003c/em\u003e mRNA expression level in the leaves of these transgenic lines increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The lines with the highest \u003cem\u003eVvLRX7\u003c/em\u003e expression (#2, #7, and #11) were selected for further experiments. To determine whether \u003cem\u003eVvLRX7\u003c/em\u003e overexpression affected salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, #2, #7, and #11 were grown in the presence of 150 mM NaCl. All \u003cem\u003eVvLRX7\u003c/em\u003e-OE lines exhibited significantly higher germination and survival rates than WT lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c, d), which suggest that \u003cem\u003eVvLRX7\u003c/em\u003e promotes plant germination and survival under salt stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLRX proteins, which are cell wall-localized chimeric extensins, are essential for modulating plant development and stress resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Comprehensive studies have been conducted to systematically catalog the \u003cem\u003eLRX\u003c/em\u003e gene family in several species, including \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], tomato (\u003cem\u003eL. esculentum\u003c/em\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], maize (\u003cem\u003eZ. mays\u003c/em\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and rice (\u003cem\u003eOryza sativa\u003c/em\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine (\u003cem\u003eV. vinifera\u003c/em\u003e. L) had not been genome-wide analyzed before this study. Therefore, a genome-wide identification of the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine was conducted, and 14 \u003cem\u003eVvLRX\u003c/em\u003e genes were detected. Then, the physicochemical properties, gene/protein structures, chromosomal locations, evolutionary relationships, tissue-specific expression profiles, and expression responses under salt stress conditions of these genes were characterized. Moreover, the role of \u003cem\u003eVvLRX7\u003c/em\u003e in augmenting salt tolerance in plants was specifically investigated. The study findings provide crucial insights and a valuable foundation for the subsequent functional analyses of \u003cem\u003eVvLRX\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLRX\u003c/em\u003e genes of higher plants are broadly categorized into two distinct clades: one chiefly expressed in vegetative tissues and the other in pollen grains and tubes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the current study, phylogenetic analyses and sequence alignments revealed that \u003cem\u003eVvLRXs\u003c/em\u003e segregate into four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), a classification deviating from the typical grouping observed in most species [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This difference underscores that species-specific characteristics influence the gene family organization [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The function of the grapevine \u003cem\u003eLRX\u003c/em\u003e gene family might be different from that of the \u003cem\u003eLRX\u003c/em\u003e gene family previously identified in other species. In grapevine, groups III and IV of \u003cem\u003eVvLRXs\u003c/em\u003e exhibited more heterogeneous compositions than groups I and II (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This diversity suggests that groups III and IV was evolutionarily older, providing extended periods for gene duplication and structural rearrangement. Notably, the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine has expanded to 14 \u003cem\u003eVvLRXs\u003c/em\u003e, which is more than the numbers reported in \u003cem\u003eArabidopsis\u003c/em\u003e (11 \u003cem\u003eAtLRX\u003c/em\u003e genes), rice (8 \u003cem\u003eOsLRX\u003c/em\u003e genes), and maize (2 \u003cem\u003eStLRX\u003c/em\u003e genes) (Table S2). Tandem and segmental duplications are vital for gene family expansion and the adaptive responses of plants to environmental fluctuations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Approximately 50% \u003cem\u003eVvLRXs\u003c/em\u003e are located within duplicated genomic blocks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Table S3), which highlights the significance of these duplication processes in promoting the diversification of the \u003cem\u003eVvLRX\u003c/em\u003e gene family in grapevine. Furthermore, our analysis indicates that the number of \u003cem\u003eLRX\u003c/em\u003e genes and genome size across species were not positively correlated (Table S2). For instance, despite having a significantly larger genome (~\u0026thinsp;2300 Mbp), \u003cem\u003eZ. mays\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] has fewer \u003cem\u003eLRX\u003c/em\u003e genes than \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] (genome size: ~135 Mbp). This contrasts with prokaryotes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and viruses [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], where the gene number and genome size are typically positively correlated. This correlation is absent in eukaryotes possibly because of the presence of a higher proportion of noncoding sequences in their genomes than in prokaryotic and viral genomes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe structural features of various gene families offer insights into their evolutionary patterns [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. By contrast, conserved motifs shed light on their specific protein functions [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In the current study, the motif arrangement and gene structure of VvLRXs changed with a change in group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. S2). This suggested that the gene function of different \u003cem\u003eVvLRX\u003c/em\u003e groups evolved over time to help organisms adapt to environmental changes. Furthermore, groups I\u0026ndash;III \u003cem\u003eVvLRXs\u003c/em\u003e are more similar in the gene structure and conserved domains than group IV \u003cem\u003eVvLRXs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. S2). Functional studies of \u003cem\u003eLRX\u003c/em\u003e genes in \u003cem\u003eArabidopsis\u003c/em\u003e have indicated that proteins with similar gene structures and conserved domains tend to play analogous regulatory roles [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, groups I\u0026ndash;III \u003cem\u003eVvLRXs\u003c/em\u003e may have somewhat similar functions. Cis-acting elements, such as inducible elements, enhancers, and promoters, play crucial role in modulating plant growth, development, and stress responses by activating or inhibiting specific gene expression [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We here identified numerous cis-acting elements related to plant growth, development, stress responses, and hormones within \u003cem\u003eVvLRX\u003c/em\u003e promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The number and types of these elements varied among different \u003cem\u003eVvLRXs\u003c/em\u003e, thereby contributing to the diverse regulatory roles of these genes in the growth, development, and environmental stress tolerance of grapes. Tissue-specific expression is a key factor for predicting the function of genes [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Comprehensive data on the tissue-specific expression of \u003cem\u003eLRX\u003c/em\u003e genes in grapevine are currently lacking. We here found that \u003cem\u003eVvLRXs\u003c/em\u003e were constitutively expressed in all tissues tested in grapevine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that these genes have diverse functions in the growth and stress resistance of grapevine. Additionally, many genes within the same group (e.g., \u003cem\u003eVvLRX10\u003c/em\u003e/\u003cem\u003e14\u003c/em\u003e), and even gene pairs (e.g., \u003cem\u003eVvLRX12\u003c/em\u003e/\u003cem\u003e13\u003c/em\u003e), exhibited different tissue-specific expression patterns, which was attributable to subfunctionalization occurring between gene pairs [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eLRX\u003c/em\u003e gene functions in model plants have been well characterized, with a specific subfamily having a specific function. \u003cem\u003eLRX\u003c/em\u003e genes have broad regulatory roles in plant growth and development. Among the vegetatively expressed \u003cem\u003eLRXs\u003c/em\u003e, \u003cem\u003eLRX1/2\u003c/em\u003e are essential for forming cell walls in root hairs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eLRX3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e also regulate cell wall formation. The \u003cem\u003elrx3lrx4lrx5\u003c/em\u003e triple mutant results in stunted growth, broader rosette leaves with defects in cell\u0026ndash;cell adhesion in the epidermal layer, cell and vacuole growth defects, and increased anthocyanin accumulation [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The pollen-expressed \u003cem\u003eLRXs\u003c/em\u003e (\u003cem\u003eLRX8\u003c/em\u003e/\u003cem\u003e9\u003c/em\u003e/\u003cem\u003e10\u003c/em\u003e/\u003cem\u003e11\u003c/em\u003e) work synergistically to maintain cell wall integrity in the pollen tube and are critical for pollen germination [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eLRX3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e are known to be vital for plant salt tolerance, with the \u003cem\u003elrx345\u003c/em\u003e triple mutant and \u003cem\u003efer\u003c/em\u003e mutant plants being hypersensitive to salt stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our current study, \u003cem\u003eVvLRX7\u003c/em\u003e was found to share the highest homology with \u003cem\u003eArabidopsis LRX3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and its expression was strongly induced under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, \u003cem\u003eVvLRX7\u003c/em\u003e was the only \u003cem\u003eVvLRXs\u003c/em\u003e with an expression level positively correlated with the salt tolerance of different grape stocks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that \u003cem\u003eVvLRX7\u003c/em\u003e is crucial in the responses of grapevine to salt stress. We then cloned \u003cem\u003eVvLRX7\u003c/em\u003e and obtained 12 \u003cem\u003eVvLRX7\u003c/em\u003e-OE \u003cem\u003eArabidopsis\u003c/em\u003e lines. The lines with the highest VvLRX7 expression (#2, #7, and #11) were selected to verify salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). \u003cem\u003eVvLRX7\u003c/em\u003e-overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e lines exhibited increased tolerance to NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c, d)), indicating that \u003cem\u003eVvLRX7\u003c/em\u003e positively regulates in the plant\u0026rsquo;s response to salt stress. In summary, this study performed an extensive analysis of the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine. qRT-PCR was performed to examine \u003cem\u003eVvLRX\u003c/em\u003e expression after the salt treatment. We then investigated the specific function of the \u003cem\u003eVvLRX7\u003c/em\u003e gene by using transgenic technology. The findings offer valuable resources for future studies on the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine and other plant species.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn current study, 14 full-length \u003cem\u003eLRX\u003c/em\u003e genes were detected in grapevine. These were then classified into four distinct groups. According to the synteny analysis, both segmental and tandem duplications were essential for \u003cem\u003eLRX\u003c/em\u003e gene family expansion in grapevine. Gene/protein structure examination indicated substantial divergence among the different \u003cem\u003eVvLRX\u003c/em\u003e groups throughout evolution. According to the results of analysis of cis-regulatory elements and tissue-specific expression patterns, \u003cem\u003eVvLRXs\u003c/em\u003e may play roles in various aspects of grapevine growth and resistance to environmental stress. Of note, several \u003cem\u003eVvLRXs\u003c/em\u003e were upregulated in the leaves of grape rootstocks after salt treatment, with \u003cem\u003eVvLRX7\u003c/em\u003e exhibiting the most pronounced increase in expression. A positive correlation was noted between \u003cem\u003eVvLRX7\u003c/em\u003e expression and salt tolerance of grape rootstocks. Additionally, \u003cem\u003eVvLRX7\u003c/em\u003e overexpression in \u003cem\u003eArabidopsis\u003c/em\u003e significantly enhanced salt tolerance of the plant. These findings offer valuable insights into the \u003cem\u003eLRX\u003c/em\u003e gene function in grapevine and the potential applications of these genes in improving stress tolerance.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eGrapevine\u003cem\u003e\u0026nbsp;LRX\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene\u003c/strong\u003e\u003cstrong\u003es i\u003c/strong\u003e\u003cstrong\u003edentification\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino acid sequences of \u003cem\u003eArabidopsis\u003c/em\u003e LRXs were obtained from TAIR[\u0026dagger;] and served as references for identifying grapevine LRX proteins by through BLAST [57] searches in Phytozome2[\u0026Dagger;] and Winberige[\u003cspan style=\"text-align: start;color: rgb(32, 33, 36);background-color: rgb(255, 255, 255);font-size: 20px;\"\u003e\u0026sect;\u003c/span\u003e] databases. Sequences with an e-value of \u0026lt;1e\u003csup\u003e⁻30\u003c/sup\u003e were selected for further analysis. Using NCBI\u0026ndash;CDD and SMART, the potential VvLRX sequences were analyzed for confirming the existence of LRR and extensin domains. The Expasy ProtParam tool[\u0026lowast;\u0026lowast;] was employed to predict the size, molecular weight, and isoelectric point of LRXs in grapevine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic and synteny analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo create an unrooted phylogenetic tree based on the alignment of the complete amino acid sequences of LRX proteins from \u003cem\u003eArabidopsis\u003c/em\u003e and grapevine, the neighbor-joining (NJ) method was employed by using MEGA-7 software. The Clustal W program allowed sequence alignments, and bootstrap values were derived from 1000 replicates to ensure reliability [58]. Using MCScanX [59], gene duplication patterns were analyzed to conduct synteny analysis. Chromosome locations were identified by referring to the Winberige website, and chromosomal positions and synteny relationships were visualized using TBtools [60].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene structure and conserved motif analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Gene Structure Display Server (GSDS)[\u0026dagger;\u0026dagger;] was used for analyzing the exon\u0026ndash;intron structures of the 14 \u003cem\u003eVvLRX\u003c/em\u003e\u003cem\u003es\u0026nbsp;\u003c/em\u003e[61]. The visualizations were produced using TBtools. Conserved motifs within the 14 VvLRXs were identified using MEME [62][\u0026Dagger;\u0026Dagger;], with motif widths of 6\u0026ndash;200 amino acids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePromoter analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing TBtools, the promoter region, identified as 2-kb sequences upstream of individual \u003cem\u003eVvLRXs\u003c/em\u003e, was extracted from the Winberige database. Cis-acting elements within these sequences were identified using PlantCARE[\u003cspan style=\"text-align: start;color: rgb(32, 33, 36);background-color: rgb(255, 255, 255);font-size: 20px;\"\u003e\u0026sect;\u003cspan style=\"text-align: start;color: rgb(32, 33, 36);background-color: rgb(255, 255, 255);font-size: 20px;\"\u003e\u0026sect;\u003c/span\u003e\u003c/span\u003e] and visualized using TBtools.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue-specific expression\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eprofile\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue-specific \u003cem\u003eVvLRX\u003c/em\u003e expression data were obtained from the BAR database[\u0026lowast;\u0026lowast;\u0026lowast;]. These data included gene expression profiles across major grapevine organs, such as roots, stems, leaves, buds, flowers, fruits, seedlings, and pollen. The FPKM values for all genes were subjected to quality control. Normalized log2 expression values were converted, and a corresponding heatmap was generated using TBtools software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSixteen-month-old potted grape rootstocks, namely 5BB (\u003cem\u003eV. berlandieri\u003c/em\u003e \u0026times; \u003cem\u003eV. riparia\u003c/em\u003e), Beta (\u003cem\u003eV. riparia\u003c/em\u003e \u0026times; \u003cem\u003eV. labrusca\u003c/em\u003e), 101-14 (\u003cem\u003eV. riparia\u003c/em\u003e \u0026times; \u003cem\u003eV. rupestris\u003c/em\u003e), 1103P (\u003cem\u003eV. berlandieri\u003c/em\u003e \u0026times; \u003cem\u003eV. rupestris\u003c/em\u003e), 520A (\u003cem\u003eV. berlandieri\u003c/em\u003e \u0026times; \u003cem\u003eV. riparia\u003c/em\u003e), and 3309C (\u003cem\u003eV. riparia\u003c/em\u003e \u0026times; \u003cem\u003eV. rupestris\u003c/em\u003e), were maintained under controlled greenhouse conditions at the Shandong Academy of Agricultural Sciences (Jinan, China; 36\u0026deg;42\u0026prime;N, 117\u0026deg;4\u0026prime;E). The greenhouse conditions were 8 h dark and 16 h light at 26\u0026deg;C under 60% relative humidity. The grape rootstocks were cultivated in a mixture of sand, peat, and soil in a 1:1:3 ratio (v/v/v). To induce salt stress for the experiment, the grape rootstocks were irrigated with 150 mM NaCl solution. For the gene expression analysis, the newly emerged and still shining leaves from the 5BB rootstock were sampled at 0, 3, 6, 12, 24, and 48 h after the treatment. Similarly, the newly emerged and still shining leaves from 520A, 1103P, 3309C, 101-14, and Beta rootstocks were sampled at 48 h after the treatment. All samples were immediately flash-frozen in liquid nitrogen and stored at \u0026minus;80 \u0026deg;C for subsequent RNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSnapGene software was used to design qRT-PCR primers (Table S1). The PrimeScript\u0026trade; RT Master Mix (Perfect Real Time) from TaKaRa was used for cDNA synthesis. TB Green Premix DimerEraser (TaKaRa) was used as a fluorescence labeling agent. The internal reference gene selected was \u003cem\u003eVvUBI\u0026nbsp;\u003c/em\u003e[21]. qRT-PCR was conducted on a CFX96\u0026trade; Real-Time System (Bio-Rad Laboratories). A 25-\u0026micro;L reaction mixture was prepared by mixing 2 \u0026micro;L template cDNA, 1 mM forward and reverse primers, and 12.5 \u0026micro;L of TB Green Premix DimerEraser.\u0026nbsp;The PCR program included initial denaturation at 95 \u0026deg;C for 1 min, followed by 40 cycles of 95 \u0026deg;C for 5 s, 58 \u0026deg;C for 30 s, and 72 \u0026deg;C for 30 s.\u0026nbsp;The 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to calculate relative gene expression levels [63]. Statistical analyses were performed using Student\u0026apos;s t-test in GraphPad Prism 9, and error bars represent standard deviations from three biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003cstrong\u003eector and transgenesis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;c\u003c/strong\u003e\u003cstrong\u003eonstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe overexpression vector was constructed by amplifying the 2259-bp coding sequence (CDS) of \u003cem\u003eVvLRX7\u003c/em\u003e from the cDNA of 5BB grape stock leaves. Gene cloning was confirmed through sequencing. Using the ABclonal MultiF Seamless Assembly Mix (ABclonal Technology), the CDS was inserted into the plant expression vector PCambia1300. The recombinant PCambia1300-VvLRX7 vector was subsequently transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105. Table S1 presents the list of amplification primers used. Transgenic \u003cem\u003eA. thaliana\u003c/em\u003e plants (Col-0 ecotype) harboring the \u003cem\u003e35S::LRX7\u003c/em\u003e expression cassette were generated through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation [64].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.L., X.J.L., K.L. conceived and planned the research. B.L. supervised the research. K.L., X.J.L., C.P.W., Y.H., Z.G.Z., and B.L. performed the experiments. K.L. conducted data analysis. K.L. wrote the manuscript. B.L. edited the manuscript. All authors have read and agreed to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land (Grant No. GYJ2023004), Science and Technology Innovation Program of Shandong Academy of Agricultural Sciences (Grant No. CXGC2024D16), Major Agricultural Technology Collaborative Promotion Program of Shandong (Grant No. SDNYXTTG-2024-26), Guide Foundation of\u0026nbsp;Shandong Academy of Grape\u0026nbsp;(Grant No. SDAG2021B08).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Shandong Academy of Grape, Shandong Academy of Agricultural Science, Jinan 250100, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land, P. R. China, Dongying 257000, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCosgrove DJ: Re-constructing our models of cellulose and primary cell wall assembly. Curr Opin Plant Biol 2014, 22:122-131.\u003c/li\u003e\n\u003cli\u003eZhang B, Gao Y, Zhang L, Zhou Y: The plant cell wall: Biosynthesis, construction, and functions. J Integr Plant Biol 2021, 63(1):251-272.\u003c/li\u003e\n\u003cli\u003eCosgrove DJ: Structure and growth of plant cell walls. Nat Rev Mol Cell Biol 2024, 25(5):340-358.\u003c/li\u003e\n\u003cli\u003eQuinn O, Kumar M, Turner S: The role of lipid-modified proteins in cell wall synthesis and signaling. Plant Physiol 2023, 194(1):51-66.\u003c/li\u003e\n\u003cli\u003eHerger A, D\u0026uuml;nser K, Kleine-Vehn J, Ringli C: Leucine-Rich Repeat Extensin Proteins and Their Role in Cell Wall Sensing. 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Hortic Res-England 2023, 10(11):uhad198.\u003c/li\u003e\n\u003cli\u003eBohn L, Huang J, Weidig S, Yang Z, Heidersberger C, Genty B, Falter-Braun P, Christmann A, Grill E: The temperature sensor TWA1 is required for thermotolerance in Arabidopsis. Nature 2024, 629(8014):1126-1132.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Footnotes","content":"† https://www.arabidopsis.org/browse/gene_family\n\n‡ https://phytozome-next.jgi.doe.gov/blast-search\n\n§ http://www.winberige.cc/ftp.html\n\n∗∗ https://web.expasy.org/protparam/\n\n†† http://gsds.cbi.pku.edu.cn/\n\n‡‡ https://meme-suite.org/meme/tools/meme\n\n§§ http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\n\n∗∗∗ https://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Genome-wide analysis, Leucine-rich repeat extensins, Grapevine, Gene expression, Gene function","lastPublishedDoi":"10.21203/rs.3.rs-4776721/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4776721/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeucine-rich repeat (LRR) extensins (LRXs), cell wall-localized chimeric extensin proteins, are essential for the development of plants and in their stress resistance. Despite their significance, an extensive genome-wide analysis of the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) is lacking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe here detected 14 grapevine\u003cem\u003e LRX\u003c/em\u003e genes and classified them into four groups through phylogenetic analysis. Their physiological and biochemical properties and gene/protein structures were also analyzed. According to synteny analysis, the expansion of the grapevine\u003cem\u003e LRX\u003c/em\u003e gene family has been appreciably affected by tandem and segmental duplications. On investigating tissue-specific expression profiles and cis-regulatory elements, \u003cem\u003eVvLRXs \u003c/em\u003ewere found to be the comprehensive regulators of grapevine growth and response to environmental stresses. Salt stress treatments induced the expression of several \u003cem\u003eVvLRX\u003c/em\u003es, and \u003cem\u003eVvLRX7\u003c/em\u003ewas the most significantly upregulated. Furthermore, \u003cem\u003eVvLRX7\u003c/em\u003eexpression was positively correlated with the salt tolerance of grape rootstocks. \u003cem\u003eVvLRX7\u003c/em\u003e overexpression in \u003cem\u003eArabidopsis\u003c/em\u003emarkedly enhanced its salt tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has provided a general understanding of the characteristics and evolution of the \u003cem\u003eLRX\u003c/em\u003e gene family in grapevine. And it has been demonstrated that \u003cem\u003eVvLRX7\u003c/em\u003e may be key regulator in plant salt response.\u003cstrong\u003e \u003c/strong\u003eThis provides a basis for future studies of the function of grapevine LRXs and the improvement of salt stress tolerance in grapevine.\u003c/p\u003e","manuscriptTitle":"Genome-wide Identification and Characterization of the LRX Gene Family in Grapevine (Vitis vinifera L.) and Functional Characterization of VvLRX7 in Plant Salt Response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-15 20:12:13","doi":"10.21203/rs.3.rs-4776721/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-22T12:35:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-22T08:26:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-22T08:24:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-07-21T12:25:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2f7a1ba4-acbb-4689-b108-1e5637145133","owner":[],"postedDate":"August 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T17:25:49+00:00","versionOfRecord":{"articleIdentity":"rs-4776721","link":"https://doi.org/10.1186/s12864-024-11087-3","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2024-11-29 15:58:22","publishedOnDateReadable":"November 29th, 2024"},"versionCreatedAt":"2024-08-15 20:12:13","video":"","vorDoi":"10.1186/s12864-024-11087-3","vorDoiUrl":"https://doi.org/10.1186/s12864-024-11087-3","workflowStages":[]},"version":"v1","identity":"rs-4776721","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4776721","identity":"rs-4776721","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}