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2024 Volume 4
Article Contents
Abstract
Introduction
Structure characteristics and tasting mechanism of marine umami peptides
Structure characteristics of marine umami peptides
The taste mechanism of marine umami peptides
Umami receptors
Recognition and transduction mechanism of marine umami peptides
Preparation and regulation techniques of marine umami peptides
Preparation technique
Microbial fermentation
Enzymatic hydrolysis
Acid hydrolysis
High-temperature processing
Solvent extraction
Regulation techniques of umami taste
Embedding methods
Amino acid addition
Application of Industry 4.0 technology in marine umami peptides
Molecular simulation
Artificial intelligence
Big data
Cloud computing
Blockchain
Conclusions and perspectives
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Identification and characterization of grape VAP27 gene family and their roles in disease resistance

  • 1.

    State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • 2.

    Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • # Authors contributed equally: Ruonan Li, Bianbian Wang

More Information
  • Received: 24 January 2024
    Revised: 03 April 2024
    Accepted: 24 April 2024
    Published online: 17 May 2024
    Fruit Research  4,  Article number: e019 (2024)  |  Cite this article
  • Vesicle-associated membrane protein (VAMP)-associated proteins (VAP27s), which are widely expressed in plants and animals, play an important role in metabolism, physiology, growth, and development, disease resistance, and immunity. While the function of this family has been elucidated in model plants like Arabidopsis thaliana and tomato, its role in grapevine remains unclear. In this present study, 12 vesicle-associated protein-membrane protein genes were identified in the grapevine genome by bioinformatics, designated as the VAP27 gene family. A phylogenetic tree, encompassing 53 genes from three model plants, Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum, revealed the subdivision of the VAP27 gene family into three subfamilies, each presumably serving different functions, besides localizing in endoplasmic reticulum, individual members also localize in nucleus. Additionally, we compared the transcriptional levels and subcellular localizations of the VvVAP27 family members across different plant tissues (flower, leaf, seed, root, fruit, tendril, and stem), indicating site-specific functionalities for different gene members. To investigate the responsiveness of the VAP27 gene family to pathogen infection, particularly Plasmopara viticola on host plants, we analyzed the expression patterns of VAP27 genes post-infection. Our findings revealed divergent expression profiles among different members at different stages of infection. The gene family responded to the infection of downy mildew on grapevine and could inhibit the spread of Phytophthora capsici lesions in Nicotiana benthamiana. These results provide an important basis for further studies delving into the functions of the VAP27 gene family in plant growth and disease resistance.

  • Introduction

    Umami has played an essential role in the Chinese diet since ancient times. References to umami can be found in many classical Chinese texts dating back to early history. For instance, during the Song Dynasty (960 AD to 1279 AD), Hong Lin mentioned in 'Shanjia Qing Offering' that bamboo shoots 'taste very fresh', and Yizun Zhu of the Ming Dynasty (1368 AD to 1644 AD) also noted in 'The Secret of Food Constitution' that 'pickled pork had a special umami taste'. These references reflect the historical appreciation and pursuit of umami by the Chinese people, although they were based on subjective perceptions at that time. It was not until Professor Ikeda's groundbreaking work that umami was studied as a scientific issue for the first time. He isolated glutamate from kelp (Laminaria japonica) and discovered its ability to enhance soup flavor. This unique taste, distinct from sour, sweet, bitter, and salty flavors, led Professor Ikeda to coin the term 'umami'[1,2]. However, due to limited systematic scientific research on umami at that time, it was not recognized as a basic taste by the scientific community; instead, it was considered a comprehensive taste associated with pleasure and increased appetite. It wasn't until 2002 when umami receptors (mGlu1/4, T1R1/T1R3) were discovered that umami began to be acknowledged as a fundamental taste[3,4].

    Umami, as a fundamental taste, is generated through complex physiological processes. Initially, umami substances bind to umami receptors, triggering an allosteric effect and producing gustatins. Subsequently, gustatins are further converted into electrical taste signals. Finally, taste signals are transmitted to the cerebral cortex by nerve fibers and sensed by the gustatory center[5,6]. Umami substances come in various forms, mainly including free amino acids and their salts, nucleotides and their salts, organic acids, organic bases, and umami peptides[7,8]. In recent years, umami peptides have emerged as a new form of umami substance that not only enhances the umami taste but also enriches the nutritional value of food, thus becoming a new research focus. Additionally, umami peptides can synergize with salt to enhance the salty taste and reduce the use of salt for better human health[9−11]. Therefore, it is highly significant to study umami peptides.

    Marine organisms have long been recognized as a crucial source of nutrients for humans, particularly high-quality dietary protein[12,13]. Oceans cover 71% of the Earth's surface area and contain vast amounts of resources. In comparison to land-sourced protein, marine protein production causes less environmental stress[14]. Various marine proteins possess a delicious and unique taste and are important raw materials for the preparation of umami peptides[15]. Furthermore, the extraction of umami peptides from marine proteins is an important method to promote the development and utilization of marine resources, which holds significant strategic importance[16−18].

    Compared with the land-sourced umami peptides, marine umami peptides have their advantages: Firstly, they are more widely sourced as the area of the ocean is nearly two and a half times larger than that of the land on earth, and the diversity of species in the ocean is far greater than that on land. Secondly, marine umami peptides have higher nutritional value as they usually contain ω-3 polyunsaturated fatty acids (PUFA), minerals, and trace elements such as iodine, zinc, and selenium, which are important for human health. Lastly, marine organisms are rich in umami substances, resulting in an intense umami taste with a more obvious umami-enhancing effect[19−21]. However, it should be noted that current research on marine umami peptides is still in its infancy. The preparation and extraction of these peptides are traditional and lack systematic summary. Therefore, based on existing research findings, summarizing the current progress of marine umami peptides and prospecting new technologies for their application would be beneficial for subsequent research and development efforts in this field.

    In this review, the sequence and structural characteristics of reported marine umami peptides are summarized and analyzed systematically. Then, the recognition mechanism of marine umami peptides and their receptors are introduced, followed by the analysis of the signal transduction mechanism. Next, the preparation and regulation techniques of marine umami peptides are summarized. Finally, the application of Industry 4.0 technologies (molecular simulation, artificial intelligence, blockchain, big data, cloud computing) in the study of marine umami peptides are introduced and their future development is prospected. This review aims to introduce the current research of marine umami peptides and prospect the future trends of the methods applied in the subsequent study of marine umami peptides.

    Structure characteristics and tasting mechanism of marine umami peptides

    Structure characteristics of marine umami peptides

    The amino acid sequence and source information of the reported marine umami peptides that have been reported were shown in Supplemental Table S1 and the distribution of the identified marine umami peptides are shown in Fig. 1. To date, 141 marine umami peptides have been identified, of which 104 are short-chain peptides (dipeptides to nonapeptides), accounting for 73.76%. The number of medium and long-chain polypeptides (decapeptides to eighteen peptides) is 37, accounting for 26.24%. It can be seen that most of the marine umami peptides are short chain peptides. This is consistent with previous studies of umami peptides from other sources[8,22,23]. Specifically, the top three types of marine umami peptides are heptapeptides (23, 16.31%), nonapeptides (17, 12.06%) and hexapeptides (14, 9.93%). The last four types of marine umami peptides are tridecanoic peptides (3, 2.13%), tetradeca peptides (3, 2.13%), hexadecapeptides (2, 1.42%) and octadecapeptides (1, 0.71%). It can be observed from this that the longer the peptide chain, the lower the likelihood of it being umami peptides. In addition, from the view of the amino acid composition of umami peptides, it is found that 85.11% of 120 marine umami peptides contain umami amino acids (glutamic acid or aspartic acid). Zhang et al.[24] summarized the amino acid composition of umami peptides and found that among all umami peptides, those containing umami amino acids accounted for 79.00%, which was lower than the 85.11% found in marine umami peptides. This suggests that marine umami peptides contain a higher proportion of umami amino acids.

    Figure 1.  The information of the identified marine umami peptides. (a) The distribution of the identified marine umami peptides. (b) The percentage of marine umami peptides with umami amino acid (D: aspartic acid, E: glutamic acid).
    DownLoad: Full-Size Img PowerPoint

    Furthermore, the types and sequences of amino acids that compose umami peptides also have a great influence on the taste characteristics of peptides[25]. The study of Arai et al.[26] found that the umami taste can only be presented when hydrophilic amino acid is located at the C-terminus. Zhang et al.[24] proposed that the proportion of alkaline amino acids in umami peptides should not be excessively high, as alkaline amino acids are typically found at the end of the polypeptide chain. The amino acid fingerprints of the reported marine umami peptides are shown in Supplemental Fig. S1. In all identified marine peptides, the top four amino acids are glutamic acid (E), accounting for 11.41%, alanine (A) (9.41%), leucine (L) (9.32%) and aspartic acid (D) (9.22%), which are mainly umami/acidic amino acids and hydrophobic amino acids. The last four amino acids are tyrosine (Y) (1.62%), cysteine (C) (1.24%), tryptophan (W) (1.24%). According to the classification of amino acid properties, the proportion of acidic/umami amino acids is 20.63%. The data for alkaline, hydrophilic and hydrophobic amino acids is 13.30%, 29.85%, and 36.23%, respectively (Fig. 2). It can be seen that among all the amino acids composing marine umami peptides, the proportion of acidic/umami amino acid is significantly higher than that of alkaline amino acids, and the proportion of hydrophobic amino acids is higher than that of hydrophilic amino acids. Regarding the order of peptide sequences, it can be observed that hydrophilic and basic amino acids are primarily distributed at the ends of the peptide chain, while hydrophobic and acidic amino acids are mainly distributed in the middle of the peptide chain.

    Figure 2.  The amino acid composition of the reported marine umami peptides according to the amino acid features.
    DownLoad: Full-Size Img PowerPoint

    The advanced structure of marine umami peptides mainly refers to its secondary structure, including α-helix, β-folding and random curling, etc. The unique marine environment (high salt, low temperature, and high pressure) contributes to the strong stability and adaptability of the structure of marine umami peptides. For instance, the α-helix structure in marine umami peptides enhance their stability, allowing them to maintain biological activity even in extreme environments. In addition, marine umami peptides may also contain special modification groups, such as phosphate groups, sugar groups, etc., which can increase their water solubility and biological activity[27,28].

    The taste mechanism of marine umami peptides

    The taste formation mechanism of marine umami peptides is similar to that of umami peptides from other sources. Initially, umami peptides and receptors (T1R1/T1R3, mGlu1/4) recognize each other in the mouth, leading to the allosteric effects[29,30]. Once the receptor is activated, it produces the corresponding umami gustducin, which further transforms into an umami signal further. Subsequently, the signal molecules bind to their respective receptors and activate taste bud ion channels located on the cell membrane. This activation leads to the release of signal molecules from the cell and their transmission to the nerve center through the taste nerve fibers, resulting in the perception of umami (Fig. 3)[31,32].

    Figure 3.  Schematic diagram of the flavor mechanism of umami peptides (adapted from[32]).
    DownLoad: Full-Size Img PowerPoint
    Umami receptors

    The receptors for umami peptides are a class of membrane proteins called G-protein-coupled receptors (GPCRS). Specifically, the umami peptide receptors mainly include T1R1 and T1R3. T1R1 is co-expressed with T1R3. T1R1/T1R3 heteromer in humans is primarily responsible for detecting and responding to umami substances[29,33,34]. When the umami substance interacts with the T1R1/T1R3 receptor, T1R1/T1R3 initiates signaling pathways that transmit taste information to the brain, resulting in the perception of umami taste[35]. Besides T1R1/T1R3, the umami receptors also include some metabotropic glutamate receptors (mGluRs). MGluRs are also members of the GPCRs family, responsible for the neurotransmitter glutamate. There are eight subtypes of mGluRs, which are classified into three groups based on sequence homology, signal transduction mechanisms, and pharmacological properties. The identified mGluRs umami receptors include mGluR1[36] and mGluR4[37].

    Recognition and transduction mechanism of marine umami peptides

    The analysis of the recognition and transduction mechanism of marine umami peptides is an effective way to study the taste mechanism of umami peptides and mine new umami peptides. Among all umami receptors, heterodimer T1R1/T1R3 receptors are mainly distributed in the front of the tongue. They come into contact with umami substances once the umami substances get into the mouth. Other receptors (mGluR1 and mGluR4) located in the back of the tongue, which results in the lag in its contact with umami substances. Therefore, heterodimer T1R1/T1R3 receptors were identified as the best candidates for umami receptors[38]. All members of the T1R class have a similar structure, that is, the seven-spanning transmembrane region (7TM) and the N-terminal extracellular terminal region[39]. The extracellular terminal region consists of a large 'venus fly trap' (VFT) and a small cysteine-rich domain (CRD). The VFT is the main binding domain for umami ligand recognition, while the CRD mainly connects the VFT domain and the 7TM domain, which is responsible for the transmembrane transmission of the structural changes that occur after VFT ligand recognition[40].

    At present, the mechanism of the interaction between umami peptides and umami receptors has not been clarified clearly[32]. Evidence from immunocytochemical and molecular studies suggest that stimulation of umami receptor heterodimers T1R1/T1R3 by umami substances activate the G protein subunit Gα, leading to the release of the Gβγ subunit and stimulating the phospholipase PLCβ2 pathway, which produces inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG)[41,42]. The increase of Ca2+ activates the TRPM5 transient receptor potential, leading to the depolarization of taste cells. Taste cells then evoke action potentials through sodium channels and release ATP, which is converted into electrical signals to activate taste nerve fibers, thereby generating umami perception[43]. Although it has been widely recognized that umami receptor heterodimers T1R1/T1R3 stimulate the G protein subunits leading to umami signal transduction, the interaction between umami receptors and ligands and the conformational changes of the receptors after binding umami substances, remain to be examined systematically.

    Preparation and regulation techniques of marine umami peptides

    Preparation technique

    Marine umami peptides refer to peptides prepared from marine proteins with an umami taste. The processing methods for these peptides can vary due to the diverse sources of marine proteins. Common processing methods include microbial fermentation, enzymatic hydrolysis, acid hydrolysis, high-temperature processing, and solvent extraction (see Table 1).

    Table 1.  Overview of the preparation methods for marine umami peptides.
    Methods Mechanism Advantages Disadvantages Ref.
    Microbial fermentation Microbial metabolism degrades marine proteins into peptides Simple operation, safety and low cost High environmental requirements [50,87]
    Enzymatic hydrolysis Proteases specifically cleave hydrolytic peptide bonds Mild reaction conditions, green environmental protection, and high yield Poor control of the reaction leads to the production of undesirable by-products [21,50,51,54]
    Acid hydrolysis Proteins are hydrolyzed under acidic conditions Low cost and high yield Toxic substances are easily produced in the reaction process [66,100]
    High-temperature
    processing    		 Protein degrades into small parts under thermal treatment Simple operation and high efficiency High energy consumption and high requirements for equipment [30,68−72]
    Solvent extraction The majority of umami peptides are soluble in water Simple operation and low cost Low efficiency [44,75]
     | Show Table
    DownLoad: CSV
    Microbial fermentation

    Microbial fermentation is a method that utilizes microbial metabolism to degrade marine proteins and obtain marine umami peptides. The vigorous metabolic reaction of microorganisms coverts macromolecular organic matter into amino acids, peptides, organic acids, and other substances[44−46]. Yang et al.[47] found that the production of umami peptides based on microbial metabolism in Chouguiyu was strongly correlated with 22 protease-producing microbial genera, among which Vagococcus, Peptostreptococcus, Acinetobacter, Psychrobacter, and Enterococcus played a major role in the formation of umami peptides. Microbial fermentation has the advantages of fast speed and low cost. At the same time, microbial fermentation does not require the separation and purification of proteins and enzymes, which reduces the production cost and simplifies the operation[48]. Therefore, the use of proteolytic microorganisms to extract umami peptides from animal and plant proteins is more cost-effective and practical than traditional enzymatic hydrolysis. However, microbial fermentation requires high environmental hygiene and is easily contaminated by other bacteria during the fermentation process. As a green and environmentally friendly method for the preparation of marine umami peptides, microbial fermentation has great potential for umami peptide preparation. Future research should focus on the screening of highly resistant strains to achieve more application prospects.

    Enzymatic hydrolysis

    Enzymatic hydrolysis is one of the commonly used methods for the preparation of marine umami peptides. Enzymes can break down proteins into peptides with different molecular weights and free amino acids, which are key umami compounds[49]. Various enzymes such as complex protease[50], pepsin, trypsin[51−54], flavourzyme, papain[21], chymotrypsin[52,53] are commonly employed in enzymatic hydrolysis. The reaction conditions for enzymatic hydrolysis are mild, safe and efficient, easily controlled, and do not produce toxic and harmful substances. This makes it a key technology for developing seafood seasoning[55,56]. Studies have shown that sequential hydrolysis of endonuclease and exonuclease can improve efficiency, reduce bitterness, and enhance the umami taste[57,58]. Flavor protease contains both endo-cut enzyme and exo-cut enzyme activities. These enzymes effectively degrade exposed hydrophobic groups and hydrolyze certain flavor precursor substances to facilitate the formation of high-quality flavor products[59]. To enhance proteolysis and prepare good flavor umami peptides, many researchers have utilized double enzyme hydrolysis methods. For example, Deng et al.[21] prepared and identified umami peptides from Trachinotus ovatus using enzymatic hydrolysis with papain and flavourzyme. Additionally, a stepwise dual-enzymatic hydrolysis process using alkaline and flavor protease was employed to prepare umami enzymatic hydroxylate from squid processing by-products[60].

    Acid hydrolysis

    Under acidic conditions, proteins can undergo hydrolysis reactions to produce peptides or amino acids[61]. Acid hydrolysis is characterized by its simple process, low cost and high-efficiency, making it a widely used method for protein hydrolysis[62−64]. Hydrochloric acid is often used to hydrolyze food proteins to produce umami substances for food flavoring[61,65]. However, during the acid hydrolysis process of preparing marine umami peptides, toxic and carcinogenic substances such as chloropropanol could be generated as by-products[66]. Compared with enzymatic hydrolysis, acid hydrolysis requires more severe reaction conditions[67], which has led to its gradual replacement by other methods.

    High-temperature processing

    Many researchers have utilized high-temperature methods for the preparation of water extracts containing umami peptides[68−72]. During the heating process, proteins degrade into smaller components[73,74]. Wang et al. obtained 29 peptide fragments through high-temperature water boiling combined with gel ultrafiltration and gel filtration chromatography and identified four of them as umami peptides[75]. Additionally, three umami peptides were isolated and purified from Leccinum extremiorientale using high-temperature cooking[76]. Alim et al. separated and purified 15 types of umami peptides from thermally treated yeast extract, with an optimum reaction temperature of 110 °C for umami peptide generation[77]. High-temperature processing is a common method for extracting nutrients from foods; therefore, the process of preparing umami peptides by this method is simple and suitable for industrial production. However, it is important to strictly control the temperature and heating time to avoid excessive hydrolysis and denaturation of the protein.

    Solvent extraction

    Because most umami peptides are water-soluble, water-based solvent extraction is commonly used for their extraction. Hot water extraction is a traditional method for obtaining umami peptides. Four umami peptides were identified from Lactarius volemus aqueous extracts, with one peptide (EVAEALDAPKTT) having a very low umami threshold of only 0.0625 mg/mL[78]. To enhance extraction efficiency, solvent extraction is often combined with high-temperature treatment, homogenization, ultrasonic treatment, and other methods[61]. Wang et al. extracted proteins and peptides from Takifugu flavidus using homogenization and a water-based solvent extraction method, resulting in the identification of four peptides with umami taste[79]. The process of solvent extraction is straightforward and does not require expensive equipment. However, it has the disadvantage of high energy consumption and long extraction time[61]. In recent years, there has been a growing interest in natural green extraction solvents, such as aqueous two-phase and deep eutectic solvent (DES), for extracting bioactive substances, making it a research hotspot[80,81]. These solvents have also been applied to the extraction of marine umami peptides. For example, DES composed of glucose, sucrose, and water was used to enhance the umami-enhancing capacity of pea protein hydrolysate[82]. Although there are currently few reports on the application of natural green extraction solvents, their potential in the extraction of marine umami peptides can be seen based on their extensive use in extracting bioactive substances[83−86].

    Regulation techniques of umami taste

    During the process of preparing marine umami protein and peptides products, it is inevitable that some unique substances from marine organisms will be incorporated into them, such as ω-3PUFA and astaxanthin. However, these substances will produce many unpleasant flavors after oxidation. Moreover, umami peptides may be decomposed by temperature, pH, and other conditions during processing or storage, leading to a significant reduction in their umami taste. Therefore, it is necessary to use embedding and adding amino acids in the actual production process to regulate the umami taste and maintain the stability of these products.

    Embedding methods

    Embedding is a technique used to immobilize active substances (such as drugs, enzymes, proteins, etc.) in a carrier material. This technique serves various purposes including protecting the active substance, prolonging its duration of action, improving stability, and controlling the release rate[87]. In the food industry, embedding can also be utilized to immobilize food additives or condiments in carriers to achieve specific release characteristics. Currently, spray drying and freeze drying are commonly employed methods for embedding umami peptides[88]. The process of microencapsulation through spray drying and freeze drying involves preparing a mixed emulsion of heartwood and wall material first, followed by spraying or freeze-drying the emulsion in a desiccator[89]. While these methods effectively prevent the oxidation of umami peptides and their reaction with other substances which could weaken their flavor profile, they do have drawbacks such as high energy consumption and low automation control level[90].

    Amino acid addition

    The amino acid addition method is a technique to regulate the umami taste by adding pure amino acids or amino acids mixture into the umami protein or peptide products. These amino acids or amino acids mixtures can cooperate with other flavorful peptides, nucleotides, and other substances contained in food to further enhance umami taste. Fu et al.[91] improved the umami taste of the umami peptides in fish head soup by adding cysteine. Similarly, Ruan et al.[92] maintained the umami taste of soy sauce by adding hydrolysate from low-value fish. The addition of amino acids is a direct method for increasing the umami flavor of products. While this process is simple, it requires exploration and optimization of the amount of added amino acids in the early laboratory stages, which may also result in additional economic costs being incurred at that time.

    Application of Industry 4.0 technology in marine umami peptides

    Recently, various global challenges, including global warming, increasing global population, overfishing, and other ecosystem damage, make it necessary for human beings to further innovate, study, and carry out sustainable development and utilization of marine resources. Therefore, Industry 4.0 technology has received great attention in recent years to improve efficiency and productivity and enhance sustainability[93,94]. Industry 4.0, categorized according to different stages of industrial development represents an era in which information technology is utilized to drive transformation within the industry. The artificial intelligence, big data analysis, Internet of Things, blockchain, smart sensors, robotic and cyber-physical systems are some of the technologies featured in Industry 4.0[95,96], and emerges as a way to improve the competitiveness of marine food[97,98]. Industry 4.0 technology has been used widely in the simulation of the interaction between the marine umami peptides and their receptors, the rapid screening and structure prediction of marine umami peptides, and the establishment and sharing of marine umami peptides database[99−101]. Furthermore, Industry 4.0 technology holds significant potential for the collection and processing of characteristic data and traceability analysis of marine umami peptides. This section introduces and prospects the application of Industry 4.0 technologies (molecular simulation, artificial intelligence, big data, cloud computing, and blockchain) in the study of marine umami peptides (Fig. 4), aiming to promote their application in the subsequent scientific research of marine umami proteins and peptides.

    Figure 4.  The application prospect of Industry 4.0 in marine umami peptides.
    DownLoad: Full-Size Img PowerPoint

    Molecular simulation

    Molecular simulation (MS) technology is a method that uses computers to simulate the behavior and properties of molecular systems at the atomic or molecular level[102]. MS technology has been applied to the study of marine umami peptides, mainly including the following aspects: (1) Interaction simulation: the interaction between marine umami peptides and other molecules (such as receptors and enzymes) can be simulated, and the mechanism of action and binding mode in vivo can be predicted, which is helpful for studying the biological activity and pharmacological effects of marine umami peptides. The interaction between shrimp umami peptide and its receptor was simulated through homology modeling and molecular docking and the results indicated that amino acid residues Arg151, Asp147 and Gln52 might be the key binding sites[103]. Five umami peptides were obtained from Meretrix lusoria, and explored the interaction between these umami peptides with T1R1/T1R3 by molecular simulation technology[19]. The results indicated that the peptides could enter the binding pocket in the Venus flytrap domain of the T1R3 cavity, wherein Asp196 and Glu128 may play key roles in the sensation of umami taste. Moreover, hydrogen bonding and electrostatic interactions are important interaction forces. (2) Structural prediction: molecular simulation technology can help researchers to predict and simulate the molecular structure of marine umami peptides, including three-dimensional structure, conformation and stability analysis of proteins, peptides, and other molecules, so as to help understand their functions and biological activities. Bu et al.[87] studied the characterization and structure-activity relationship of novel umami peptides isolated from Thai fish sauce by molecular simulation technology combined with quantitative structure–activity relationship (QSAR). The results indicated that the umami peptide chains were rotated and folded obviously, and hydroxyphenyl group of the N-terminal tyrosine residue side chain rotated significantly, which may enhance the umami taste. Although molecular simulation technology can effectively simulate the structural change process of biological macromolecules dynamically, the internal structure of biological systems, with uncertainties that change over time, is much more complex than imagined[104]. Therefore, the simulation process of biomacromolecules also has certain limitations, which require the improvement of computing software and hardware as much as possible to meet the needs of solving practical problems.

    Artificial intelligence

    Artificial intelligence (AI) is the technology of creating and applying intelligent machines or software that can mimic, augment, and extend human intelligence. The research field of artificial intelligence covers robotics, language recognition, image recognition, natural language processing, etc. The goal is to make computers have the ability to think and make decisions like humans[105,106]. The application of AI in marine umami peptides mainly includes the following aspects: (1) Discovering new marine umami peptides involves the use of advanced techniques such as machine learning and deep learning. By analyzing large quantities of known peptides, researchers can model and predict their structure and taste. This approach holds great potential for advancing our understanding of marine umami peptides and their applications in various fields. Meanwhile, virtual screening combined with AI could be used to determine their biological activities and taste characteristics, helping to accelerate the discovery of new marine peptides. Zhang et al.[107] investigated an interpretable BERT-based AI model to realize rapid screening of new umami peptides with a computational accuracy of 93.23%. Qi et al.[108] developed a novel peptide sequence-based umami peptide predictor, namely Umami-MRNN, which was based on multi-layer perceptron and recurrent neural network. The independent tests have shown that Umami-MRNN achieved an accuracy of 90.5%. (2) Prediction of allosteric effects can be achieved by integrating and analyzing a substantial amount of experimental data, in combination with methodologies such as machine learning and network analysis. This approach allows for the elucidation of the mechanism of action and signaling pathways associated with marine umami peptides. This helps to understand the function of marine umami peptides in cells and provides a theoretical basis for further application and development. Cui et al.[109] predicted the conserved sites and recognition mechanisms of T1R1 through ensemble docking combined with machine learning. The results indicated that residues 107S-109S, 148S-154T, and 247F-249A mainly form hydrogen bonding contacts, which might be the key binding sites during the sensation of umami. The binding processing of two umami peptides (MTLERPW and MNLHLSF) with T1R1 was predicated by Li et al.[110] through phage display combined with AI, and the key binding sites (MW-7 and MF-7) in the VFT were identified. (3) Utilizing AI technology, the analysis of large-scale marine umami peptide sequences can be conducted through bioinformatics techniques such as sequence alignment, structure prediction, and functional annotation. This approach is beneficial for predicting the characteristics of marine umami peptides and establishing a reference threshold for their properties. An AI system for taste analysis based on a graph neural network was developed by Lee et al.[33], which could predict the taste threshold of marine umami peptides based on their structure, providing a new method for the perception research of marine umami peptides. AI relies on a large amount of high-quality data for training and verification, with the database serving as the foundation for AI model testing and training[111]. AI relies on a large amount of high-quality data for training and verification, with the database serving as the foundation for AI model testing and training[111]. Therefore, AI requires a substantial volume of accurate data and computational resources to train, optimize, and support research related to umami peptides. The collection of such data has become a limiting factor in the application of AI in the development of umami peptides. However, as the model improves and more data is accumulated, AI will create new opportunities for the advancement of marine umami peptides.

    Big data

    Big data technology is a data processing technique that utilizes computer systems and network technology to process massive amounts of data. It aims to extract useful information from the data through association analysis and prediction. Big data technology is characterized by its volume, velocity, variety, and value. The primary function of big data is to record, describe, and predict various phenomena[112−114]. In the context of marine umami peptides, the application of big data encompasses several key aspects. Firstly, it involves the establishment and sharing of databases. By integrating and sharing characteristic data related to marine umami peptides, a specialized database can be developed. This database is a valuable resource for studying marine umami peptides, ultimately expediting research processes and fostering collaboration within the field. Gradinaru et al.[115] and Rojas et al.[116] collected a large number of characteristic information of umami substances through big data technology and established two open access umami databases (PlantMolecularTasteDB and ChemTastesDB), which greatly facilitated the subsequent research on marine umami peptides. Secondly, by utilizing big data analytics, marine biological scientists and researchers can effectively process large-scale marine biological data to gain a better understanding of the genomic, proteomic, and metabolomic features of marine organisms. This will enable them to explore the biosynthetic pathways and properties of umami peptides[117]. Finally, the promotion and marketing of marine umami peptide products can benefit from the application of big data technology for consumer behavior analysis. By analyzing consumer data and market trends, a better understanding of consumer needs and preferences can be obtained, which in turn can guide the promotion and marketing strategies of marine umami peptide products. This approach allows for a more targeted and effective marketing strategy that is based on empirical evidence and insights into consumer behavior[118]. From a functional perspective, the application of big data in marine umami peptides primarily involves the collection of various relevant data. Therefore, the challenges of applying big data in marine umami peptides lie in the capacity, source, and quality of the data[119].

    Cloud computing

    Cloud computing technology is an internet-based computing model, which provides a variety of computing services through the network, including storage, database, software, analysis and processing capabilities. The application of cloud computing in the development and utilization of marine umami peptides are still in their early stages. The application might include the following aspects: (1) Data storage and processing: the study of marine umami peptides requires a large amount of data storage and processing, including gene data and protein data of marine biological samples. Cloud computing can provide efficient data storage and processing capabilities to help researchers quickly analyze the components and characteristics of marine umami peptides. (2) Virtual experiments and simulations: through the cloud computing platform, researchers can conduct virtual experiments and simulations to explore the biological activities, pharmacological effects and other characteristics of marine umami peptides, to accelerate research progress. Although the function of cloud computing has greatly facilitated the discovery of new marine umami peptides, the nature of cloud computing also brings challenges for cloud computing applications[120,121]. The main challenges of cloud computing in marine umami peptide is data security[19]. Because the cloud computing provider has complete control over all operations, malicious detection, service manipulation and economic denial-of-service attacks by the provider are the potential threats of data being damaged or breached[121].

    Blockchain

    Blockchain technology is a distributed database-based technology that is capable of recording and storing transaction data. It possesses the characteristics of decentralization, non-tampering, and transparency. At its core, blockchain technology consists of a chain structure composed of a series of blocks, with each block containing a specific amount of transaction data. The security and consistency of the data are ensured through encryption algorithms and consensus mechanisms[122]. Blockchain technology has a wide range of applications in the downstream management of marine umami peptides. Firstly, through the use of blockchain technology, it is possible to achieve full traceability of marine umami peptide products. This means that every stage in the production, processing, and transportation of each batch of products can be recorded on the blockchain. As a result, consumers can access detailed information about the product such as its source, production date, and production process by scanning the two-dimensional code on the product or querying relevant information. This increased transparency and credibility serves to enhance consumer confidence in the product[123]. Secondly, the authenticity of marine umami peptide products can be verified using blockchain technology. Each batch of products is assigned a unique blockchain identity, allowing consumers to verify the product's authenticity by scanning the two-dimensional code or entering relevant information. This ensures that the purchased product is genuine[124]. Thirdly, supply chain management: Blockchain technology has the potential to enhance the supply chain management of marine umami peptide products[125]. By integrating all aspects of the supply chain into a blockchain network, real-time monitoring and transparent management can be achieved. This reduces information asymmetry and risk within the supply chain while improving efficiency and reliability[126]. The high-cost problem is a significant challenge when applying blockchain technology in the marine umami industry. Embedding blockchain technology into the marine umami peptide traceability system requires a substantial investment of both time and money for participants. As the complexity of the blockchain increases, so does the need for additional computing power to confirm more blocks, resulting in higher power consumption[127]. Furthermore, as the number of transactions within the marine umami peptide tracking system grows, so does the volume of data. This increase in data creates challenges related to storage and computation, ultimately reducing the capacity scale of the system and increasing synchronization time for new users[127,128].

    Conclusions and perspectives

    As a treasure house of natural resources, the ocean contains a huge amount of marine proteins. The preparation of umami peptides from these marine proteins is an important way to utilize ocean resources. This review summarized the amino acid composition, characteristics, and sequence information of the reported 141 marine umami peptides. Additionally, the taste mechanism and preparation methods of marine umami peptides were introduced. According to the current research results, most marine peptides are composed of four to ten amino acids and abundant in umami/hydrophobic amino acids. The preparation methods of marine umami peptides include microbial fermentation, enzymatic hydrolysis, acid hydrolysis, high-temperature processing, and solvent extraction. Each method has its advantages and disadvantages, better results may be obtained by using the combined methods. At the same time, embedding methods and amino acid addition were commonly used to adjust umami taste, which had the advantages of simple operation and was suitable for industrial production. Finally, the existing ongoing projects regarding the application of Industry 4.0 technology in marine umami peptides were introduced and the future trends of its application prospected. Based on the current state of research, several Industry 4.0 technologies (molecular simulation, artificial intelligence, and big data) have been widely utilized in simulating the interaction between the marine umami peptides and their receptors, rapidly screening and predicting the structure of marine umami peptides, as well as establishing and sharing a database of marine umami peptides. The use of cloud computing and blockchain is still in its early stages. Prospective applications may include the collection and processing of characteristic data, traceability analysis of marine umami peptides etc. Essentially, the core technology of Industry 4.0 primarily serves as information storage and data processing tools that greatly enhance researchers' work efficiency. Furthermore, in order to better understand the properties and applied research of marine umami peptides, it is necessary to encourage and stimulate more investment in biotechnology to promote the sustainable development and utilization of these marine resources in the future.

    Author contributions

    The authors confirm their contribution to the paper as follows: data curation: Hu D, Zheng Z, Liang B, Jin Y, Shi C, Chen Q, Wei L; writing-original draft: Hu D, Zheng Z; writing-review & editing: Dong X, Lu Y; conceptualization: Jin Y, Shi C, Lu Y; literature arrangement: Chen Q, Wei L; software, investigation: Li D, Li C; project administration, resources: Ye J, Dai Z; funding acquisition: Lu Y. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article.

    Acknowledgments

    The authors are grateful to the National Key Research and Development Program of China (2023YFD2100203), the Natural Science Fund of Zhejiang Province (LQ22C200008) and Basic research funds for provincial colleges and universities (FR2401ZD).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
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Identification and characterization of grape VAP27 gene family and their roles in disease resistance

  • 1.

    State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • 2.

    Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • Received: 24 January 2024
  • Revised: 03 April 2024
  • Accepted: 24 April 2024
  • Published online: 17 May 2024
Fruit Research  4,  Article number: e019  (2024)  |  Cite this article

Abstract: Vesicle-associated membrane protein (VAMP)-associated proteins (VAP27s), which are widely expressed in plants and animals, play an important role in metabolism, physiology, growth, and development, disease resistance, and immunity. While the function of this family has been elucidated in model plants like Arabidopsis thaliana and tomato, its role in grapevine remains unclear. In this present study, 12 vesicle-associated protein-membrane protein genes were identified in the grapevine genome by bioinformatics, designated as the VAP27 gene family. A phylogenetic tree, encompassing 53 genes from three model plants, Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum, revealed the subdivision of the VAP27 gene family into three subfamilies, each presumably serving different functions, besides localizing in endoplasmic reticulum, individual members also localize in nucleus. Additionally, we compared the transcriptional levels and subcellular localizations of the VvVAP27 family members across different plant tissues (flower, leaf, seed, root, fruit, tendril, and stem), indicating site-specific functionalities for different gene members. To investigate the responsiveness of the VAP27 gene family to pathogen infection, particularly Plasmopara viticola on host plants, we analyzed the expression patterns of VAP27 genes post-infection. Our findings revealed divergent expression profiles among different members at different stages of infection. The gene family responded to the infection of downy mildew on grapevine and could inhibit the spread of Phytophthora capsici lesions in Nicotiana benthamiana. These results provide an important basis for further studies delving into the functions of the VAP27 gene family in plant growth and disease resistance.

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    Introduction

    • The vesicle-associated membrane protein (VAMP)-associated protein (VAPs) family has been identified as a highly conserved group of proteins identified both in plants[1] and animals[2]. It was first identified in animals participating in the transportation of related substances by forming SNARE protein complexes[3]. Their plant homologues are named VAP27 because the first member identified had a molecular weight of 27 kDa[4]. The structure of VAPs usually contains an N-terminal major sperm domain (aa 1–129), a coiled-coil domain (aa 178–234), and a C-terminal transmembrane domain (aa 234–253)[4]. It is reported that the N-terminal sperm domain is crucial for the interaction between VAP27-1 and NET3C, and the fixation of VAP27-1 in the ER-PM contact site[5]. In Arabidopsis, 10 VAP homologs have been identified[1]. To date, numerous research on VAPs have been reported in the plant kingdom, and increasing proteins relevant to VAPs have also been discovered[5−8]. For example, NET3C and VAP27 form homo-dimers or homo-oligomers that function in mediating the communication of PM and ER via interacting with actin and microtubules for lipid transport, calcium influx, and other vital biological processes[5]. VAP27-1 and VAP27-3 have been revealed to interact with clathrin and play a central role in maintaining clathrin homeostatic dynamics at endocytic membranes and regulating endocytosis[8].

      According to existing studies, VAP proteins family localize to the endoplasmic reticulum (ER) and ER/plasma membrane (PM) contact sites and are tightly linked to the cytoskeleton that plays a supporting role[9−12]. The endoplasmic reticulum (ER), as one of the important components of the intimal system, plays an indispensable role in protein synthesis, folding and quality control, protein secretion, lipid biosynthesis, and calcium storage[13,14]. The ER is also actively involved in endocytosis with mechanisms that have not been clearly defined[15]. The transportation and translocation of various proteins, steroids, lipids, and other synthesized molecules typically depend on ER-centered traditional vesicular trafficking pathways[16,17]. The close association between the ER and the PM, facilitated by proteins like VAPs, is essential for vesicle trafficking from the ER. Given the intimate connection between the VAP family and the plasma membrane, researchers propose VAP interactions with proteins involved in plant development and maintaining structural stability. This explains the requirement of VAPs for growth, cell division, and abiotic stress responses[18−22]. Recent research in Arabidopsis thaliana also supports this idea, revealing that SYT1, an ER-resident protein[23], plays a vital role in stabilizing the ER network and connection between VAP27-1-enriched ER and plasma membrane[24]. VAMP721/722 are components of the default secretory pathway and can transport substances required for cell growth, suggesting a potential role in plant autoimmune regulation.

      There have been studies demonstrating that the plasma membrane participates in the secretion of immune protein for the activation of plant immune defense against pathogen invasion. For instance, the antimicrobial proteins secreted through vesicle trafficking was targeted and destroyed by the RxLR effector of Phytophthora brassicae by working together with host RABA-type GTPase, subsequently compromising the immune system[25]. There are also reports indicating that the VAP protein family could influence the development of various plants and defense networks. The immune mechanism of the VAP protein family in Arabidopsis thaliana and tomato has been confirmed[20,22].

      Grapevine (Vitis vinifera L.) is distinguished as one economically valuable fruit, appreciated both for fresh consumption and the production of various processed items such as wine and grape juice. Environmental stresses can seriously affect grapevine growth and development in cropland. For instance, high humidity on prolonged rainy days during critical maturation stages can compromise the quality of grapevine, while drought conditions can drastically reduce fruit yields. Additionally, biotic stresses, such as downy mildew, powdery mildew, anthracnose, and others[26,27], pose threats by impeding normal leaf growth and causing yield losses. Given its substantial economic importance, grapevine cultivation is widespread across various countries. The identification of significant functional genes becomes of utmost interest.

      At present, research on the VAP27 protein is limited, particularly in the context of grapevine. Therefore, in this study, we identified and analyzed the VAP27 protein family through bioinformatic analysis of genomic and transcriptomic data. The structure and function of the VAP27 gene were preliminarily analyzed, laying the foundation for further study of gene functionality.

    Materials and methods

      Identification and annotation of VAP27s in grape

    • The grape genome sequences of Vitis vinifera cv. 'Pinot Noir' (PN40024.v4) were downloaded from Ensemble Plants (https://plants.ensembl.org/Vitis_vinifera/Info/Index). Initial identification involved querying the grape genome database using the Arabidopsis VAPs protein sequences through BLAST. Next, an HMM file was constructed using the seed alignment file for the VAP domain (PF00635) obtained from the Pfam database, utilizing the HMMER3 software package. HMM searches were then performed against local protein databases of grape sequencing using HMMER3. To ensure accuracy, the physical localizations of all candidate Vitis vinifera VAP27s (VvVAP27s) on chromosomes were examined, and redundant sequences with identical chromosome locations were excluded. All obtained VAP27 protein sequences were subjected to Pfam analysis (http://pfam.xfam.org/) to verify the DBD domain. The presence of DBD domains and coiled-coil structures was confirmed using SMART (http://toolkit.tuebingen.mpg.de/marcoil) and MARCOIL (http://toolkit.tuebingen.mpg.de/marcoil). Sequences lacking the DBD domain or a coiled-coil structure were eliminated from further analysis.

      Phylogenetic, exon-intron structure, and motif analysis of VAP27 Genes

    • Genome sequences, CDS sequences, and protein sequences of the VAP27 family were downloaded for analysis. An unrooted phylogenetic tree was constructed for sequences from grapes, Arabidopsis thaliana, tomatoes, and rice using the Neighbor-Joining (NJ) method with the bootstrap test replicated 1,000 times. The software used for creating these phylogenetic trees was MEGA5. The exons and introns of grape VAP27 genes were determined based on alignments of transcribed sequences and corresponding genomic sequences, and the visualization of VAP27 gene structures was performed with the online Gene Structure Display Server 2.0. Conserved motifs and domains of grape VAP27 genes were identified using MEME 4.11.2 (http://meme-suite.org/tools/meme) and SMART (http://toolkit.tuebingen.mpg.de/marcoil) software.

      Transcriptome analysis of VvVAP27 gene

    • Using published data[28], the expression patterns of VvVAP27 gene family at 54 stages of grape plant development were analyzed using the average expression values of three biological replicates. Clustering analysis plots from RNA-seq datasets were created using FPKM (fragments mapped per kilobase read per million times) values. The expression heatmap of the grape VvVAP27 gene family was drawn using TBtools.

      Plant materials and treatments

    • The grapevine materials used in this study are Vitis vinifera 'Pinot Noir' and Vitis piasezkii 'Liuba-8', cultured in the Grape Repository of Northwest A&F University, Yangling, Shaanxi, China. The P. viticola population was collected from the susceptible Vitis plants as per previous studies[29−31]. Briefly, infected leaves were collected and washed in sterile distilled water three times. The leaves were positioned with the abaxial side facing up on sterile moist filter papers in trays and incubated overnight at room temperature to allow P. viticola sporulation. Leaves on which fresh sporangia developed were transferred into a large Petri dish and washed gently with sterile distilled water. The sporangium suspension was filtered with three-layer sterilized gauze. The concentration was adjusted to 5 × 104 sporangia/mL using a hemocytometer under a light microscope.

      For inoculation, the third to fifth fully expanded leaves from the top were detached and washed three times in sterile distilled water, and inoculated with 10 μL drops of the sporangia suspension on the abaxial leaf surface. The inoculated leaves were placed on sterile Petri dishes (90 mm in diameter) containing three-layer sterile moist filter papers and incubated in an incubator at 23 ± 1 °C, 90% relative humidity, and a photoperiod of 16 h light and 8 h dark. Samples were collected at 0, 6, 12, 24, 48, 96, and 120 h post-inoculation (hpi), with 0 hpi as the control samples. The collected samples were promptly frozen in liquid nitrogen and stored at −80 °C. Each biological replicate was a pool of three independent leaves. The tobacco plant material, N. benthamiana, was routinely grown at 25/20 °C in a greenhouse under white light (18 h light/6 h dark).

      RNA extraction and RT–qPCR

    • Total RNA was extracted from grapevine leaves using the RNA Mini Kit (Omega, USA) following the manufacturer's instructions. The EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, China) was used to perform reverse transcription and synthesize double-stranded cDNAs. In the reverse transcription, 500 ng RNA was used with the Anchored Oligo (dT) 18 as the primer. The remaining reaction components included 10 μL of 2× ES Reaction Mix, 1.0 μL of gDNA Remover, 1.0 μL of EasyScript® RT Enzyme Mix, and sterile distilled H2O were added to reach a final volume of 20 μL. The reaction was carried out at 42 °C for 15 min, 85 °C for 10 s. Quantitative PCR was performed on an Applied Biosystems QuantStudio 6 (Thermo Fisher Scientific, USA) with PerfectStart® Green qPCR SuperMix (TransGen Biotech, China), according to the recommended protocol. In brief, each reaction mixture contained 10 μL of 2× TransStart Top Green qPCR SuperMix, 2.0 μL of cDNA template, 0.5 μL of each primer, and 8.0 μL of sterile distilled H2O. Cycling parameters included an initial step at 50 °C for 2 min and 94 °C for 30 s, followed by 45 cycles at 95 °C for 5 s, 59 °C for 15 s, and 72 °C for 30 s. Melt-curve analyses were performed with a program starting at 95 °C for 15 s and then a constant increase from 60 to 95 °C. Data were analyzed by the 2−ΔΔCT method for calculating gene relative expression levels with three biological replicates. Gene-specific primers were designed using Primer Premier 5.0 software and gene transcripts were normalized to VvActin as internal standards.

      Transient transformation of Agrobacterium tumefaciens

    • The pCambia2300-VAP27s-GFP construct was introduced into the A. tumefaciens GV3101 strain, and recombinant colonies were verified through growth on a selective medium and PCR analysis. For Agrobacterium-mediated transient transformation assays, bacterial cells were collected by centrifugation and then resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone). The bacterial suspension was diluted to a concentration of OD600 = 0.6 and incubated for 3 h at 28 °C before infiltration. Infiltration on tobacco leaves was carried out using a syringe without a needle.

      Location analysis

    • Laser confocal microscopy was employed to determine the localization of the VAP27 gene family in plants. The VAP27 gene was inserted into the pCambia2300-GFP vector and the recombinant plasmid was confirmed by Sanger sequencing. Then the recombinant plasmid was transformed into the Agrobacterium strain GV3101. The monoclonal plaque was amplified in liquid culture and confirmed by PCR analysis. For visualization, the ER-RK marker was co-injected with VAP27-GFP into the N. benthamiana leaves. The transformed N. benthamiana leaves were observed using confocal microscopy (TCS SP8 of Leica). The excitation wavelength for green fluorescent protein was set to 488 nm.

      Phytophthora capsici inoculation

    • The mycelium of P. capsici was first cultivated on 10% V8 juice agar medium and then was transferred into 10% liquid V8 medium and cultured in 25 °C darkness for 5 d. The developed hyphae were collected and resuspended in sterile water at 4 °C for 30 min, followed by incubation at room temperature for 30 min to allow the release of zoospores from sporangia. The resulting sporangial suspension was adjusted to 1.0 × 104 sporangia/mL.

      For the P. capsica infection experiment, the A. tumefaciens containing certain plasmids were injected into N. benthamiana. At 48 h post-infection, the inoculated leaves were detached, and their petioles were first wrapped in sterile cotton and then wrapped in two layers of sterile wet filter paper. Then the leaves were first treated using 0.1% Tween-20, followed by inoculation with 30 μL zoospore suspension of P. capsica. The infected leaf samples were kept in the dark at 25 °C to allow the P. capsica development. The lesion area was statistically analyzed.

    Results

      Identification and genomic distribution of the VAP27 family in the grape genome

    • A total of 12 VAP27 genes in the grape genome were identified, designated as Vitis vinifera VAP27 (VvVAP27)1−12 according to their chromosomal positions (Table 1). The genomic distribution revealed an uneven mapping of VAP27s on eight out of the 19 grape chromosomes. Specifically, VvVAP27-1, VvVAP27-2, VvVAP27-3, VvVAP27-4, VvVAP27-7, and VvVAP27-10 were located on Chromosome 2, Chromosome 5, Chromosome 8, Chromosome 12, Chromosome 14, and Chromosome 19, respectively. VvVAP27-5 and VvVAP27-6 were situated on Chromosome 13, while VvVAP27-8 and VvVAP27-9 were positioned on Chromosome 15. VvVAP27-11 and VvVAP27-12 were putatively located on the 'Chromosome Unknown'. Further study will delve into unraveling the biological functions of these 12 VAP27 genes.

      Table 1.  Chromosome distribution of identified 12 grapevine VAP27 genes. Detailed information, including gene locus, gene symbol, length, chromosome, and site is available in the Ensembl Plants Database.

      Protein nameGene IDChrLength (aa)Annotation
      VvVAP27-1Vitvi02g005452238PREDICTED: vesicle-associated protein 1-1
      VvVAP27-2Vitvi05g003605293PREDICTED: vesicle-associated protein 1-2
      VvVAP27-3Vitvi08g001378532PREDICTED: ankyrin-1
      VvVAP27-4Vitvi12g0063812259PREDICTED: vesicle-associated protein 4-1
      VvVAP27-5Vitvi13g0009913470PREDICTED: ankyrin repeat, PH and SEC7 domain containing protein secG
      VvVAP27-6Vitvi13g0085013136PREDICTED: hypothetical protein VITISV_015240
      VvVAP27-7Vitvi14g0034714336PREDICTED: vesicle-associated protein 2-2
      VvVAP27-8Vitvi15g0067715239PREDICTED: vesicle-associated protein 1-2
      VvVAP27-9Vitvi15g0071315239PREDICTED: vesicle-associated protein 1-3
      VvVAP27-10Vitvi19g0030419264PREDICTED: vesicle-associated protein 4-1
      VvVAP27-11Vitvi10g04245Un264PREDICTED: vesicle-associated protein 4-2
      VvVAP27-12Vitvi00g04146Un348PREDICTED: LOW QUALITY PROTEIN: vesicle-associated protein

      Phylogenetic analysis of the VAP27 gene family

    • To elucidate the evolutionary relationships within the VAP27 gene family, we conducted a comprehensive analysis involving 53 VAPs, including 10 from Arabidopsis, 17 from rice, 15 from tomato, and 12 from grape, and the result was visualized by constructing a phylogenetic tree (Fig. 1). The 53 VAP27 members across these four species fell into three distinct groups (Fig. 1 Clade I−III). Clade I, consisted of VvVAP27-1, VvVAP27-6, VvVAP27-8 and VvVAP27-9 gene. Clade II contained 3 VvVAP27 members: VvVAP27-2, VvVAP27-7 and VvVAP27-12. Clade III emerged as the most populated, encompassing five VvVAP27 members: VvVAP27-3, VvVAP27-4, VvVAP27-5, VvVAP27-10, and VvVAP27-11.

      Figure 1. 

      Unrooted phylogenetic tree of VAP27s in grape, Arabidopsis, rice, and tomato. Vv: Vitis vinifera L. grape species; AT: Arabidopsis thaliana; OS: Oryza sativa; Soly: Solanum lycopersicum.

      The clustering patterns suggest a closer evolutionary proximity of the VAP family in grapes to that of dicotyledon tomatoes compared to rice. This supports the reliability of the analysis results.

      Structural analysis of grape VAP27 genes

    • The gene structures of the 12 grapevine VAP27s were explored through a comprehensive examination of exon/intron boundaries. The varying length and splicing patterns observed among the 12 VAP27s are depicted in Fig. 2a. The structural analysis showed a range of intron numbers from 1 to 7. Notably, VvVAP27-3, VvVAP27-6, and VvVAP27-7 were absent of introns, while VvVAP27-5 exhibited a singular intron. The remaining VAP27 member's genes had between six and seven introns (Fig. 2a). The results revealed significant diversity within the VAP27 family.

      Figure 2. 

      (a) Intron-exon structure and (b) conserved motifs of VvVAP27.

      According to previous studies, motifs recognized as playing an important role in interaction and signal transduction within the transcriptional complex[32,33] were analyzed using MEME for the 12 conservative VAP27 genes. This was conducted also because these motifs are closely related to gene classification. Among these VAP27s, a total of 10 motifs were identified (Fig. 2b), with Motif 1 present in all 11 members except VAP27-12, which indicates its high conservation within the VAP27 gene family. Motif 8 and Motif 9 were the least conserved, found only in VvVAP27-4, VvVAP27-10, and VvVAP27-11 (Motif 8), and VvVAP27-1, VvVAP27-8, and VvVAP27-9 (Motif 9). The high sequence similarity among genes within the same branch suggests shared functions and roles in plants. The analysis of motif and gene structure analysis enrich our understanding of the VAP27 family's classification, providing a robust theoretical foundation.

      Expression profiles of VAP27s in different tissues

    • VAP27 RNAi induces various defects in plant morphology, pollen, seed, and root development in Arabidopsis[1], we anticipated a similar involvement of the VvVAP27 gene in grape growth and development. To investigate this, we examined the expression profiles of the 12 VAP27 genes across different tissues of grapevine (Fig. 3). These tissues represented distinct growth and development stages of grapevine, including root, young stem, leaf, inflorescence, skin, veraison berry, and tendril. Examination of transcriptome data from the VvVAP27 family revealed significant variations across different tissues. The majority of family members (VvVAP27-1 to VvVAP27-10) exhibited comparable expression levels in tissues including flowers, berries, leaves, stems, seeds, and shoots, suggesting their involvement throughout various stages of plant growth and development. Only a subset of genes (VvVAP27-11 and VvVAP27-12) showed significant differences in expression among tissues. Expression of the VvVAP27-11 gene was higher in the berries than in the other tissues that maintained relatively consistent levels. In contrast, VvVAP27-12 showed transcriptional peaks exclusively in seeds and flowers, suggesting a potential association of VvVAP27-12 with flowering and fruit development.

      Figure 3. 

      Expression profiles of the grape VvVAP27s gene. Hierarchical clustering of expression profiles of grape VAP27 genes across different tissues.

      Expression patterns of VAP27 genes post-plasmopara viticola inoculation

    • It has been documented that the VAP27 gene family is involved in regulating plant disease resistance against external pathogen infection[6,18−22]. Therefore, we explored whether the VAP27 family exhibits similar functionality in grapevine downy mildew resistance. Our investigation focused on the expression levels of 12 VAP27 gene members at eight time points post-downy mildew inoculation (0, 6, 12, 24, 48, 72, 96, and 120 hpi). Utilizing RT-qPCR, we assessed whether the VAP27 gene responded to the induction of Grape downy mildew (Fig. 4). Vvactin1 was used as a grapevine internal reference gene for normalization[25].

      Figure 4. 

      Heat map showing the expression profiles of VvVAP27 genes at different time points post-downy mildew infection. The color scale represents expression levels, with red indicating high expression level and blue indicating low expression level. The expression was normalized and the data are displayed as log2 values.

      We discovered that genes in the VvVAP27 family genes exhibited specificity in responses to downy mildew induction (Fig. 4). There were a few members significantly overexpressed at the early stage of downy mildew infection in 'Liuba-8'. Only VvVAP27-6 had the highest expression level at 48 hpi compared to other time points. VvVAP27-2 was highly expressed at 120 hpi, with no difference found at other time points. VvVAP27-3, VvVAP27-6, VvVAP27-11, and VvVAP27-12 genes were highly expressed throughout the downy mildew infection period in 'Pinot Noir'. This indicated that these four genes were positively responsive to the induction of downy mildew, suggesting an important role in the grapevine's defense against downy mildew invasion. The expression levels of the other eight genes remained unchanged across different infection periods. We postulate that the varying expression patterns among different members may be related to the regulation of VAP27-mediated plant disease resistance, possibly involving distinct mechanisms of immunity. However, further experimental verification is needed to substantiate these hypotheses.

      Subcellular localization of VvVAP27 genes

    • To better explore the function of the VvVAP27 gene family, subcellular localization analysis was conducted on some selected VvVAP27 genes. By detecting GFP-tagged proteins, we found that most of the genes were localized to the endoplasmic reticulum (ER). This localization aligns with previous literature reports indicating that membrane proteins of vesicle-associated proteins function by participating in the formation and regulation of plant cell membranes. The endoplasmic reticulum participates in the formation of cell membranes and is closely related to secretory vesicles that function in transporting secretory proteins to various parts of the plant to contribute to plant growth, development, and disease resistance[34,35]. VvVAP27-2, VvVAP27-4, VvVAP27-6, and VvVAP27-9 are all mapped to the endoplasmic reticulum. However, VvVAP27-2, VvVAP27-4, and VvVAP27-6 are also detected in the nucleus in addition to the ER (Fig. 5). Based on the analysis of the expression pattern induced by downy mildew and the Phytophthora capsici infection experiment, we speculate that the subcellular localization may affect the gene expression.

      Figure 5. 

      Subcellular localization analysis of VvVAP27 members.

      Response of VAP27 gene to P. capsici inoculation

    • To further study the role of VAP27 genes in disease resistance, the VvVAP27 genes that were induced by grape downy mildew in tobacco leaves were screened. These tobacco leaves were transiently transformed by A. tumefaciens that carry a high-level expression vector with an individual VvVAP27 gene insert before being inoculated with P.capsici spore suspension. The findings revealed that VAP27 gene members inhibited the occurrence of the pathogenicity and significantly enhanced the resistance of tobacco leaves to the pathogen. However, the efficacy of pathogen inhibition varied among different VAP27 members. This result is consistent with previous studies on gene responses to downy mildew infection.

      Specifically, both VvVAP27-6 and VvVAP27-9 exhibited a consistent phenotype, inhibiting infection by pathogens (Fig. 6a). Leaf lesion areas were smaller in VvVAP27-6 and VvVAP27-9 expressing leaves compared with controls, suggesting that they effectively promoted plant immunity (Fig. 6b) and that VvVAP27-6 had a higher inhibitory capacity than VvVAP27-9. VvVAP27-2 and VvVAP27-4, did not differ significantly in the size of the lesion area compared with empty-carrier controls. The heterogeneous functions within this family underscore the need for further experimental studies to elucidate the roles of the remaining genes.

      Figure 6. 

      Responses of VvVAP27 induced by Phytophthora capsici infection. The inoculation of Phytophthora capsici was conducted on Nicotiana benthamiana leaves transiently transformed with VvVAP27. (a) Observation of the phenotypes of VvVAP27 family members and the control after inoculation with Phytophthora capsici, visualized by trypan blue staining. (b) Statistical analysis of the lesion areas caused by Phytophthora capsici infection on Nicotiana benthamiana leaves transiently transformed by VvVAP27. (c) The expression of VvVAP27 family members and GFP protein was detected by Western blot. The experiment was repeated three times and asterisks represent the level of significant differences (* p < 0.05, ** p < 0.01).

    Discussion

    • Vesicle-associated membrane proteins (VAMP-associated proteins) (VAPs) are a family of proteins widely expressed in plants, which play a key role in plant defense against both biotic and abiotic stresses. In this study, a family of 12 VAP27 genes were identified in grapes using bioinformatic methods. Consistent with previous studies, the VAMP gene family has demonstrated multifaceted involvement in diverse defense processes across different plant tissues. VAP27-1 and VAP27-3, as non-plant VAP homologs[5], have been localized extensively to the ER and EPCS[1,5,16]. These proteins were identified to promote plant endocytosis and play a role in endocytosis. In Arabidopsis thaliana, VAMP721/722 have been identified as essential factors for growth, cell division, and responses to abiotic stress[18−22]. The PEN1-SNAP33-VAMP721/722 pathway in Arabidopsis thaliana facilitates the transport of vital materials for cell viability to the endoplasmic reticulum[36,37]; VAMP721 and VAMP722 are involved in the secretory transport of substances to endosomal compartments of the plasma membrane to promote the formation of cell plates during plant cytokinesis[6]. The phylogenetic tree analysis revealed a notable similarity in the quantity and gene structure of the VAP27 gene family between grape, Arabidopsis thaliana, and tomato. Hence, it can be inferred that the VAP27 gene family in grape shares similar functions with those in Arabidopsis thaliana[36], and plays comparable roles in growth, development, and immune resistance mechanisms. Additionally, we analyzed the gene structure of 12 identified VvVAP27 genes using MEME. Results (Fig. 2) showed that most of the VvVAP27 genes (VvVAP27-1, VvVAP27-2, VvVAP27-3, VvVAP27-6, VvVAP27-7, VvVAP27-8, VvVAP27-9) contain conserved domains, especially VvVAP27-3 and VvVAP27-5; VvVAP27-1, VvVAP27-8 and VvVAP27-9; VvVAP27-4, VvVAP27-10 and VvVAP27-11 placed within the same group in the phylogenetic tree classification (Fig. 1). This observation may explain the specific biological functions associated with each subfamily. The analysis of motif and gene structure provides a further theoretical basis for the classification of the VvVAP27 subfamily, guiding subsequent in-depth functional studies within these identified groups.

      To understand the regulatory mechanisms of the VAP27 gene family on the growth and development of grapevine, we analyzed the regulatory effect of the VvVAP27 gene family on the growth and development of grapevine using transcriptome content assay (Fig. 3) across different grape tissues (flowers, seeds, leaves, buds, berries, tendrils, stems, roots). Our transcriptome data analysis showed the highest expression of VvVAP27-11 in berries than in other tissues, implying its potential role in regulating fruit development and quality and promoting fruit setting. Similarly, the expression levels of VvVAP27-12 were higher in flower and seed than in other tissues, indicating that VvVAP27-12 plays an important role in flower induction, seed setting, and growth regulation. This study provides a theoretical basis for further understanding the function of the VAP27 family members in the process of grape growth and development.

      It has been found that the VAP gene family can induce plant cell autonomous immunity, acting on the cell surface or post-pathogen entry, thereby impeding pathogenesis. This phenomenon has been well-documented in various plant species, including tomato, Arabidopsis, and tobacco. In tobacco, VAPB proteins interact with proteins in the intestine of tobacco whitefly (Bemisia tabaci) during the transmission of tomato yellow leaf curl virus (TYLCV), and silencing VAPB results in an increase in virus number and transmission rate, demonstrating that VAPB can play a key role in resistance to TYLCV[4]. Moreover, in Arabidopsis, the SNARE proteins VAMP 721/722 direct secretory vesicles to pathogen-attack sites during immune responses, indicating that these vesicles deliver immune molecules and function in immune responses[36]. Additionally, SYP132, an essential protein for defense against bacterial pathogens, specifically interacts with VAMP721/722 in response to the immune control of P. syringae[21]. Given these findings, we were intrigued by the possibility that the VAP27 gene family might play a similar role in disease resistance in grapes. Grapevine downy mildew, caused by the oomycete P. viticola, is one of the most serious diseases in grape production. P. viticola was originally endemic to North America, but it has now spread to all major grape-producing regions worldwide[37]. This study revealed that the majority of VvVAP27 members exhibited responses to downy mildew infection. Notably, VvVAP27-1, VvVAP27-2, VvVAP27-4, and VvVAP27-6 were significantly up-regulated at 0, 6, and 12 h after infection, and VvVAP27-12 was highly expressed at 24, 48, and 72 h after infection with P. viticola, while other genes were highly expressed only at specific stages. These findings suggest that members of the VAP27 gene family are likely to respond to downy mildew infection. Our results provide a new idea to study the effect of the VAP27 gene on grapevine downy mildew, but further research is needed to study the mechanisms of VAP27 gene action.

      In this study, we identified a new grape gene family, named the VAP27 gene family. Through bioinformatic analysis and transcriptome sequencing, we uncovered striking structural and functional similarities between the VAP27 gene family in grape and those in Arabidopsis thaliana and tomato. The current findings suggest a potentially significant role for this gene family in the growth and development of the grape, as well as in orchestrating immune responses against downy mildew.

    Conclusions

    • The vesicle-associated protein-membrane protein gene family (VAP27) in the grape genome was identified, which consists of 12 gene members. Within this family, some members exhibit localization on the endoplasmic reticulum, and a minority reside within the nucleus. The present results demonstrate the induction of the gene family in response to downy mildew in grape and their ability to inhibit the infection of P. capsici, thus playing an important role in plant disease resistance.

    Author contributions

    • The authors confirm contribution to the paper as follows: conceptualization, writing – review & editing: Xu Y, Liu G; validation, visualization, writing – original draft: Li R, Wang B; investigation: Li R, Wang B, Zha M, Zhang K, Li M, Xie L, Chen X, Liu G; funding acquisition: Liu G. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article.

      Acknowledgments

      • This work was supported by grants from the National Natural Science Foundation of China (31972374, 32372660, and 31601716).

      Conflict of interest

      • The authors declare that they have no conflict of interest. Yan Xu is the Editorial Board member of Vegetable Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      • # Authors contributed equally: Ruonan Li, Bianbian Wang

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (1) References (37)
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    Cite this article
    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
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  • Introduction
  • Materials and methods

  • Identification and annotation of VAP27s in grape

  • Phylogenetic, exon-intron structure, and motif analysis of VAP27 Genes

  • Transcriptome analysis of VvVAP27 gene

  • Plant materials and treatments

  • RNA extraction and RT–qPCR

  • Transient transformation of Agrobacterium tumefaciens

  • Location analysis

  • Phytophthora capsici inoculation

  • Results

  • Identification and genomic distribution of the VAP27 family in the grape genome

  • Phylogenetic analysis of the VAP27 gene family

  • Structural analysis of grape VAP27 genes

  • Expression profiles of VAP27s in different tissues

  • Expression patterns of VAP27 genes post-plasmopara viticola inoculation

  • Subcellular localization of VvVAP27 genes

  • Response of VAP27 gene to P. capsici inoculation

  • Discussion
  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
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