Identification, Classification, and Transcriptional Analysis of Rab GTPase Genes from Tomato (Solanum lycopersicum) Reveals Salt Stress Response Genes.

Salinity in plants generates an osmotic and ionic imbalance inside cells that compromises the viability of the plant. Rab GTPases, the largest family within the small GTPase superfamily, play pivotal roles as regulators of vesicular trafficking in plants, including the economically important and globally cultivated tomato (Solanum lycopersicum). Despite their significance, the specific involvement of these small GTPases in tomato vesicular trafficking and their role under saline stress remains poorly understood. In this work, we identified and classified 54 genes encoding Rab GTPases in cultivated tomato, elucidating their genomic distribution and structural characteristics. We conducted an analysis of duplication events within the S. lycopersicum genome, as well as an examination of gene structure and conserved motifs. In addition, we investigated the transcriptional profiles for these Rab GTPases in various tissues of cultivated and wild tomato species using microarray-based analysis. The results showed predominantly low expression in most of the genes in both leaves and vegetative meristem, contrasting with notably high expression levels observed in seedling roots. Also, a greater increase in gene expression in shoots from salt-tolerant wild tomato species was observed under normal conditions when comparing Solanum habrochaites, Solanum pennellii, and Solanum pimpinellifolium with S. lycopersicum. Furthermore, an expression analysis of Rab GTPases from Solanum chilense in leaves and roots under salt stress treatment were also carried out for their characterization. These findings revealed that specific Rab GTPases from the endocytic pathway and the trans-Golgi network (TGN) showed higher induction in plants exposed to saline stress conditions. Likewise, disparities in gene expression were observed both among members of the same Rab GTPase subfamily and between different subfamilies. Overall, this work emphasizes the high degree of conservation of Rab GTPases, their high functional diversification in higher plants, and the essential role in mediating salt stress tolerance and suggests their potential for further exploration of vesicular trafficking mechanisms in response to abiotic stress conditions.

Stress salinity in plants: New strategies to cope with in the foreseeable scenario.

The excess of salts in soils causes stress in most plants, except for some halophytes that can tolerate higher levels of salinity. The excess of Na+ generates an ionic imbalance, reducing the K+ content and altering cellular metabolism, thus impacting in plant growth and development. Additionally, salinity in soil induces water stress due to osmotic effects and increments the production of reactive oxygen species (ROS) that affect the cellular structure, damaging membranes and proteins, and altering the electrochemical potential of H+, which directly affects nutrient absorption by membrane transporters. However, plants possess mechanisms to overcome the toxicity of the sodium ions, such as internalization into the vacuole or exclusion from the cell, synthesis of enzymes or protective compounds against ROS, and the synthesis of metabolites that help to regulate the osmotic potential of plants. Physiologic and molecular mechanisms of salinity tolerance in plants will be addressed in this review. Furthermore, a revision of strategies taken by researchers to confer salt stress tolerance on agriculturally important species are discussed. These strategies include conventional breeding and genetic engineering as transgenesis and genome editing by CRISPR/Cas9.

A SNARE-like protein from Solanum lycopersicum increases salt tolerance by modulating vesicular trafficking in tomato.

Intracellular vesicular trafficking ensures the exchange of lipids and proteins between endomembrane compartments. This is relevant under high salinity conditions, since both the removal of transporters and ion channels from the plasma membrane and the compartmentalization of toxic ions require the formation of vesicles, which can be maintained as multivesicular bodies or be fused to the central vacuole. SNARE proteins (Soluble N-ethylmaleimide-sensitive factor attachment receptor) participate in the vesicle fusion process and give specificity to their destination. Plant genome studies have revealed a superfamily of genes that encode for proteins called SNARE-like. These proteins appear to be participating in vesicular trafficking with similar functions to those of SNARE proteins. A SNARE-like, named SlSLSP6, in Solanum lycopersicum plants has been shown to be induced under high salinity conditions. A phylogenetic relationship of SlSLSP6 with SNARE-like proteins of salinity-tolerant plants, including Salicornia brachiata, Zostera marina and Solanum pennelli, was determined. Considering its amino acid sequence, a putative clathrin adapter complex domain and palmitoylation site was predicted. Subcellular localization analysis evidenced that SlSLSP6 is mostly localized in the plasma membrane. Using transgenic tomato plants, we identified that overexpression of SlSLSP6 increased tolerance to salt stress. This tolerance was evident when we quantified an improvement in physiological and biochemical parameters, such as higher chlorophyll content, performance index, efficiency of photosystem II and relative water content, and lower malondialdehyde content, compared to control plants. At the subcellular level, the overexpression of SlSLSP6 reduced the presence of H2O2 in roots and increased the compartmentalization of sodium in vacuoles during salt stress. These effects appear to be associated with the higher endocytic rate of FM4-64, determined in the plant root cells. Taken together, these results indicate that SlSLSP6 increases tolerance to salt stress by modulating vesicular trafficking through over-induction of the endocytic pathway. This work contributes to understanding the role of this type of SNARE-like protein during salt stress and could be a potential candidate in breeding programs for tolerance to salt stress in tomato plants.

The Exocytosis Associated SNAP25-Type Protein, SlSNAP33, Increases Salt Stress Tolerance by Modulating Endocytosis in Tomato.

In plants, vesicular trafficking is crucial for the response and survival to environmental challenges. The active trafficking of vesicles is essential to maintain cell homeostasis during salt stress. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are regulatory proteins of vesicular trafficking. They mediate membrane fusion and guarantee cargo delivery to the correct cellular compartments. SNAREs from the Qbc subfamily are the best-characterized plasma membrane SNAREs, where they control exocytosis during cell division and defense response. The Solanum lycopersicum gene SlSNAP33.2 encodes a Qbc-SNARE protein and is induced under salt stress conditions. SlSNAP33.2 localizes on the plasma membrane of root cells of Arabidopsis thaliana. In order to study its role in endocytosis and salt stress response, we overexpressed the SlSNAP33.2 cDNA in a tomato cultivar. Constitutive overexpression promoted endocytosis along with the accumulation of sodium (Na+) in the vacuoles. It also protected the plant from cell damage by decreasing the accumulation of hydrogen peroxide (H2O2) in the cytoplasm of stressed root cells. Subsequently, the higher level of SlSNAP33.2 conferred tolerance to salt stress in tomato plants. The analysis of physiological and biochemical parameters such as relative water content, the efficiency of the photosystem II, performance index, chlorophyll, and MDA contents showed that tomato plants overexpressing SlSNAP33.2 displayed a better performance under salt stress than wild type plants. These results reveal a role for SlSNAP33.2 in the endocytosis pathway involved in plant response to salt stress. This research shows that SlSNAP33.2 can be an effective tool for the genetic improvement of crop plants.

Identification and transcriptional analysis of SNARE vesicle fusion regulators in tomato (Solanum lycopersicum) during plant development and a comparative analysis of the response to salt stress with wild relatives.

Intracellular vesicular trafficking ensures the exchange of lipids and proteins between the membranous compartments. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) play a central role in membrane fusion and they are key factors for vesicular trafficking in plants, including crops economically important such as tomato (Solanum lycopersicum). Taking advantage of the complete genome sequence available of S. lycopersicum, we identified 63 genes that encode putative SNARE proteins. Then, phylogenetic analysis allowed the classification of SNAREs in five main groups and recognizing their possible functions. A structure analysis of the genes, their syntenic relationships and their location in the chromosomes were also carried out for their characterization. In addition, the expression profiles of SNARE genes in different tissues were investigated using microarray-based analysis. The results indicated that specific SNAREs had a higher induction in leaf, root, flower and mature green fruit. S. lycopersicum is characterized for being a crop sensitive to saline stress unlike its wild relatives, such as Solanum pennellii, Solanum pimpinellifolium, Solanum habrochaites or Solanum chilense, which are tolerant. In this context, we analyzed different microarrays and evaluated and validated the transcript levels through qRT-PCR experiments. The results showed that SlGOS12.2, SlVAMP727 and SlSYP51.2 could have a positive relationship with salt stress and probably an important role in their tolerance. All these data increase our knowledge and can also be utilized to identify potential molecular targets for conferring tolerance to various stresses in tomato.

The RabGAP Gene Family in Tomato (Solanum lycopersicum) and Wild Relatives: Identification, Interaction Networks, and Transcriptional Analysis during Plant Development and in Response to Salt Stress.

RabGTPase activating proteins (RabGAP) are responsible for directing the deactivation of vesicular trafficking master regulators associated to plant development, the RabGTPase proteins. Recently, RabGAPs were identified in Arabidopsis and rice, but studies were not yet reported in tomato. Herein, we identified 24 RabGAP-encoding genes in cultivated tomato (Solanum lycopersicum) and its wild relative genomes (Solanum pimpinellifolium and Solanum pennellii). We analyzed them based on their exon-intron structures, conserved protein motifs, putative subcellular localizations, phylogenetic and gene duplications analyses, interaction networks, and gene expression patterns in tomato. Phylogenetic relationship analysis also indicated that RabGAP family is classified into seven subclasses, of which subclasses I and II are plant-exclusive. Furthermore, segmental duplication events and positive evolutionary forces are associated with the maintenance of the number and function of their members. On the other hand, the protein-protein interaction networks on tomato suggested that members of subclasses I, II, and III could be associated to endocytic traffic routes. In addition, the qRT-PCR experiments in S. lycopersicum and Solanum chilense exposed to a salt stress treatment validated the differential expression patterns of 20 RabGAP genes in different tissues, development stages, and stress conditions obtained through extensive microarray-based analyses. This work suggests the critical role of RabGAP family in the context of intracellular vesicular trafficking in tomato, particularly under conditions of abiotic stress. It also contributes to the breeding programs associated with the development of crops tolerant to salt stress.

Genome-Wide Identification and Transcriptional Regulation of Aquaporin Genes in Bread Wheat (Triticum aestivum L.) under Water Stress.

Aquaporins (AQPs) are transmembrane proteins essential for controlling the flow of water and other molecules required for development and stress tolerance in plants, including important crop species such as wheat (Triticum aestivum). In this study, we utilized a genomic approach for analyzing the information about AQPs available in public databases to characterize their structure and function. Furthermore, we validated the expression of a suite of AQP genes, at the transcriptional level, including accessions with contrasting responses to drought, different organs and water stress levels. We found 65 new AQP genes, from which 60% are copies expanded by polyploidization. Sequence analysis of the AQP genes showed that the purifying selection pressure acted on duplicate genes, which was related to a high conservation of the functions. This situation contrasted with the expression patterns observed for different organs, developmental stages or genotypes under water deficit conditions, which indicated functional divergence at transcription. Expression analyses on contrasting genotypes showed high gene transcription from Tonoplast Intrinsic Protein 1 (TIP1) and 2 (TIP2), and Plasma Membrane Intrinsic Protein 1 (PIP1) and 2 (PIP2) subfamilies in roots and from TIP1 and PIP1 subfamilies in leaves. Interestingly, during severe drought stress, 4 TIP genes analyzed in leaves of the tolerant accession reached up to 15-fold the level observed at the susceptible genotype, suggesting a positive relationship with drought tolerance. The obtained results extend our understanding of the structure and function of AQPs, particularly under water stress conditions.

Involvement of SchRabGDI1 from Solanum chilense in endocytic trafficking and tolerance to salt stress.

Physiological responses of plants to salinity stress requires the coordinated activation of many genes. A salt-induced gene was isolated from roots of the wild tomato species Solanum chilense and named SchRabGDI1 because it encodes a protein with high identity to GDP dissociation inhibitors of plants. These proteins are regulators of the RabGTPase cycle that play key roles in intracellular vesicular trafficking. The expression pattern of SchRabGDI1 showed an early up-regulation in roots and leaves under salt stress. Functional activity of SchRabGDI1 was shown by restoring the defective phenotype of the yeast sec19-1 mutant and the capacity of SchRabGDI1 to interact with RabGTPase was demonstrated through BiFC assays. Expression of SchRabGDI1 in Arabidopsis thaliana plants resulted in increased salt tolerance. Also, the root cells of transgenic plants showed higher rate of endocytosis under normal growth conditions and higher accumulation of sodium in vacuoles and small vesicular structures under salt stress than wild type. Our results suggest that in salt tolerant species such as S. chilense, bulk endocytosis is one of the early mechanisms to avoid salt stress, which requires the concerted expression of regulatory genes involved in vesicular trafficking of the endocytic pathway.

Differential Roles for VviGST1, VviGST3, and VviGST4 in Proanthocyanidin and Anthocyanin Transport in Vitis vinífera.

In plant cells, flavonoids are synthesized in the cytosol and then are transported and accumulated in the vacuole. Glutathione S-transferase-mediated transport has been proposed as a mechanism involved in flavonoid transport, however, whether binding of flavonoids to glutathione S-transferase (GST) or their transport is glutathione-dependent is not well understood. Glutathione S-transferases from Vitis vinífera (VviGSTs) have been associated with the transport of anthocyanins, however, their ability to transport other flavonoids such as proanthocyanidins (PAs) has not been established. Following bioinformatics approaches, we analyzed the capability of VviGST1, VviGST3, VviGST4, and Arabidopsis TT19 to bind different flavonoids. Analyses of protein-ligand interactions indicate that these GSTs can bind glutathione and monomers of anthocyanin, PAs and flavonols. A total or partial overlap of the binding sites for glutathione and flavonoids was found in VviGST1, and a similar condition was observed in VviGST3 using anthocyanin and flavonols as ligands, whereas VviGST4 and TT19 have both sites for GSH and flavonoids separated. To validate the bioinformatics predictions, functional complementation assays using the Arabidopsis tt19 mutant were performed. Overexpression of VviGST3 in tt19-1 specifically rescued the dark seed coat phenotype associated to correct PA transport, which correlated with higher binding affinity for PA precursors. VviGST4, originally characterized as an anthocyanin-related GST, complemented both the anthocyanin and PA deposition, resembling the function of TT19. By contrast, VviGST1 only partially rescued the normal seed color. Furthermore the expression pattern of these VviGSTs showed that each of these genes could be associated with the accumulation of different flavonoids in specific tissues during grapevine fruit development. These results provide new insights into GST-mediated PA transport in grapevine and suggest that VviGSTs present different specificities for flavonoid ligands. In addition, our data provide evidence to suggest that GST-mediate flavonoid transport is glutathione-dependent.

New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.

In grapevine, anthocyanins and proanthocyanidins are the main flavonoids in berries, which are associated to organoleptic properties in red wine such as color and astringency. Flavonoid pathway is specifically regulated at transcriptional level and several R2R3-MYB proteins have shown to act as positive regulators. However, some members of this family have shown to repress the flavonoid biosynthesis. In this work, we present the characterization of VvMYB4-like gene, which encodes a putative transcriptional factor highly expressed in the skin of berries at the pre veraison stage in grapevine. Its over-expression in tobacco resulted in the loss of pigmentation in flowers due a decrease in anthocyanin accumulation. Severity in anthocyanin suppression observed in petals could be associated with the expression level of the VvMYB4-like transgene. Expression analysis of flavonoid structural genes revealed the strong down-regulation of the flavonoid-related genes anthocyanidin synthase (ANS) and dihydroflavonol reductase (DFR) genes and also the reduction of the anthocyanin-related gene UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), which was dependent of the transgene expression. In addition, expression of VvMYB4-like in the model plant Arabidopsis showed similar results, with the higher down-regulation observed in the AtDFR and AtLDOX genes. These results suggest that VvMYB4-like may play an important role in regulation of anthocyanin biosynthesis in grapevine acting as a transcriptional repressor of flavonoid structural genes.