Tissue-Specific Regulation of Vesicular Trafficking Mediated by Rab-GEF Complex MON1/CCZ1 From Solanum chilense Increases Salt Stress Tolerance in Arabidopsis thaliana.

Salt stress constrains the development and growth of plants. To tolerate it, mechanisms of endocytosis and vacuolar compartmentalization of Na+ are induced. In this work, the genes that encode a putative activator of vesicular trafficking called MON1/CCZ1 from Solanum chilense, SchMON1 and SchCCZ1, were co-expressed in roots of Arabidopsis thaliana to determine whether the increase in prevacuolar vesicular trafficking also increases the Na+ compartmentalization capacity and tolerance. Initially, we demonstrated that both SchMON1 and SchCCZ1 genes rescued the dwarf phenotype of both A. thaliana mon1-1 and ccz1a/b mutants associated with the loss of function, and both proteins colocalized with their functional targets, RabF and RabG, in endosomes. Transgenic A. thaliana plants co-expressing these genes improved salt stress tolerance compared to wild type plants, with SchMON1 contributing the most. At the sub-cellular level, co-expression of SchMON1/SchCCZ1 reduced ROS levels and increased endocytic activity, and number of acidic structures associated with autophagosomes. Notably, greater Na+ accumulation in vacuoles of cortex and endodermis was evidenced in the SchMON1 genotype. Molecular analysis of gene expression in each genotype supported these results. Altogether, our analysis shows that root activation of prevacuolar vesicular trafficking mediated by MON1/CCZ1 emerges as a promising physiological molecular mechanism to increase tolerance to salt stress in crops of economic interest.

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.

AtZAT4, a C2H2-Type Zinc Finger Transcription Factor from Arabidopsis thaliana, Is Involved in Pollen and Seed Development.

Pollen plays an essential role in plant fertility by delivering the male gametes to the embryo sac before double fertilization. In several plant species, including Arabidopsis, C2H2-type zinc-finger transcription factors (TFs) have been involved in different stages of pollen development and maturation. ZINC FINGER of Arabidopsis thaliana 4 (AtZAT4) is homologous to such TFs and subcellular localization analysis has revealed that AtZAT4 is located in the nucleus. Moreover, analysis of AtZAT4 expression revealed strong levels of it in flowers and siliques, suggesting a role of the encoded protein in the regulation of genes that are associated with reproductive development. We characterized a T-DNA insertional heterozygous mutant Atzat4 (+/−). The relative gene expression analysis of Atzat4 (+/−) showed significant transcript reductions in flowers and siliques. Furthermore, the Atzat4 (+/−) phenotypic characterization revealed defects in the male germline, showing a reduction in pollen tube germination and elongation. Atzat4 (+/−) presented reduced fertility, characterized by a smaller silique size compared to the wild type (WT), and a lower number of seeds per silique. Additionally, seeds displayed lower viability and germination. Altogether, our data suggest a role for AtZAT4 in fertilization and seed viability, through the regulation of gene expression associated with reproductive development.

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.

Root-knot nematodes induce gall formation by recruiting developmental pathways of post-embryonic organogenesis and regeneration to promote transient pluripotency.

Root-knot nematodes (RKNs; Meloidogyne spp.) induce new post-embryogenic organs within the roots (galls) where they stablish and differentiate nematode feeding cells, giant cells (GCs). The developmental programmes and functional genes involved remain poorly defined. Arabidopsis root apical meristem (RAM), lateral root (LR) and callus marker lines, SHORT-ROOT/SHR, SCARECROW/SCR, SCHIZORIZA/SCZ, WUSCHEL-RELATED-HOMEOBOX-5/WOX5, AUXIN-RESPONSIVE-FACTOR-5/ARF5, ARABIDOPSIS-HISTIDINE PHOSPHOTRANSFER-PROTEIN-6/AHP6, GATA-TRANSCRIPTION FACTOR-23/GATA23 and S-PHASE-KINASE-ASSOCIATED-PROTEIN2B/SKP2B, were analysed for nematode-dependent expression. Their corresponding loss-of-function lines, including those for LR upstream regulators, SOLITARY ROOT/SLR/IAA14, BONDELOS/BDL/IAA12 and INDOLE-3-ACETIC-ACID-INDUCIBLE-28/IAA28, were tested for RKN resistance/tolerance. LR genes, for example ARF5 (key factor for root stem-cell niche regeneration), GATA23 (which specifies pluripotent founder cells) and AHP6 (cytokinin-signalling-inhibitor regulating pericycle cell-divisions orientation), show a crucial function during gall formation. RKNs do not compromise the number of founder cells or LR primordia but locally induce gall formation possibly by tuning the auxin/cytokinin balance in which AHP6 might be necessary. Key RAM marker genes were induced and functional in galls. Therefore, the activation of plant developmental programmes promoting transient-pluripotency/stemness leads to the generation of quiescent-centre and meristematic-like cell identities within the vascular cylinder of galls. Nematodes enlist developmental pathways of new organogenesis and/or root regeneration in the vascular cells of galls. This should determine meristematic cell identities with sufficient transient pluripotency for gall organogenesis.

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.

Transcriptome analysis reveals potential roles of a barley ASR gene that confers stress tolerance in transgenic rice.

Control of gene expression and induction of cellular protection mechanisms are two important processes that plants employ to protect themselves against abiotic stresses. ABA-, stress, and ripening-induced (ASR) proteins have been identified to participate in such responses. Previous studies have proposed that these proteins can act as transcription factors and as molecular chaperones protecting transgenic plants against stresses; however a gene network regulated by ASRs has not been explored. To expand our knowledge on the function of these proteins in cereals, we present the functional characterization of a barley ASR gene. Expression of HvASR5 was almost ubiquitous in different organs and responded to ABA and to different stress treatments. When expressed ectopically, HvASR5 was able to confer drought and salt stress tolerance to Arabidopsis thaliana and to improve growth performance of rice plants under stress conditions. A transcriptomic analysis with two transgenic rice lines overexpressing HvASR5 helped to identify potential downstream targets and understand ASR-regulated cellular processes. HvASR5 up-regulated the expression of a distinct set of genes associated with stress responses, metabolic processes (particularly carbohydrate metabolism), as well as reproduction and development. These data, together with the confirmed nuclear and cytoplasmic localization of HvASR5, further support the hypothesis that HvASR5 can also carry out roles as molecular protector and transcriptional regulator.

Overexpression of a SDD1-Like Gene From Wild Tomato Decreases Stomatal Density and Enhances Dehydration Avoidance in Arabidopsis and Cultivated Tomato.

Stomata are microscopic valves formed by two guard cells flanking a pore, which are located on the epidermis of most aerial plant organs and are used for water and gas exchange between the plant and the atmosphere. The number, size and distribution of stomata are set during development in response to changing environmental conditions, allowing plants to minimize the impact of a stressful environment. In Arabidopsis, STOMATAL DENSITY AND DISTRIBUTION 1 (AtSDD1) negatively regulates stomatal density and optimizes transpiration and water use efficiency (WUE). Despite this, little is known about the function of AtSDD1 orthologs in crop species and their wild stress-tolerant relatives. In this study, SDD1-like from the stress-tolerant wild tomato Solanum chilense (SchSDD1-like) was identified through its close sequence relationship with SDD1-like from Solanum lycopersicum and AtSDD1. Both Solanum SDD1-like transcripts accumulated in high levels in young leaves, suggesting that they play a role in early leaf development. Arabidopsis sdd1-3 plants transformed with SchSDD1-like under a constitutive promoter showed a significant reduction in stomatal leaf density compared with untransformed sdd1-3 plants. Additionally, a leaf dehydration shock test demonstrated that the reduction in stomatal abundance of transgenic plants was sufficient to slow down dehydration. Overexpression of SchSDD1-like in cultivated tomato plants decreased the stomatal index and density of the cotyledons and leaves, and resulted in higher dehydration avoidance. Taken together, these results indicate that SchSDD1-like functions in a similar manner to AtSDD1 and suggest that Arabidopsis and tomatoes share this component of the stomatal development pathway that impinges on water status.

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.

Root-endophytes improve the ecophysiological performance and production of an agricultural species under drought condition.

Throughout many regions of the world, climate change has limited the availability of water for irrigating crops. Indeed, current models of climate change predict that arid and semi-arid zones will be places where precipitation will drastically decrease. In this context, plant root-associated fungi appear as a new strategy to improve ecophysiological performance and yield of crops under abiotic stress. Thus, use of fungal endophytes from ecosystems currently subjected to severe drought conditions could improve the ecophysiological performance and quantum yield of crops exposed to drought. In this study, we evaluated how the inoculation of fungal endophytes isolated from Antarctic plants can improve the net photosynthesis, water use efficiency and production of fresh biomass in a lettuce cultivar, grown under different water availability regimes. In addition, we assessed if the presence of biochemical mechanisms and gene expression related with environmental tolerance are improved in presence of fungal endophytes. Overall, those individuals with presence of endophytes showed higher net photosynthesis and maintained higher water use efficiency in drought conditions, which was correlated with greater fresh and dry biomass production as well as greater root system development. In addition, presence of fungal endophytes was correlated with a higher proline concentration, lower peroxidation of lipids and up-/down-regulation of ion homeostasis. Our results suggest that presence of fungal endophytes could minimize the negative effect of drought by improving drought tolerance through biochemical mechanisms and improving nutritional status. Thus, root-endophytes might be a successful biotechnological tool to maintain high levels of ecophysiological performance and productivity in zones under drought.

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.

GC-MS metabolic profiling of Cabernet Sauvignon and Merlot cultivars during grapevine berry development and network analysis reveals a stage- and cultivar-dependent connectivity of primary metabolites.

Information about the total chemical composition of primary metabolites during grape berry development is scarce, as are comparative studies trying to understand to what extent metabolite modifications differ between cultivars during ripening. Thus, correlating the metabolic profiles with the changes occurring in berry development and ripening processes is essential to progress in their comprehension as well in the development of new approaches to improve fruit attributes. Here, the developmental metabolic profiling analysis across six stages from flowering to fully mature berries of two cultivars, Cabernet Sauvignon and Merlot, is reported at metabolite level. Based on a gas chromatography-mass spectrometry untargeted approach, 115 metabolites were identified and relative quantified in both cultivars. Sugars and amino acids levels show an opposite behaviour in both cultivars undergoing a highly coordinated shift of metabolite associated to primary metabolism during the stages involved in growth, development and ripening of berries. The changes are characteristic for each stage, the most pronounced ones occuring at fruit setting and pre-Veraison. They are associated to a reduction of the levels of metabolites present in the earlier corresponding stage, revealing a required catabolic activity of primary metabolites for grape berry developmental process. Network analysis revealed that the network connectivity of primary metabolites is stage- and cultivar-dependent, suggesting differences in metabolism regulation between both cultivars as the maturity process progresses. Furthermore, network analysis may represent an appropriate method to display the association between primary metabolites during berry developmental processes among different grapevine cultivars and for identifying potential biologically relevant metabolites.

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.

Pollen Morphology and Boron Concentration in Floral Tissues as Factors Triggering Natural and GA-Induced Parthenocarpic Fruit Development in Grapevine.

Parthenocarpic fruit development (PFD) reduces fruit yield and quality in grapevine. Parthenocarpic seedless berries arise from fruit set without effective fertilization due to defective pollen germination. PFD has been associated to micronutrient deficiency but the relation of this phenomenon with pollen polymorphism has not been reported before. In this work, six grapevine cultivars with different tendency for PFD and grown under micronutrient-sufficient conditions were analyzed to determine pollen structure and germination capability as well as PFD rates. Wide variation in non-germinative abnormal pollen was detected either among cultivars as well as for the same cultivar in different growing seasons. A straight correlation with PFD rates was found (R2 = 0.9896), suggesting that natural parthenocarpy is related to defective pollen development. Such relation was not observed when PFD was analyzed in grapevine plants exposed to exogenous gibberellin (GA) or abscissic acid (ABA) applications at pre-anthesis. Increase (GA treatment) or reduction (ABA treatment) in PFD rates without significative changes in abnormal pollen was determined. Although these plants were maintained at sufficient boron (B) condition, a down-regulation of the floral genes VvBOR3 and VvBOR4 together with a reduction of floral B content in GA-treated plants was established. These results suggest that impairment in B mobility to reproductive tissues and restriction of pollen tube growth could be involved in the GA-induced parthenocarpy.

VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L.

KEY MESSAGE: VvMATE1 and VvMATE2 encode putative PA transporters expressed during seed development in grapevine. The subcellular localization of these MATE proteins suggests different routes for the intracellular transport of PAs. Proanthocyanidins (PAs), also called condensed tannins, protect plants against herbivores and are important quality components of many fruits. PAs biosynthesis is part of the flavonoid pathway that also produces anthocyanins and flavonols. In grape fruits, PAs are present in seeds and skin tissues. PAs are synthesized in the cytoplasm and accumulated into the vacuole and apoplast; however, little is known about the mechanisms involved in the transport of these compounds to such cellular compartments. A gene encoding a Multidrug And Toxic compound Extrusion (MATE) family protein suggested to transport anthocyanins-named VvMATE1-was used to identify a second gene of the MATE family, VvMATE2. Analysis of their deduced amino acid sequences and the phylogenetic relationship with other MATE-like proteins indicated that VvMATE1 and VvMATE2 encode putative PA transporters. Subcellular localization assays in Arabidopsis protoplasts transformed with VvMATE-GFP fusion constructs along with organelle-specific markers revealed that VvMATE1 is localized in the tonoplast whereas VvMATE2 is localized in the Golgi complex. Major expression of both genes occurs during the early stages of seed development concomitant with the accumulation of PAs. Both genes are poorly expressed in the skin of berries while VvMATE2 is also expressed in leaves. The presence of putative cis-acting elements in the promoters of VvMATE1 and VvMATE2 may explain the differential transcriptional regulation of these genes in grapevine. Altogether, these results suggest that these MATE proteins could mediate the transport and accumulation of PAs in grapevine through different routes and cellular compartments.

Organ- and stress-specific expression of the ASR genes in rice.

KEY MESSAGE: Rice ASR genes respond distinctly to abscisic acid, dehydration and cold stress. Their tissue-specific expression provides new hints about their possible roles in plant responses to stress. Plant ASR proteins have emerged as an interesting distinct group of proteins with apparent roles in protecting cellular structures as well as putative regulators of gene expression, both important responses of plants to environmental stresses. Regardless of the possible functions proposed by different studies, little is known about their role in cereals. To further understand the function of these proteins in the Gramineae, we investigated the expression pattern of the six ASR genes present in the rice genome in response to ABA, stress conditions and in different organs. Although transcription of most OsASRs is transiently enhanced by ABA treatment, the genes present a differential response under cold and drought stress as well as specific expression in certain tissues and organs. Analysis of their promoters reveals regulatory cis-elements associated to hormonal, sugar and stress responses. The promoters of two genes, OsASR1 and OsASR5, direct the expression of the GUS reporter gene especially to leaf vascular tissue in response to dehydration and low temperature. In control conditions, a GUS reporter assay also indicates specific expression of these two genes in roots, anthers and seed scutellar tissues. These results provide new clues about the possible role of ASRs in plant stress responses and development.

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco.

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.