BackgroundWild plants of Vitis closely related to the cultivated grapevine (V. v. vinifera) are believed to have been first domesticated 10,000 years BC around the Caspian Sea. V. v. vinifera is hermaphrodite whereas V. v. sylvestris is a dioecious species. Male flowers show a reduced pistil without style or stigma and female flowers present reflexed stamens with infertile pollen. V. vinifera produce perfect flowers with all functional structures. The mechanism for flower sex determination and specification in grapevine is still unknown.ResultsTo understand which genes are involved during the establishment of male, female and complete flowers, we analysed and compared the transcription profiles of four developmental stages of the three genders. We showed that sex determination is a late event during flower development and that the expression of genes from the ABCDE model is not directly correlated with the establishment of sexual dimorphism. We propose a temporal comprehensive model in which two mutations in two linked genes could be players in sex determination and indirectly establish the Vitis domestication process. Additionally, we also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role involved in sex differentiation. Also, the detection of differentially transcribed regions that extended existing gene models (intergenic regions) between sexes suggests that they may account for some of the variation between the subspecies.ConclusionsThere is no evidence of differences of expression levels in genes from the ABCDE model that could explain the shift from hermaphroditism to dioecy. We propose that sex specification occurs after floral organ identity has been established and therefore, sex determination genes might be having an effect downstream of the ABCDE model genes.For the first time a full transcriptomic analysis was performed in different flower developmental stages in the same individual. Our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across developmental stages and genders that will contribute for future work in sex determination in seed plants.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1095) contains supplementary material, which is available to authorized users.

Heat stress is a major limiting factor of grapevine production and quality. Acclimation and recovery are essential to ensure plant survival, and the recovery mechanisms can be independent of the heat response mechanisms. An experimental set up with and without acclimation to heat followed by recovery [stepwise acclimation and recovery (SAR) and stepwise recovery (SR), respectively] was applied to two grapevine varieties, Touriga Nacional (TN), and Trincadeira (TR), with different tolerance to abiotic stress. Major differences were found between leaves of SAR and SR, especially after recovery; in SAR, almost all parameters returned to basal levels while in SR they remained altered. Acclimation led to a swifter and short-term antioxidative response, affecting the plant to a lesser extent than SR. Significant differences were found among varieties: upon stress, TN significantly increased ascorbate and glutathione reduction levels, boosting the cell's redox-buffering capacity, while TR needed to synthesize both metabolites, its response being insufficient to keep the redox state at working levels. TR was affected by stress for a longer period and the up-regulation pattern of antioxidative stress genes was more obvious. In TN, heat shock proteins were significantly induced, but the canonical heat-stress gene signature was not evident probably because no shutdown of the housekeeping metabolism was needed.

Worldwide, extensive agricultural losses are attributed to drought, often in combination with heat in Mediterranean climate regions, where grapevine traditionally grows. The available scenarios for climate change suggest increases in aridity in these regions. Under natural conditions plants are affected by a combination of stresses, triggering synergistic or antagonistic physiological, metabolic or transcriptomic responses unique to the combination. However the study of such stresses in a controlled environment can elucidate important mechanisms by allowing the separation of the effects of individual stresses. To gather those effects, cuttings of two grapevine varieties, Touriga Nacional (TN) and Trincadeira (TR), were grown under controlled conditions and subjected to three abiotic stresses (drought - WS, heat - HS and high light - LS) individually and in combination two-by-two (WSHS, WSLS, HSLS) or all three (WSHSLS). Photosynthesis, water status, contents of H2 O2 , abscisic acid and metabolites of the ascorbate-glutathione cycle were measured in the leaves. Common and distinct response features were identified in the different stress combinations. Photosynthesis was not hindered in TN by LS, while even individual stresses severely affect photosynthesis in TR. Abscisic acid may be implicated in grapevine osmotic responses since it is correlated with tolerance parameters, especially in combined stresses involving drought. Overall, the responses to drought-including treatments were clearly distinct to those without drought. From the specific behaviours of the varieties, it can be concluded that TN shows a higher capacity for heat dissipation and for withstanding high light intensities, indicating better adjustment to warm conditions, provided that water supply is plentiful.

Background: Predicted climate changes announce an increase of extreme environmental conditions including drought and excessive heat and light in classical viticultural regions. Thus, understanding how grapevine responds to these conditions and how different genotypes can adapt, is crucial for informed decisions on accurate viticultural actions. Global transcriptome analyses are useful for this purpose as the response to these abiotic stresses involves the interplay of complex and diverse cascades of physiological, cellular and molecular events. The main goal of the present work was to evaluate the response to diverse imposed abiotic stresses at the transcriptome level and to compare the response of two grapevine varieties with contrasting physiological trends, Trincadeira (TR) and Touriga Nacional (TN).

BackgroundQuantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes.ResultsUsing the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both.ConclusionsEach method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis.

Grapevine is an extremely important crop worldwide. In Southern Europe, post flowering phases of the growth cycle can occur under high temperatures, excessive light and drought conditions at soil and/or atmospheric level. In this study we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress (WDS) and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high throughput transcriptomic grapevine array, the 23K custom made Affymetrix Vitis GeneChip. We identified 1594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, a cross-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors.

Vitis vinifera vinifera is a hermaphrodite subspecies, while its ancestor, Vitis vinifera sylvestris, is dioecious. We have identified two genes that together allow the discrimination between male, female and hermaphrodite Vitis plants. The sex locus region on chromosome 2 was screened resulting in the discovery of a new gene, VviFSEX. The same screening revealed another gene, VviAPRT3, located in the sex region, that be used as a sex marker. Both genes are good candidates to be involved in flower sex differentiation in grapevine. To assess their role in sex specification, spatial and temporal expression analysis was performed. The expression of VviFSEX is detected in petals, stamens and carpel primordia of all flower types, making its putative function unclear; however, female plants display a single allele for this gene, while male and hermaphrodites display two alleles. On the other hand, the specific expression of VviAPRT3 in the carpel primordial of male plants suggests a possible role in the abortion of pistil structures. We propose a model to explain the carpel abortion in male flowers and the absence of stamen viability in female flowers. In addition, this work reinforces the presence of a sex locus on Vitis chromosome 2.

The most discriminating characteristic between the cultivated Vitis vinifera subsp. vinifera and the wild-form Vitis vinifera subsp. sylvestris is their sexual system. Flowers of cultivars are mainly hermaphroditic, whereas wild plants have female and male individuals whose flowers follow a hermaphroditic pattern during early stages of development and later develop non-functional reproductive organs. In angiosperms, the basic developmental system for floral organ identity is explained by the ABCDE model. This model postulates that regulatory gene functions work in a combinatorial way to confer organ identity in each whorl. In wild Vitis nothing is known about the function and expression profile of these genes. Here we show an overall view of the temporal and spatial expression pattern of the ABCDE genes as well as the pattern of VviSUPERMAN that establishes a boundary between the stamen and the carpel whorls, in the male, female and complete flower types. The results show a similar pattern in Vitis species suggesting that the pathway leading to unisexuality acts independently and/or downstream of B- and C- function genes.