Genome Wide Identification, Molecular Characterization, and Gene Expression Analyses of Grapevine NHX Antiporters Suggest Their Involvement in Growth, Ripening, Seed Dormancy, and Stress Response.

Plant NHX antiporters are critical for cellular pH, Na+, and K+ homeostasis and salt tolerance. Even though their genomic and functional studies have been conducted in many species, the grapevine NHX family has not been described yet. Our work highlights the presence of six VvNHX genes whose phylogenetic analysis revealed their classification in two distinct groups: group I vacuolar (VvNHX1-5) and group II endosomal (VvNHX6). Several cis-acting regulatory elements related to tissue-specific expression, transcription factor binding, abiotic/biotic stresses response, and light regulation elements were identified in their promoter. Expression profile analyses of VvNHX genes showed variable transcription within organs and tissues with diverse patterns according to biochemical, environmental, and biotic treatments. All VvNHXs are involved in berry growth, except VvNHX5 that seems to be rather implicated in seed maturation. VvNHX4 would be more involved in floral development, while VvNHX2 and 3 display redundant roles. QPCR expression analyses of VvNHX1 showed its induction by NaCl and KNO3 treatments, whereas VvNHX6 was induced by ABA application and strongly repressed by PEG treatment. VvNHX1 plays a crucial role in a bunch of grape developmental steps and adaptation responses through mechanisms of phyto-hormonal signaling. Overall, VvNHX family members could be valuable candidate genes for grapevine improvement.

Quality selection and genetic diversity of Tunisian durum wheat varieties using SSR markers / Seleção de qualidade e diversidade genética de vari edades de trigo duro da tunísia usando marcadores SSR

Our study focuses on the molecular analysis of the genetic diversity within 15 Tunisian durum wheat varieties and the assessment of the efficiency of some available markers to select valuable genotypes for technological proprieties of semolina (i.e. parameters related to SDS-sedimentation, mixing time and breakdown resistance of mixograph, grain protein content and yellow colour). While several markers were validated, others were not informative within the genotypes used. A high level of polymorphic information content (PIC) was detected, with an average of 5.2 polymorph alleles per locus and 0.6 average. Old varieties have high protein content however; modern varieties display strong gluten strength. Our results thus open the opportunity to choose valuable parents on the base of pedigrees, technological properties and genetic distances; and lead us to select efficient markers for the Regional Indigenous Land Strategy (Rils) selection strategy

Nosso estudo enfoca a análise molecular da diversidade genética em 15 variedades de trigo duro tunisiano e a avaliação da eficiência de alguns marcadores disponíveis para selecionar genótipos valiosos para propriedades tecnológicas de semolina (ou seja, parâmetros relacionados à sedimentação sds, tempo de mistura e resistência à degradação do mixógrafo, teor de proteína dos grãos e cor amarela). Enquanto vários marcadores foram validados, outros não foram informativos dentro dos genótipos utilizados. Foi detectado um alto nível de conteúdo de informação polimórfica (pic), com uma média de 5,2 alelos polimórficos por locus e 0,6 média. Variedades antigas têm alto teor de proteína no entanto; variedades modernas exibem forte força de glúten. Nossos resultados abrem, assim, a oportunidade de escolher pais valiosos com base em pedigrees, propriedades tecnológicas e distâncias genéticas; e nos levam a selecionar marcadores eficientes para a estratégia de seleção da estratégia regional de terras indígenas (rils).

Computational Approach for Structural Feature Determination of Grapevine NHX Antiporters.

Plant NHX antiporters are responsible for monovalent cation/H+ exchange across cellular membranes and play therefore a critical role for cellular pH regulation, Na+ and K+ homeostasis, and salt tolerance. Six members of grapevine NHX family (VvNHX1-6) have been structurally characterized. Phylogenetic analysis revealed their organization in two groups: VvNHX1-5 belonging to group I (vacuolar) and VvNHX6 belonging to group II (endosomal). Conserved domain analysis of these VvNHXs indicates the presence of different kinds of domains. Out of these, two domains function as monovalent cation-proton antiporters and one as the aspartate-alanine exchange; the remaining are not yet with defined function. Overall, VvNHXs proteins are typically made of 11-13 putative transmembrane regions at their N-terminus which contain the consensus amiloride-binding domain in the 3rd TM domain and a cation-binding site in between the 5th and 6th TM domain, followed by a hydrophilic C-terminus that is the target of several and diverse regulatory posttranslational modifications. Using a combination of primary structure analysis, secondary structure alignments, and the tertiary structural models, the VvNHXs revealed mainly 18 α helices although without ß sheets. Homology modeling of the 3D structure showed that VvNHX antiporters are similar to the bacterial sodium proton antiporters MjNhaP1 (Methanocaldococcus jannaschii) and PaNhaP (Pyrococcus abyssi).

The grapevine VvWRKY2 gene enhances salt and osmotic stress tolerance in transgenic Nicotiana tabacum.

Our study aims to assess the implication of WRKY transcription factor in the molecular mechanisms of grapevine adaptation to salt and water stresses. In this respect, a full-length VvWRKY2 cDNA, isolated from a Vitis vinifera grape berry cDNA library, was constitutively over-expressed in Nicotiana tabacum seedlings. Our results showed that transgenic tobacco plants exhibited higher seed germination rates and better growth, under both salt and osmotic stress treatments, when compared to wild type plants. Furthermore, our analyses demonstrated that, under stress conditions, transgenic plants accumulated more osmolytes, such as soluble sugars and free proline, while no changes were observed regarding electrolyte leakage, H2O2, and malondialdehyde contents. The improvement of osmotic adjustment may be an important mechanism underlying the role of VvWRKY2 in promoting tolerance and adaptation to abiotic stresses. Principal component analysis of our results highlighted a clear partition of plant response to stress. On the other hand, we observed a significant adaptation behaviour response for transgenic lines under stress. Taken together, all our findings suggest that over-expression of VvWRKY2 gene has a compelling role in abiotic stress tolerance and, therefore, would provide a useful strategy to promote abiotic stress tolerance in grape via molecular-assisted breeding and/or new biotechnology tools.

Identification and characterization of single nucleotide polymorphism markers in FADS2 gene associated with olive oil fatty acids composition.

BACKGROUND: Genotyping of the FAD2.1 and FAD2.3 polymorphisms in the fatty acid desaturase 2 gene (FADS2) shows that they are associated with the fatty acids composition of olive oil samples. However, these associations require further confirmation in the Tunisian olive oil cultivars, and little is known about the effect of polymorphisms in fatty acid-related genes on olive oil mono- and poly- unsaturated fatty acids distribution. METHODS: A set of olive oils from 12 Tunisian cultivars was chosen. The fatty acid composition of each olive oil sample was determined by gas chromatography. Statistical and modeling Bayesian analyses were used to assess whether the FAD2.1 and FAD2.3 genotypes were associated with fatty acids composition. RESULTS: The TT-FAD2.1 and the GG-FAD2.3 genotypes were found to be associated with a lower proportion of oleic acid (C18:1) (r = -0.778, p = 0.003; r = -0.781, p= 0.003) as well as higher proportion of linoleic (C18:2) (r = 0.693, p = 0.012; r = -0.759, p= 0.004) and palmitic acids (C16:0) (r = 0.643, p = 0.024; r = -0.503, p= 0.095), making varieties with this haplotype (i.e. Chemlali Sfax and Meski) producing more saturated (C16: 0) and polyunsaturated acids than oleic acid. The latter plays a major role in preventing several diseases. CONCLUSION: The two associations FADS2 FAD2.1 and FADS2 FAD2.3 with the fatty acid compositions of olive oil samples were identified among the studied olive cultivars. These associations differed between studied cultivars, which might explain variability in lipidic composition among them and consequently reflecting genetic diversity through differences in gene expression and biochemical pathways. FADS2 locus would constitute thus a good marker for detecting interesting lipidic chemotypes among commercial olive oils.

Isolation and molecular characterization of ERF1, an ethylene response factor gene from durum wheat (Triticum turgidum L. subsp. durum), potentially involved in salt-stress responses.

As food crop, wheat is of prime importance for human society. Nevertheless, our understanding of the genetic and molecular mechanisms controlling wheat productivity conditions has been, so far, hampered by the lack of sufficient genomic resources. The present work describes the isolation and characterization of TdERF1, an ERF gene from durum wheat (Triticum turgidum L. subsp. durum). The structural features of TdERF1 supported the hypothesis that it is a novel member of the ERF family in durum wheat and, considering its close similarity to TaERF1 of Triticum aestivum, it probably plays a similar role in mediating responses to environmental stresses. TdERF1 displayed an expression pattern that discriminated between two durum wheat genotypes contrasted with regard to salt-stress tolerance. The high number of cis-regulatory elements related to stress responses present in the TdERF1 promoter and the ability of TdERF1 to regulate the transcription of ethylene and drought-responsive promoters clearly indicated its potential role in mediating plant responses to a wide variety of environmental constrains. TdERF1 was also regulated by abscisic acid, ethylene, auxin, and salicylic acid, suggesting that it may be at the crossroads of multiple hormone signalling pathways. Four TdERF1 allelic variants have been identified in durum wheat genome, all shown to be transcriptionally active. Interestingly, the expression of one allelic form is specific to the tolerant genotype, further supporting the hypothesis that this gene is probably associated with the susceptibility/tolerance mechanism to salt stress. In this regard, the TdERF1 gene may provide a discriminating marker between tolerant and sensitive wheat varieties.

The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco.

Previous work has shown that transgenic tobacco plants constitutively over-expressing the Vitis vinifera L. transcription factor VvWRKY2 exhibit reduced susceptibility to necrotrophic fungal pathogens, suggesting that this transcription factor plays a role in grapevine response to phytopathogens. The work presented here characterizes the modifications in cell wall structure observed in the stems and petioles of these transgenic plants. Histochemical stainings of stem and petiole cross-sections using phloroglucinol or Maüle reagents revealed a delay in xylem formation, particularly in the petioles, and differences in lignin composition. Evaluation of lignin quantity and quality showed a decrease in the syringyl/guaiacyl ratio in both stem and petioles. Expression analysis using RT-PCR and potato microarrays showed that tobacco plants over-expressing VvWRKY2 exhibited altered expression of genes involved in lignin biosynthesis pathway and cell wall formation. The ability of VvWRKY2 to activate the promoter of the VvC4H gene, which is involved in the lignin biosynthetic pathway, was confirmed by transient transcriptional activation assays in tobacco protoplasts. Moreover, in situ hybridization revealed that VvWRKY2 is specifically expressed in cells undergoing lignification in young grapevine stems. Together, these results confirm that VvWRKY2 plays a role in regulating lignification in grapevine, possibly in response to biotic or abiotic stresses.

Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants.

Pathogen attack represents a major problem for viticulture and for agriculture in general. At present, the use of phytochemicals is more and more restrictive, and therefore it is becoming essential to control disease by having a thorough knowledge of resistance mechanisms. The present work focused on the trans-regulatory proteins potentially involved in the control of the plant defence response, the WRKY proteins. A full-length cDNA, designated VvWRKY1, was isolated from a grape berry library (Vitis vinifera L. cv. Cabernet Sauvignon). It encodes a polypeptide of 151 amino acids whose structure is characteristic of group IIc WRKY proteins. VvWRKY1 gene expression in grape is regulated in a developmental manner in berries and leaves and by various signal molecules involved in defence such as salicylic acid, ethylene, and hydrogen peroxide. Biochemical analysis indicates that VvWRKY1 specifically interacts with the W-box in various nucleotidic contexts. Functional analysis of VvWRKY1 was performed by overexpression in tobacco, and transgenic plants exhibited reduced susceptibility to various fungi but not to viruses. These results are consistent with a possible role for VvWRKY1 in grapevine defence against fungal pathogens.

Overexpression of VvWRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens.

WRKY genes encode proteins belonging to a large family of transcription factors that are involved in various developmental and physiological processes and in plant responses to pathogen infections. In the present work, a full-length cDNA from a Vitis vinifera L. cv. Cabernet Sauvignon grape berry library was isolated and characterized. The cDNA, designated VvWRKY2, encodes a polypeptide of 536 amino acids that shows the structural features of group I of WRKY protein family. VvWRKY2 is expressed in the different organs of healthy grapevine plants. In leaves, VvWRKY2 is induced by wounding and after infection with Plasmopara viticola. Constitutive expression of VvWRKY2 in tobacco reduced the susceptibility of transgenic tobacco to three types of fungal pathogens infecting different parts of the plant: Botrytis cinerea (leaves), Pythium spp. (roots) and Alternaria tenuis (seeds). The results indicate that VvWRKY2 may be involved in the resistance of grapevine against the pathogens.