BackgroundTerpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.ResultsWe present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation.ConclusionsThe highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

Background: Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents.

Ethylene evolution occurs concomitantly with the progression of disease symptoms in response to many virulent pathogen infections in plants. A tomato mutant impaired in ethylene perception-Never ripe -exhibited a significant reduction in disease symptoms in comparison to the wild type after inoculations of both genotypes with virulent bacterial ( Xanthomonas campestris pv vesicatoria and Pseudomonas syringae pv tomato ) and fungal ( Fusarium oxysporum f sp lycopersici ) pathogens. Bacterial spot disease symptoms were also reduced in tomato genotypes impaired in ethylene synthesis (1-aminocyclopropane-1-carboxylic acid deaminase) and perception (14893), thereby corroborating a reducing effect for ethylene insensitivity on foliar disease development. The reduction in foliar disease symptoms in Never ripe plants was a specific effect of ethylene insensitivity and was not due to reductions in bacterial populations or decreased ethylene synthesis. PR-1B1 mRNA accumulation in response to X. c. vesicatoria infection was not affected by ethylene insensitivity, indicating that ethylene is not required for defense gene induction. Our findings suggest that broad tolerance of diverse vegetative diseases may be achieved via engineering of ethylene insensitivity in tomato. INTRODUCTIONSusceptible host plants can incur extensive disease damage from infections by virulent pathogens. Although plant pathologists have focused considerable attention on mechanisms of host and non-host resistance to avirulent pathogens in numerous systems (Bent, 1996), our understanding of the physiology underlying disease development in response to infections by virulent pathogens is relatively limited. Understanding how disease development is regulated in compatible plant-microbe interactions is a first step toward engineering plants that are tolerant to virulent pathogen infections. Plants that are disease tolerant have the ability to produce ample yields even when they are infected by pathogens (Agrios, 1997).The mechanisms underlying the widespread cell death that occurs during susceptible responses are not understood. Numerous studies have focused on the importance of pathogen-derived compounds to compatible interactions and have demonstrated that phytotoxins produced by some pathogens are critical for host recognition and infection; however, there are only a limited number of cases in which phytotoxins have been demonstrated to directly control disease symptom development (DeVay, 1988;Sigee, 1993; Walton, 1996). It is important to note that infections by organisms that produce phytotoxins, such as Pseudomonas syringae pv tomato (Palmer and Bender, 1995) and Fusarium oxysporum f sp lycopersici (DeVay, 1988), and those that do not, such as Xanthomonas campestris pv vesicatoria (R.E. Stall, unpublished data), cause extensive necroses in susceptible tissues. Pathogen-derived ethylene has been suggested to act as a phytotoxin that accelerates the chlorosis, necrosis, and foliar abscission associated with disease development; however, under in vitr...

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.

SummaryEthylene and salicylic acid (SA) are key intermediates in a host's response to pathogens. Previously, we have shown using a tomato compatible interaction that ethylene and SA act sequentially and are essential for disease symptom production. Here, we have examined the relationship between the two signals in the Arabidopsis-Xanthomonas campestris pv. campestris (Xcc) compatible interaction. Preventing SA accumulation by expression of the nahG gene reduced subsequent ethylene production and altered the development of disease symptoms, with plants showing no visible chlorosis. The ethylene insensitive lines, etr1-1 and etr2-1, on the other hand, accumulated SA and exhibited normal but precocious symptom development. Therefore, Arabidopsis, like tomato, was found to exhibit co-operative ethylene and SA action for the production of disease symptoms. However, in Arabidopsis, SA was found to act upstream of ethylene. Jasmonic acid and indole-3-acetic acid levels were also found to increase in response to Xcc. In contrast to ethylene, accumulation of these hormones was not found to be dependent on SA action. These results indicate that the plants response to a virulent pathogen is a composite of multiple signaling pathways.