Physiology and Gene Expression Analysis of Tomato (Solanum lycopersicum L.) Exposed to Combined-Virus and Drought Stresses.

Crop productivity can be obstructed by various biotic and abiotic stresses and thus these stresses are a threat to universal food security. The information on the use of viruses providing efficacy to plants facing growth challenges owing to stress is lacking. The role of induction of pathogen-related genes by microbes is also colossal in drought-endurance acquisition. Studies put forward the importance of viruses as sustainable means for defending plants against dual stress. A fundamental part of research focuses on a positive interplay between viruses and plants. Notably, the tomato yellow leaf curl virus (TYLCV) and tomato chlorosis virus (ToCV) possess the capacity to safeguard tomato host plants against severe drought conditions. This study aims to explore the combined effects of TYLCV, ToCV, and drought stress on two tomato cultivars, Money Maker (MK, UK) and Shalala (SH, Azerbaijan). The expression of pathogen-related four cellulose synthase gene families (CesA/Csl) which have been implicated in drought and virus resistance based on gene expression analysis, was assessed using the quantitative real-time polymerase chain reaction method. The molecular tests revealed significant upregulation of Ces-A2, Csl-D3,2, and Csl-D3,1 genes in TYLCV and ToCV-infected tomato plants. CesA/Csl genes, responsible for biosynthesis within the MK and SH tomato cultivars, play a role in defending against TYLCV and ToCV. Additionally, physiological parameters such as "relative water content," "specific leaf weight," "leaf area," and "dry biomass" were measured in dual-stressed tomatoes. Using these features, it might be possible to cultivate TYLCV-resistant plants during seasons characterized by water scarcity.

Dual domestications and origin of traits in grapevine evolution.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

Salt stress induces endoplasmic reticulum stress-responsive genes in a grapevine rootstock.

Grapevines, although adapted to occasional drought or salt stress, are relatively sensitive to growth- and yield-limiting salinity stress. To understand the molecular mechanisms of salt tolerance and endoplasmic reticulum (ER) stress and identify genes commonly regulated by both stresses in grapevine, we investigated transcript profiles in leaves of the salt-tolerant grapevine rootstock 1616C under salt- and ER-stress. Among 1643 differentially expressed transcripts at 6 h post-treatment in leaves, 29 were unique to ER stress, 378 were unique to salt stress, and 16 were common to both stresses. At 24 h post-treatment, 243 transcripts were unique to ER stress, 1150 were unique to salt stress, and 168 were common to both stresses. GO term analysis identified genes in categories including 'oxidative stress', 'protein folding', 'transmembrane transport', 'protein phosphorylation', 'lipid transport', 'proteolysis', 'photosynthesis', and 'regulation of transcription'. The expression of genes encoding transporters, transcription factors, and proteins involved in hormone biosynthesis increased in response to both ER and salt stresses. KEGG pathway analysis of differentially expressed genes for both ER and salt stress were divided into four main categories including; carbohydrate metabolism, amino acid metabolism, signal transduction and lipid metabolism. Differential expression of several genes was confirmed by qRT-PCR analysis, which validated our microarray results. We identified transcripts for genes that might be involved in salt tolerance and also many genes differentially expressed under both ER and salt stresses. Our results could provide new insights into the mechanisms of salt tolerance and ER stress in plants and should be useful for genetic improvement of salt tolerance in grapevine.

Genome composition analysis of multipartite BNYVV reveals the occurrence of genetic re-assortment in the isolates of Asia Minor and Thrace.

Beet necrotic yellow vein virus (BNYVV) is the cause of rhizomania, an important disease of sugar beet around the world. The multipartite genome of the BNYVV contains four or five single-stranded RNA that has been used to characterize the virus. Understanding genome composition of the virus not only determines the degree of pathogenicity but also is required to development of resistant varieties of sugar beet. Resistance to rhizomania has been conferred to sugar beet varieties by conventional breeding methods or modern genome engineering tools. However, over time, viruses undergo genetic alterations and develop new variants to break crop resistance. Here, we report the occurrence of genetic reassortment and emergence of new variants of BNYVV among the isolates of Thrace and Asia Minor (modern-day Turkey). Our findings indicate that the isolates harbor European A-type RNA-2 and RNA-3, nevertheless, RNA-5 is closely related to East Asian J-type. Furthermore, RNA-1 and RNA-4 are either derived from A, B, and P-types or a mixture of them. The RNA-5 factor which enhance the pathogenicity, is rarely found in the isolates studied (20%). The creation of new variants of the virus emphasizes the necessity to develop new generation of resistant crops. We anticipate that these findings will be useful for future genetic characterization and evolutionary studies of BNYVV, as well as for developing sustainable strategies for the control of this destructive disease.

Eastern Anatolian apples with a unique population structure are genetically different from Anatolian apples.

The origin of the apple is known to be the Transcaucasian region. Eastern Anatolia, which is located on the migration routes from Asia to Europe, has a rich and an uncharacterized apple germplasm and the characterization of apple genetic sources from this region is important for both evolutionary studies and apple breeding. In this study, 94 M. domestica accessions originated from seven diverse regions within Eastern Anatolia were studied using 16 SSR (simple sequence repeat) loci. SSR markers we used produced high allele numbers in all loci and CH02d11 (PI: 0.059) with 18 alleles was the most informative locus. In addition, 14 identical accession groups were identified. Most likely due to self-incompatibility, relatively high levels of heterozygosity (Ho: 0.696) was found for Eastern Anatolia apples. Structure Harvester analyses of East Anatolian apple accessions showed that although each group seems to be somewhat distinct, some levels of admixture with other populations might also exist. Due to a significant gene flow between all pairs of seven apple populations, a limited (low) differentiation was found between the populations. Comparisons using 16 common SSR loci revealed that Eastern Anatolia accessions were genetically different from Anatolian accessions. In addition, based on FCA, and Nei's genetic distance analyses, Eastern Anatolian apples were found to be genetically different from the commercial apple cultivars Golden Delicious and Florina. Our results suggesting that Eastern Anatolia apple populations have a unique structure will be useful for future genetic and evolutionary studies on apples.

Profiling of proteasome activity in Alyssum species on serpentine soils in Turkey reveals possible insight into nickel tolerance and accumulation.

In crops and most plants, nickel induces oxidative stress resulting in oxidized and misfolded proteins. Proteasomes maintain cellular homeostasis during stress by removing these damaged proteins. Although mild stress tolerance is mediated by proteasomal proteolysis of misfolded and oxidized proteins, previous studies have observed that severe nickel stress decreases proteasome activity in nickel-sensitive plants. Whether or not proteasome function is impaired in nickel-tolerant plants is not know. Therefore, we tested the hypothesis that proteasome activity is elevated in nickel-tolerant Alyssum species capable of accumulating nickel to unusually high levels. Our field studies examined Alyssum sibiricum and Alyssum caricum, a moderate nickel accumulator and hyper-accumulator respectively, growing on their native serpentine soil in Turkey. A. sibiricum had higher proteasome activity on serpentine soil compared to non-serpentine soil; these plants also had elevated levels of nickel accumulation and higher proteasome activity compared to other low accumulating plants in the genus Festuca or Astragalus. In A. caricum, proteasome activity was very weakly correlated with nickel soil bioavailability or accumulation in leaf tissue, suggesting that proteasome function was not impaired in plants that accumulated the highest concentration of nickel. We discuss if maintained proteasome activity might underpin nickel tolerance and the unique ecophysiology of nickel hyper-accumulation in plants.

Determination of genetic diversity of Vitis vinifera cv. Kabarcik populations from the Coruh Valley using SSR markers.

Northeastern Turkey is recognized as one of the most important germplasm centers for the grape in the world. In the present study, simple sequence repeat markers were used to investigate the genetic diversity between four Vitis vinifera cv. Kabarcik populations sampled from the Coruh Valley in Turkey, at altitudes of 800-1,150 m. The mean observed number of alleles per locus varied from 2 (loci VVMD7 and VVMD24) to 6 (VVS2) among populations. The population from the highest altitude showed the greatest average number of alleles, 4.5. With regard to the six loci examined in all populations, the mean observed heterozygosity was higher than the expected heterozygosity. Among the loci, VVS2 (probability of identity = 0.137) was found to be the most informative among populations. Genetic distances between populations ranged from 0.072 to 0.216. Genetic differentiation among populations was strongly related to geographic distances in all populations.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets.

BACKGROUND: Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses. RESULTS: A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues. CONCLUSIONS: The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods.

Transcript abundance profiles reveal larger and more complex responses of grapevine to chilling compared to osmotic and salinity stress.

Cabernet Sauvignon grapevines were exposed to sudden chilling (5 degrees C), water deficit (PEG), and an iso-osmotic salinity (120 mM NaCl and 12 mM CaCl(2)) for 1, 4, 8, and 24 h. Stomatal conductance and stem water potentials were significantly reduced after stress application. Microarray analysis of transcript abundance in shoot tips detected no significant differences in transcript abundance between salinity and PEG before 24 h. Chilling stress relates to changes in membrane structure, and transcript abundance patterns were predicted to reflect this. Forty-three percent of transcripts affected by stress vs control for 1 through 8 h were affected only by chilling. The functional categories most affected by stress included metabolism, protein metabolism, and signal transduction. Osmotic stress affected more protein synthesis and cell cycle transcripts, whereas chilling affected more calcium signaling transcripts, indicating that chilling has more complex calcium signaling. Stress affected many hormone (ABA, ethylene, and jasmonate) and transcription factor transcripts. The concentrations and transporter transcripts of several anions increased with time, including nitrate, sulfate, and phosphate. The transcript abundance changes in this short-term study were largely the same as a gradually applied long-term salinity and water-deficit study (Cramer et al. Funct Integr Genomics 7:111-134, 2007), but the reverse was not true, indicating a larger and more complex response in the acclimation process of a gradual long-term stress.

Proteomic analysis reveals differences between Vitis vinifera L. cv. Chardonnay and cv. Cabernet Sauvignon and their responses to water deficit and salinity.

The impact of water deficit and salt stress on two important wine grape cultivars, Chardonnay and Cabernet Sauvignon, was investigated. Plants were exposed to increasing salinity and water deficit stress over a 16 d time period. Measurements of stem water potentials, and shoot and leaf lengths indicated that Chardonnay was more tolerant to these stresses than Cabernet Sauvignon. Shoot tips were harvested every 8 d for proteomic analysis using a trichloroacetic acid/acetone extraction protocol and two-dimensional gel electrophoresis. Proteins were stained with Coomassie Brilliant Blue, quantified, and then 191 unique proteins were identified using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry. Peptide sequences were matched against both the NCBI nr and TIGR Vitis expressed sequence tag (EST) databases that had been implemented with all public Vitis sequences. Approximately 44% of the protein isoforms could be identified. Analysis of variance indicated that varietal difference was the main source of protein expression variation (40%). In stressed plants, reduction of the amount of proteins involved with photosynthesis, protein synthesis, and protein destination was correlated with the inhibition of shoot elongation. Many of the proteins up-regulated in Chardonnay were of unclassified or of unknown function, whereas proteins specifically up-regulated in Cabernet Sauvignon were involved in protein metabolism.

Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles.

Grapes are grown in semiarid environments, where drought and salinity are common problems. Microarray transcript profiling, quantitative reverse transcription-PCR, and metabolite profiling were used to define genes and metabolic pathways in Vitis vinifera cv. Cabernet Sauvignon with shared and divergent responses to a gradually applied and long-term (16 days) water-deficit stress and equivalent salinity stress. In this first-of-a-kind study, distinct differences between water deficit and salinity were revealed. Water deficit caused more rapid and greater inhibition of shoot growth than did salinity at equivalent stem water potentials. One of the earliest responses to water deficit was an increase in the transcript abundance of RuBisCo activase (day 4), but this increase occurred much later in salt-stressed plants (day 12). As water deficit progressed, a greater number of affected transcripts were involved in metabolism, transport, and the biogenesis of cellular components than did salinity. Salinity affected a higher percentage of transcripts involved in transcription, protein synthesis, and protein fate than did water deficit. Metabolite profiling revealed that there were higher concentrations of glucose, malate, and proline in water-deficit-treated plants as compared to salinized plants. The metabolite differences were linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Water-deficit-treated plants appear to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals (reactive oxygen species), and cope with photoinhibition.

AFLP analysis of genetic variation within the two economically important Anatolian grapevine (Vitis vinifera L.) varietal groups.

The Anatolian region of modern-day Turkey is believed to have played an important role in the history of grapevine (Vitis vinifera L.) domestication and spread. Despite this, the rich grape germplasm of this region is virtually uncharacterized genetically. In this study, the amplified fragment length polymorphisms (AFLP)-based genetic relations of the grapevine accessions belonging to the 2 economically important Anatolian table grape varietal groups known as V. vinifera 'Misket' (Muscat) and V. vinifera 'Parmak' were studied. Thirteen AFLP primer combinations used in the analyses revealed a total of 1495 (35.5% polymorphic) and 1567 (34.6% polymorphic) DNA fragments for the 'Misket' and 'Parmak' varietal groups, respectively. The unweighted pair-group method with arthimetic averaging (UPGMA) cluster analysis and principal coordinate analysis (PCA) conducted on polymorphic AFLP markers showed that both varietal groups contain a number of synonymous (similar genotypes known by different names) as well as homony mous (genetically different genotypes known by the same name) accessions. Our results also showed that 6 of the Anatolian 'Misket' genotypes were genetically very similar to V. vinifera 'Muscat of Alexandria', implying that these genotypes might have played some role in the formation of this universally known grape cultivar. Finally, the close genetic similarities found here between 'Muscat of Alexandria' and V. vinifera 'Muscat of Hamburg' support the recent suggestion that 'Muscat of Hamburg' probably originated from 'Muscat of Alexandria' through spontaneous hybridizations. Overall, the results of this study have implications for not only preservation and use of the Anatolian grape germplasm, but also better understanding of the historical role that this region has played during the domestication of grapes.

Characterizing the grape transcriptome. Analysis of expressed sequence tags from multiple Vitis species and development of a compendium of gene expression during berry development.

We report the analysis and annotation of 146,075 expressed sequence tags from Vitis species. The majority of these sequences were derived from different cultivars of Vitis vinifera, comprising an estimated 25,746 unique contig and singleton sequences that survey transcription in various tissues and developmental stages and during biotic and abiotic stress. Putatively homologous proteins were identified for over 17,752 of the transcripts, with 1,962 transcripts further subdivided into one or more Gene Ontology categories. A simple structured vocabulary, with modules for plant genotype, plant development, and stress, was developed to describe the relationship between individual expressed sequence tags and cDNA libraries; the resulting vocabulary provides query terms to facilitate data mining within the context of a relational database. As a measure of the extent to which characterized metabolic pathways were encompassed by the data set, we searched for homologs of the enzymes leading from glycolysis, through the oxidative/nonoxidative pentose phosphate pathway, and into the general phenylpropanoid pathway. Homologs were identified for 65 of these 77 enzymes, with 86% of enzymatic steps represented by paralogous genes. Differentially expressed transcripts were identified by means of a stringent believability index cutoff of > or =98.4%. Correlation analysis and two-dimensional hierarchical clustering grouped these transcripts according to similarity of expression. In the broadest analysis, 665 differentially expressed transcripts were identified across 29 cDNA libraries, representing a range of developmental and stress conditions. The groupings revealed expected associations between plant developmental stages and tissue types, with the notable exception of abiotic stress treatments. A more focused analysis of flower and berry development identified 87 differentially expressed transcripts and provides the basis for a compendium that relates gene expression and annotation to previously characterized aspects of berry development and physiology. Comparison with published results for select genes, as well as correlation analysis between independent data sets, suggests that the inferred in silico patterns of expression are likely to be an accurate representation of transcript abundance for the conditions surveyed. Thus, the combined data set reveals the in silico expression patterns for hundreds of genes in V. vinifera, the majority of which have not been previously studied within this species.