Caterina Marè

Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley

WRKY proteins constitute a large family of plant specific transcription factors implicated in many different processes. Here we describe Hv-WRKY38,a barley gene coding for a WRKY protein, whose expression is involved in cold and drought stress response. Hv-WRKY38 was early and transiently expressed during exposure to low non-freezing temperature, in ABA-independent manner. Furthermore, it showed a continuous induction during dehydration and freezing treatments. A WRKY38:YFP fusion protein was found to localise into the nucleus upon introduction into epidermal onion cells. Bacterially expressed Hv-WRKY38 was able to bind in vitro to the W-box element (T)TGAC(C/T) also recognisable by other WRKY proteins. Hv-WRKY38genomic DNA was sequenced and mapped onto the centromeric region of the barley chromosome 6H. Arabidopsis and rice sequences homologous to Hv-WRKY38 were also identified. Our results indicate that Hv-WRKY38 transcription factor may play a regulatory role in abiotic stress response.

Different stress responsive strategies to drought and heat in two durum wheat cultivars with contrasting water use efficiency

BackgroundDurum wheat often faces water scarcity and high temperatures, two events that usually occur simultaneously in the fields. Here we report on the stress responsive strategy of two durum wheat cultivars, characterized by different water use efficiency, subjected to drought, heat and a combination of both stresses.ResultsThe cv Ofanto (lower water use efficiency) activated a large set of well-known drought-related genes after drought treatment, while Cappelli (higher water use efficiency) showed the constitutive expression of several genes induced by drought in Ofanto and a modulation of a limited number of genes in response to stress. At molecular level the two cvs differed for the activation of molecular messengers, genes involved in the regulation of chromatin condensation, nuclear speckles and stomatal closure. Noteworthy, the heat response in Cappelli involved also the up-regulation of genes belonging to fatty acid β-oxidation pathway, glyoxylate cycle and senescence, suggesting an early activation of senescence in this cv. A gene of unknown function having the greatest expression difference between the two cultivars was selected and used for expression QTL analysis, the corresponding QTL was mapped on chromosome 6B.ConclusionOfanto and Cappelli are characterized by two opposite stress-responsive strategies. In Ofanto the combination of drought and heat stress led to an increased number of modulated genes, exceeding the simple cumulative effects of the two single stresses, whereas in Cappelli the same treatment triggered a number of differentially expressed genes lower than those altered in response to heat stress alone. This work provides clear evidences that the genetic system based on Cappelli and Ofanto represents an ideal tool for the genetic dissection of the molecular response to drought and other abiotic stresses.

Cold-regulated gene expression during winter in frost tolerant and frost susceptible barley cultivars grown under field conditions

Winterhardiness is a basic trait for successful winter survival barley (Hordeum vulgare L.) crop. Freezing tolerance, a fundamental component of winterhardiness, is based on an inducible process known as hardening or cold acclimation that occurs when plants are exposed to low non-freezing temperatures. In the recent years, many temperature-dependent genes specifically expressed during hardening have been isolated. Current data on relationship between gene expression and cold tolerance are mostly based on plants grown and hardened under environmentally controlled conditions and, usually, over a short period of time. In order to verify whether variations in the molecular response to cold are likely to be of significant adaptive value under natural environments, we have followed the accumulation of several COR genes ( pt59, pao86 and paf93) and proteins (COR14a and COR14b) during the 1996/97 and 97/98 winter seasons in barley cultivars with contrasting winterhardiness capacity grown under field conditions. In the 1996/97 experiment, a winter cultivar Onice and a spring cultivar Gitane were tested for the accumulation of the cold-regulated genes and proteins. The ability of the plants to promote a strong molecular response to cold was found to be associated with the winterhardiness capacity of the two cultivars. This result was further tested in the winter season 1997/98 using 10 barley varieties. All winter cultivars showed high accumulation of the cold-regulated proteins COR14a and COR14b, while some variations for this character were detected in the spring cultivars suggesting that the selection for winter survival has been effective to fix the high COR14 accumulation capacity. We conclude that a high level of COR14 may be a component the winter survival capacity of barley.

Transcriptomic and proteomic analyses of a pale-green durum wheat mutant shows variations in photosystem components and metabolic deficiencies under drought stress

BackgroundLeaf pigment content is an important trait involved in environmental interactions. In order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant (Triticum turgidum L. var. durum) under contrasting water availability conditions.ResultsThe pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. Under well-watered (control) conditions the mutant had lower levels of chlorophylls and carotenoids, but higher levels of xanthophyll de-epoxidation compared to wild-type. Transcriptomic analysis under control conditions showed that defense genes (encoding e.g. pathogenesis-related proteins, peroxidases and chitinases) were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. Transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. We also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction.ConclusionsThis study provides insight on the effect of pigment content in the molecular response to drought. Identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat.

The cold dependent accumulation of COR TMC-AP3 in cereals with contrasting, frost tolerance is regulated by different mRNA expression and protein turnover

The accumulation of specific cold-regulated (COR) proteins is a component of the hardening process and different amount of COR proteins has been related to different degrees of cold tolerance. A number of different mechanisms controls the accumulation of the COR proteins in the plant cells. In this work we describe the mechanisms controlling the accumulation of the COR protein TMC-AP3, a putative chloroplastic amino acid selective channel protein [1] in barley, durum, wheat, emmer and bread wheat. Winter barley and, to less extent, winter bread wheat showed a higher cor tmc-ap3 expression at low temperature than the spring one while no significant differences were detected between the emmer and the durum. wheat genotypes. After 2 days of de-hardening the transcript level dropped down in the same way in all tested genotypes, nevertheless the decrease in protein content was genotype dependent. In all frost resistant genotypes the amount of COR TMC-AP3 after 9 days of de-hardening was higher compared with that of susceptible ones. These findings suggest that resistant and susceptible genotypes have different protein degradation rate and/or mRNA translational efficiency. Differences in the protein degradation rate were not dependent from the amino acidic sequence of the protein, being extremely similar in all tested genotypes. A genetic study based on Chinese spring/Cheyenne chromosome substitution lines showed that the turnover of TMC-AP3 is a polygenic trait controlled by a number of loci being the most important located on chromosomes 1B, 2B, 2D and 4D.

Cytoplasmic genome substitution in wheat affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations

BackgroundAlloplasmic lines provide a unique tool to study nuclear-cytoplasmic interactions. Three alloplasmic lines, with nuclear genomes from Triticum aestivum and harboring cytoplasm from Aegilops uniaristata, Aegilops tauschii and Hordeum chilense, were investigated by transcript and metabolite profiling to identify the effects of cytoplasmic substitution on nuclear-cytoplasmic signaling mechanisms.ResultsIn combining the wheat nuclear genome with a cytoplasm of H. chilense, 540 genes were significantly altered, whereas 11 and 28 genes were significantly changed in the alloplasmic lines carrying the cytoplasm of Ae. uniaristata or Ae. tauschii, respectively. We identified the RNA maturation-related process as one of the most sensitive to a perturbation of the nuclear-cytoplasmic interaction. Several key components of the ROS chloroplast retrograde signaling, together with the up-regulation of the ROS scavenging system, showed that changes in the chloroplast genome have a direct impact on nuclear-cytoplasmic cross-talk. Remarkably, the H. chilense alloplasmic line down-regulated some genes involved in the determination of cytoplasmic male sterility without expressing the male sterility phenotype. Metabolic profiling showed a comparable response of the central metabolism of the alloplasmic and euplasmic lines to light, while exposing larger metabolite alterations in the H. chilense alloplasmic line as compared with the Aegilops lines, in agreement with the transcriptomic data. Several stress-related metabolites, remarkably raffinose, were altered in content in the H. chilense alloplasmic line when exposed to high light, while amino acids, as well as organic acids were significantly decreased. Alterations in the levels of transcript, related to raffinose, and the photorespiration-related metabolisms were associated with changes in the level of related metabolites.ConclusionThe replacement of a wheat cytoplasm with the cytoplasm of a related species affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations. The extent of these modifications was limited in the alloplasmic lines with Aegilops cytoplasm, and more evident in the alloplasmic line with H. chilense cytoplasm. We consider that, this finding might be linked to the phylogenetic distance of the genomes.

Expression of Cold-Regulated ( cor ) Genes in Barley

Barley is grown either in the northern countries close to the polar circle or on the Himalayan mountains up to 4500 m on the sea level. Such a great diffusion, despite the differences in the climatic conditions, already suggests that the barley gene pool should contain characters for wide environmental adaptability and good stress resistance. The genetic adaptation to cold climate can be achieved either by evolving a powerful frost tolerance ability or by limiting the life cycle to the short summer season (escape strategy). It is a known fact that the winter barley varieties are less hardy than winter wheat, rye and triticale, nevertheless barley is grown till the Polar Circle because spring early maturity cultivars are able to run their life cycle in the short summer season. Plant growth habit and heading date can therefore be considered as the basic traits involved in barley adaptation to environments since they allow to synchronise the plant life cycle with seasonal changes. Nevertheless because winter barley has a higher yielding potential than spring ones, there is a great interest to improve its frost resistance capacity.