Alessio Aprile

Transcriptional profiling in response to terminal drought stress reveals differential responses along the wheat genome

BackgroundWater stress during grain filling has a marked effect on grain yield, leading to a reduced endosperm cell number and thus sink capacity to accumulate dry matter. The bread wheat cultivar Chinese Spring (CS), a Chinese Spring terminal deletion line (CS_5AL-10) and the durum wheat cultivar Creso were subjected to transcriptional profiling after exposure to mild and severe drought stress at the grain filling stage to find evidences of differential stress responses associated to different wheat genome regions.ResultsThe transcriptome analysis of Creso, CS and its deletion line revealed 8,552 non redundant probe sets with different expression levels, mainly due to the comparisons between the two species. The drought treatments modified the expression of 3,056 probe sets. Besides a set of genes showing a similar drought response in Creso and CS, cluster analysis revealed several drought response features that can be associated to the different genomic structure of Creso, CS and CS_5AL-10. Some drought-related genes were expressed at lower level (or not expressed) in Creso (which lacks the D genome) or in the CS_5AL-10 deletion line compared to CS. The chromosome location of a set of these genes was confirmed by PCR-based mapping on the D genome (or the 5AL-10 region). Many clusters were characterized by different level of expression in Creso, CS and CS_AL-10, suggesting that the different genome organization of the three genotypes may affect plant adaptation to stress. Clusters with similar expression trend were grouped and functional classified to mine the biological mean of their activation or repression. Genes involved in ABA, proline, glycine-betaine and sorbitol pathways were found up-regulated by drought stress. Furthermore, the enhanced expression of a set of transposons and retrotransposons was detected in CS_5AL-10.ConclusionBread and durum wheat genotypes were characterized by a different physiological reaction to water stress and by a substantially different molecular response. The genome organization accounted for differences in the expression level of hundreds of genes located on the D genome or controlled by regulators located on the D genome. When a genomic stress (deletion of a chromosomal region) was combined with low water availability, a molecular response based on the activation of transposons and retrotransposons was observed.

The expression of several Cbf genes at the Fr-A2 locus is linked to frost resistance in wheat

The C-repeat binding factor (Cbf) gene family has been shown to have a critical role in the regulation of low-temperature stress response in Arabidopsis. In Triticum monococcum, a locus carrying a family of Cbf-like genes, orthologs of Arabidopsis Cbf genes, is tightly linked to the frost tolerance locus Fr-Am2, representing candidates for the differences in frost tolerance mapped at this locus. In this work we show that several Cbf genes have dramatically different levels of induction after cold exposure in hexaploid wheat. The Cbf-transcription levels differ between substitution and single chromosome recombinant lines carrying different 5A chromosomes or chromosome segments of the chromosome 5A from frost-tolerant and frost-sensitive wheat varieties. When the expression of eight Cbf genes, previously mapped at the Fr-A2 locus was investigated with gene specific primers using real-time RT-PCR, three Cbf sequences (Cbf1A, Cbf1C, Cbf7) showed a significantly higher relative transcription level (more than fourfold change) in lines differing for the Fr-A2 region. Differences in Cbf expression were also associated with a variation in frost tolerance. These results suggest that the amount of some Cbf mRNAs might be a critical factor for determining the level of frost tolerance in wheat.

Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

BackgroundReactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli.ResultsTo investigate this role further, we exposed wild type Arabidopsis thaliana plants and the double mutant npq1lut2 to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (1O2) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by 1O2 which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in npq1lut2 is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the flu mutant was the only biological system adopted to define the regulation of gene expression by 1O2. In this work, we propose the use of mutants accumulating 1O2 by mechanisms different from those activated in flu to better identify ROS signalling.ConclusionsWe propose that the lack of zeaxanthin and lutein leads to 1O2 accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to 1O2 accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed.

Novel durum wheat genes up-regulated in response to a combination of heat and drought stress

We report the effect of heat, drought and combined stress on the expression of a group of genes that are up-regulated under these conditions in durum wheat (Triticum turgidum subsp. durum) plants. Modulation of gene expression was studied by cDNA-AFLP performed on RNAs extracted from flag leaves. By this approach, we identified several novel durum wheat genes whose expression is modulated under different stress conditions. We focused on a group of hitherto undescribed up-regulated genes in durum wheat, among these, 7 are up-regulated by heat, 8 by drought stress, 15 by combined heat and drought stress, 4 are up-regulated by both heat and combined stress, and 3 by both drought and combined stress. The functional characterization of these genes will provide new data that could help the developing of strategies aimed at improving durum wheat tolerance to field stress.

Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues

We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature.

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.

Expression of the H+-ATPase AHA10 proton pump is associated with citric acid accumulation in lemon juice sac cells

The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a “sweet” lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit (Faris acid; FA) on different branches; it is apparently a graft chimera with layer L1 derived from Millsweet limetta and layer L2 from a standard lemon. The transcription profiles of Faris sweet lemon were compared with Faris acid lemon and Frost Lisbon (L), which is a standard sour lemon genetically indistinguishable from Faris in prior work with SSR markers. Analysis of microarray data revealed that the transcriptomes of the two sour lemon genotypes were nearly identical. In contrast, the transcriptome of Faris sweet lemon was very different from those of both sour lemons. Among about 1,000 FNA-specific, presumably pH-related genes, the homolog of Arabidopsis H+-ATPase proton pump AHA10 was not expressed in FNA, but highly expressed in FA and L. Since Arabidopsis AHA10 is involved in biosynthesis and acidification of vacuoles, the lack of expression of the AHA10 citrus homolog represents a very conspicuous molecular feature of the FNA sweet phenotype. In addition, high expression of several 2-oxoglutarate degradation-related genes in FNA suggests activation of the GABA shunt and degradation of valine and tyrosine as components of the mechanism that reduces the level of citric acid in sweet lemon.

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.

Cadmium Concentration in Grains of Durum Wheat (Triticum turgidum L. subsp. durum)

Heavy metal excess in soil represents a critical problem for crop productivity. Among these pollutants, cadmium (Cd) is one of the most dangerous in terms of food-chain contamination. Two durum wheat near-isogenic lines (NILs) and 12 commercial varieties (cultivars Arcangelo, Aureo, Aziziah, Cappelli, Cirillo, Creso, Iride, Maestrale, Parsifal, Russello, Strongfield, and Svevo) of durum wheat were exposed to a nontoxic level of Cd to evaluate its concentration in grains, roots, and shoots, as well as effects on biomass production. Cultivar Iride showed the most interesting behavior because it stored large amounts of Cd in the roots, preventing its translocation to grains. On the contrary, Cirillo and Svevo genotypes were characterized by a high Cd concentration in the grains. Furthermore, a molecular characterization employing the ScOPC20 marker associated with the Cd uptake locus has shown the absence of the expected fragment in the Iride variety and in other varieties characterized by low Cd concentration, as well as the presence of it in high Cd-accumulating cultivars.

Wounding tomato fruit elicits ripening-stage specific changes in gene expression and production of volatile compounds

Summary As tomato fruit ripen, the wounding-elicited transcriptome and VOC profiles alter. This shift is consistent with a change from protection of developing seeds to attraction of frugivores for seed dispersal.