Changes in mRNA expression following abiotic stresses have been extensively analyzed in plant species using microarrays. Different stress conditions, tissues and plant species, from Arabidopsis to cereal crops [10
], have been investigated with microarray tools to find drought-regulated genes. Roots and leaves from seedlings were analyzed in wheat, barley and rice to describe the variation in gene expression induced in response to a dehydration shock imposed for few hours [16
]. A slow-drying treatment was applied to study the transcriptome changes in wheat leaves at booting stage [63
], or in developing kernels in maize and rice [64
]. The comparative analysis of these data highlight that the conservation of the molecular response to dehydration across species and across experiments is generally low despite the presence of common regulatory mechanisms. For instance, the comparison between the genes found to be up- or down-regulated in Arabidopsis in response to dehydration by Matsui et al. [10
] (more than 4,000 genes) with those found in the experiment here described highlighted only 68 and 180 common genes for CS and Creso, respectively. When Talamè et al. [17
] compared the expression changes in leaves of barley plants subjected to slow or rapid drying, only a small portion of differentially expressed transcripts (about 10%) showed similar expression profile regardless of the dynamics of the water stress treatment. Variations in the response to drought depending on stress dynamics and on the stage of development were also reported for specific stress-responsive genes in durum wheat by De Leonardis et al. [66
]. These results underline the importance of selecting stress conditions and tissues representing a physiological status that has a relevant role during field growth to identify pathways with a field relevant role in stress tolerance. In the present work, bread and durum wheat plants were subjected to a slow drought stress during grain filling, a critical stage for yield determination. The expression analysis was carried out on glumes, the last green and photosynthetically active tissues during grain filling. For these reasons our work, more than others, should give a close representation of a yield-relevant drought response.
The durum and the bread wheat genotypes considered in this work showed different reactions to the water stress treatment when grown in soils with the same amount of available water. CS and CS_5AL-10 were characterized by less negative leaf water potential values and they took much longer than durum wheat to reach these values, suggesting that a moderate water stress can already induce in these genotypes a response leading to a lower water loss. Differences in response to water stress between hexaploid and tetraploid genotypes were already described in previous reports. Gavuzzi et al. [67
] compared 6 bread wheat, 6 durum wheat and 6 barley genotypes for physiological parameters following water stress, and found that bread wheat had the smallest water loss values. In a similar experiment, two hexaploid genotypes exhibited a higher level of proline with respect to two tetraploids in response to drought [68
]. In our experiment, bread and durum wheat were characterized by a significantly different drought response. 106 probe sets were above the induction threshold when MS and CTRL samples were compared in CS. On the contrary, in Creso no probe set was above the induction threshold in the same comparison and only in SS vs
MS and SS vs
CTRL comparisons was possible to identify significantly induced/repressed genes. For instance, a set of genes encoding microtubule subunits and cell wall degradation enzymes was found down-regulated in Creso after the SS only (Figure , clusters. 15 and 19). These transcriptional changes suggest a block in cell division and/or elongation supporting a smaller transpiring surface, a typical component of the plant drought response [42
]. These observations indicate that the ability of CS to maintain a higher water potential during drought stress is associated to a more prompt molecular response, while Creso needs a more severe drought stress to activate any transcriptional response.
Although durum wheat and bread wheat are two distinct species with a different genome organization (tetraploid -AABB, and hexaploid -AABBDD, respectively), their share the same A and B genomes. The similarity between bread and durum wheat for sequences carried on A and B genomes is very high. Chantret et al. [69
], studying the Hardeness
locus (GenBank accession number AY491681
, ca 100 Kb) in the A and B genomes of durum and bread wheat, highlighted that the two species share 97% and 99% of nucleotide identity for A and B genome, respectively. Furthermore, in a preliminary bioinformatic experiment, 104 randomly selected durum wheat ESTs (48,530 bp in total) were blasted to find the corresponding bread wheat sequences. Considering the BLAST best matches of all queries a mean identity of 98.5% (SD 0.02%) and a mean coverage of 95.2% were calculated, a results in agreement with the data of Chantret et al. The high level of genome identity between bread and durum wheat sustains the use of the same microarray for comparison of the transcriptomes of the two species, although the estimated 2% of sequence polymorphisms might lead to a small over-estimation of the transcriptomic differences.
In well-irrigated conditions, the CS (hexaploid wheat) and Creso (tetraploid wheat) transcriptomes were very different. About 6.6 thousand genes were found to be differentially expressed between the two wheat species (Table ) on a total amount of approximately 30,000 expressed genes detected in bread wheat. Although about 80% of the genes expressed in absence of stress were common between Creso and CS, the drought response in the two genotypes was significantly different. 1,470 genes were above the threshold in the SS vs CTRL in Creso, while only 842 were detected in the same comparison in CS (Table ) and among these genes only 201 were in common.
The analysis of the molecular response to drought revealed both common and genotype-specific features. A set of 556 genes were clustered in groups showing a very similar expression profile in Creso and CS (Figure and ). Notably, many genes involved in well known drought-responsive pathways (i.e. ABA, proline, sorbitol and glycine-betaine) were commonly activated in all genotypes. The expression levels of the NCED
-related probe sets, the key enzyme of ABA biosynthesis [35
], were strongly up-regulated by water stress in all genotypes suggesting that the drought treatment imposed during the experiment entailed the activation of the ABA synthesis. In Arabidopsis, the accumulation of ABA in response to water deficit leads to the induction of the transcription factor At-HB7
(homeobox-leucine zipper) that, in turn, activates the expression of At-RD20
]. The probe sets coding for HB7
wheat homologous sequences (TaAffx.108538.1.S1_at, Ta.9830.2.1.S1_at, respectively) were co-regulated with NCED
and grouped in cluster 3 and 14, respectively, suggesting that this specific response is conserved in wheat.
Other genes grouped in commonly up- or down-regulated clusters play a role in primary metabolism, energy regulation, cell rescue or interaction with environment.
On the contrary, evidences for drought-responsive features associated to the different genomic structure of Creso, CS and CS_5A-10 were also present. Some drought-related genes were expressed at lower level (or not expressed) in Creso or in CS_5A-10 compared to CS (see clusters 2, 4 and, to less extent, 21), this finding can, to some extent, be associated to the absence of the D genome (or 5AL-10). Consequently, these genes could be located on the D genome (or 5AL-10), as demonstrated for some of them by expression based mapping, or could be controlled by genetic factors located on the D genome (or 5AL-10). Furthermore, several clusters were characterized by a higher expression level in Creso or in CS_5A-10 than in CS (see clusters 5 and 6), underlining that the different genome organization have a direct consequence on plant adaptation to stress. The 5AL-10 deleted region carries the Cbf
]. Although originally described as cold-regulated, the Cbf
transcription factors are also induced during exposure to drought in wheat [66
]. The absence of an important class of stress-related transcription factors can lead to modifications in the expression of many other genes located overall the genome. Furthermore, the analysis of the genes involved in the proline biosynthetic pathway have suggested an up-regulation of the ornithine-dependent pathway in bread wheat (Figure ), while the enhanced expression of a set of transposons and retrotransposons was detected in CS_5AL-10 only. It is known that transposon and retrotransposon expression can be activated by biotic/abiotic stresses [73
], our data suggest that the combination of abiotic stress with a "genetic stress" due to chromosomal deletion represent a suitable condition for a general up-regulation of transposon and retrotransposon-related mRNAs.