Background: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion: The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

High temperatures occurring during grain filling are known to affect wheat grain yield and quality considerably. In this paper we report the results of experiments carried out with two cultivars of bread wheat ( Triticum aestivum L.) and two cultivars of durum wheat ( Triticum durum Desf.). The plants, cultivated in pots, were subjected to 13 heat treatments (temperature up to 40°C) differing in duration and timing and starting 7 days after anthesis. Heat treatments were applied by temporary transfer of the pots to a glasshouse where the temperature rose to 40°C as a consequence of solar radiation for periods ranging from 5 to 30 days. The applied heat shocks substantially affected dry matter and protein accumulation in the different parts of the plant. Early heat shock (5 days with a total of 18 h of temperature in the range 35–40°C) caused a small reduction of kernel mass and no effect on protein per kernel; the damage was greater in the central and in the final stage of grain filling. Plants subjected to a progressive increase of temperature, or to an early heat shock, acquired thermotolerance to further heat shocks. Continuous exposure to very high temperatures from 27 days after pollination to maturity did not negatively affect grain yield and it facilitated the remobilisation of nitrogen from vegetative to reproductive organs. Rheological properties were severely affected by heat shocks at all stages of grain filling: 5 days of heat shock were sufficient to reduce mixing tolerance by 40–60%. These variations in rheological properties were accompanied by modification of the level of protein aggregation: soluble polymeric proteins and low molecular weight gliadins progressively increased according to the intensity of the stress, while insoluble polymeric proteins decreased. Our experiments, carried out in conditions close to the Mediterranean climate, indicate that the occurrence of very high temperature in the range 35–40°C during grain filling substantially affects dry matter and protein accumulation in the different parts of the plant. The formation of the complex protein aggregates responsible for positive dough mixing properties is significantly reduced by very high temperature. When heat shock came late in grain filling, grain yield and protein concentration were not negatively affected but a ‘dough weakening’ effect, which may reduce the commercial value of the production, is to be expected.

The effect of iron (Fe) and sulphur (S) deprivation on sulphate uptake and assimilation pathways was investigated in durum wheat by analysing the expression of genes coding for major transporters and enzymes involved in sulphate assimilation and reduction: high-affinity sulphate transporters (TdSultr1.1 and TdSultr1.3), ATP sulphurylase (TdATPSul1 and TdATPSul2), APS reductase (TdAPR), sulphite reductase (TdSiR), O-acetylserine(thiol)lyase (TdOASTL1 and TdOASTL2), and serine acetyltransferase (TdSAT1 and TdSAT2). Further experiments were carried out to detect changes in the activities of these enzymes, together with the evaluation of growth parameters (fresh biomass accumulation, leaf green values, and total S, thiol, and Fe concentrations). Fe shortage in wheat plants under adequate S nutrition resulted in an S deficiency-like response. Most of the genes of the S assimilatory pathway induced by S deprivation (TdATPSul1, TdAPR, TdSir, TdSAT1, and TdSAT2) were also significantly up-regulated after the imposition of the Fe limitation under S-sufficient conditions. However, the differential expression of genes encoding the two high-affinity transporters (TdSultr1.1 and TdSultr1.3) indicates that the mechanisms of sulphate uptake regulation under Fe and S deficiency are different in wheat. Moreover, it was observed that the mRNA level of genes encoding ATPS, APR, and OASTL and the corresponding enzyme activities were often uncoupled in response to Fe and S availability, indicating that most probably their regulation involves a complex interplay of transcriptional, translational, and/or post-translational mechanisms induced by S and/or Fe deficiency.