Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

<p>Abstract</p> <p>Background</p> <p>Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from <it>Triticum aestivum </it>cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways.</p> <p>Findings</p> <p>Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: <it>TaFNRII </it>(ferredoxin-NADP(H) oxidoreductase; AJ457980.1), <it>ACT2 </it>(actin 2; TC234027), and <it>rrn26 </it>(a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: <it>CYP18-2 </it>(Cyclophilin A, AY456122.1) and <it>TaWIN1 </it>(14-3-3 like protein, AB042193) were most consistently stably expressed.</p> <p>Furthermore, we showed that <it>TaFNRII, ACT2</it>, and <it>CYP18-2 </it>are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus.</p> <p>Conclusions</p> <p>This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.</p

Effect of the down-regulation of the high Grain Protein Content (GPC) genes on the wheat transcriptome during monocarpic senescence

<p>Abstract</p> <p>Background</p> <p>Increasing the nutrient concentration of wheat grains is important to ameliorate nutritional deficiencies in many parts of the world. Proteins and nutrients in the wheat grain are largely derived from the remobilization of degraded leaf molecules during monocarpic senescence. The down-regulation of the NAC transcription factor <it>Grain Protein Content </it>(<it>GPC</it>) in transgenic wheat plants delays senescence (>3 weeks) and reduces the concentration of protein, Zn and Fe in the grain (>30%), linking senescence and nutrient remobilization.</p> <p>Based on the early and rapid up-regulation of <it>GPC </it>in wheat flag leaves after anthesis, we hypothesized that this transcription factor is an early regulator of monocarpic senescence. To test this hypothesis, we used high-throughput mRNA-seq technologies to characterize the effect of the <it>GPC </it>down-regulation on the wheat flag-leaf transcriptome 12 days after anthesis. At this early stage of senescence <it>GPC </it>transcript levels are significantly lower in transgenic GPC-RNAi plants than in the wild type, but there are still no visible phenotypic differences between genotypes.</p> <p>Results</p> <p>We generated 1.4 million 454 reads from early senescing flag leaves (average ~350 nt) and assembled 1.2 million into 30,497 contigs that were used as a reference to map 145 million Illumina reads from three wild type and four GPC-RNAi plants. Following normalization and statistical testing, we identified a set of 691 genes differentially regulated by <it>GPC </it>(431 ≥ 2-fold change). Transcript level ratios between transgenic and wild type plants showed a high correlation (<it>R </it>= 0.83) between qRT-PCR and Illumina results, providing independent validation of the mRNA-seq approach. A set of differentially expressed genes were analyzed across an early senescence time-course.</p> <p>Conclusions</p> <p>Monocarpic senescence is an active process characterized by large-scale changes in gene expression which begins considerably before the appearance of visual symptoms of senescence. The mRNA-seq approach used here was able to detect small differences in transcript levels during the early stages of senescence. This resulted in an extensive list of <it>GPC</it>-regulated genes, which includes transporters, hormone regulated genes, and transcription factors. These <it>GPC</it>-regulated genes, particularly those up-regulated during senescence, provide valuable entry points to dissect the early stages of monocarpic senescence and nutrient remobilization in wheat.</p

Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle, glumes and roots. Expression of GS genes in leaves was developmentally regulated, with both GS2 and GS1 assimilating or recycling ammonia in leaves during the period of grain development and filling. During leaf senescence the cytosolic isozymes, GS1 and GSr, were the predominant forms, suggesting major roles in assimilating ammonia during the critical phases of remobilisation of nitrogen to the grain. A preliminary analysis of three different wheat genotypes showed that the ratio of leaf GS2 protein to GS1 protein was variable. Use of this genetic variation should inform future efforts to modulate this enzyme for pre-breeding efforts to improve nitrogen use in wheat