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

Abstract

BackgroundIncreasing 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 Grain Protein Content (GPC) 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.Based on the early and rapid up-regulation of GPC 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 GPC down-regulation on the wheat flag-leaf transcriptome 12 days after anthesis. At this early stage of senescence GPC 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.ResultsWe 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 GPC (431 ≥ 2-fold change). Transcript level ratios between transgenic and wild type plants showed a high correlation (R = 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.ConclusionsMonocarpic 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 GPC-regulated genes, which includes transporters, hormone regulated genes, and transcription factors. These GPC-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.

Functional characterization of GPC-1 genes in hexaploid wheat

“…qRT-PCR-The transcription profiles of eight GPC-regulated genes identified in our previous study (Cantu et al 2011) were compared in gpc-1 double mutant and control plants from the UCD 2011 greenhouse pot experiment. RNA was extracted from the flag leaves of ten biological replicates of each genotype harvested at four time points; heading date, 2, 12 and 22 DAA.…”

“…RNA was extracted from the flag leaves of ten biological replicates of each genotype harvested at four time points; heading date, 2, 12 and 22 DAA. All sample preparation and qRT-PCR reactions were performed as described by Cantu et al (2011). Transcript levels are expressed as fold-ACTIN levels and are the average of ten biological replicates ± SE.…”

“…Silencing of all homoeologous GPC-1 and GPC-2 genes using RNA interference (RNAi) results in a 24-30 day delay in senescence and a reduction in grain protein, zinc and iron content of more than 30% in comparison to the wild type control plants (Uauy et al 2006b). An RNAseq experiment was carried out to compare the transcriptomes of the GPCRNAi and control lines at an early stage of senescence and identified 691 differentially expressed genes, which included transporters, hormone-regulated genes and transcription factors, many of which were up-regulated during senescence (Cantu et al 2011). These results suggest that the GPC genes are early regulators of senescence and that senescence is an active process that involves the precise regulation of hundreds of genes.…”

PvNAC1 and PvNAC2 Are Associated with Leaf Senescence and Nitrogen Use Efficiency in Switchgrass

“…Although there have been some microarray-based transcriptomic studies of senescence in Arabidopsis [13][14][15], Populus [16], wheat [17], and barley [18], little is known about processes that are regulated directly by NAC TFs. Recently, the effect of downregulation of TtNAM-B1 on wheat flag-leaf gene expression during senescence (12 days after anthesis) was investigated using RNA-seq technology [19]. The list of putative TtNAM-B1-regulated genes included transporters, hormone-regulated genes, and other transcription factors.…”

Divergent functions of orthologous NAC transcription factors in wheat and rice

“…The senescence-associated expression pattern of dozens of NAC family members (Buchanan-Wollaston et al 2005;Breeze et al 2011;Guo et al 2004;Cantu et al 2011) suggests that they play a prominent role in senescence. Several members of the NAC family from Arabidopsis and rice such as ORE1/ANAC092/AtNAC2, ORS1, AtNAP and OsNAC5 have been functionally characterized and shown to be involved in the regulation of leaf senescence (Balazadeh et al 2008;Sperotto et al 2009;Guo and Gan 2006;Kim et al 2009;Balazadeh et al 2011).…”