Significance of light, sugar, and amino acid supply for diurnal gene regulation in developing barley caryopses.

The caryopses of barley (Hordeum vulgare), as of all cereals, are complex sink organs optimized for starch accumulation and embryo development. While their early to late development has been studied in great detail, processes underlying the caryopses' diurnal adaptation to changes in light, temperature, and the fluctuations in phloem-supplied carbon and nitrogen have remained unknown. In an attempt to identify diurnally affected processes in developing caryopses at the early maturation phase, we monitored global changes of both gene expression and metabolite levels. We applied the 22 K Barley1 GeneChip microarray and identified 2,091 differentially expressed (DE) genes that were assigned to six major diurnal expression clusters. Principal component analysis and other global analyses demonstrated that the variability within the data set relates to genes involved in circadian regulation, storage compound accumulation, embryo development, response to abiotic stress, and photosynthesis. The correlation of amino acid and sugar profiles with expression trajectories led to the identification of several hundred potentially metabolite-regulated DE genes. A comparative analysis of our data set and publicly available microarray data disclosed suborgan-specific expression of almost all diurnal DE genes, with more than 350 genes specifically expressed in the pericarp, endosperm, or embryo tissues. Our data reveal a tight linkage between day/night cycles, changes in light, and the supply of carbon and nitrogen. We present a model that suggests several phases of diurnal gene expression in developing barley caryopses, summarized as starvation and priming, energy collection and carbon fixation, light protection and chaperone activity, storage and growth, and embryo development.

Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare) WRKY transcription factor family reveals putatively retained functions between monocots and dicots.

BACKGROUND: WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far as regulators in sucrose signaling, pathogen defense, and in response to cold and drought. However, their phylogenetic relationship remained unresolved. RESULTS: In this study, we used available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features, the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 to 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. CONCLUSION: HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in monocot and dicot species.

Transcriptome analysis of high-temperature stress in developing barley caryopses: early stress responses and effects on storage compound biosynthesis.

High-temperature stress, like any abiotic stress, impairs the physiology and development of plants, including the stages of seed setting and ripening. We used the Affymetrix 22K Barley1 GeneChip microarray to investigate the response of developing barley (Hordeum vulgare) seeds, termed caryopses, after 0.5, 3, and 6 h of heat stress exposure; 958 induced and 1122 repressed genes exhibited spatial and temporal expression patterns that provide a detailed insight into the caryopses' early heat stress responses. Down-regulation of genes related to storage compound biosynthesis and cell growth provides evidence for a rapid impairment of the caryopsis' development. Increased levels of sugars and amino acids were indicative for both production of compatible solutes and feedback-induced accumulation of substrates for storage compound biosynthesis. Metadata analysis identified embryo and endosperm as primary locations of heat stress responses, indicating a strong impact of short-term heat stress on central developmental functions of the caryopsis. A comparison with heat stress responses in Arabidopsis shoots and drought stress responses in barley caryopses identified both conserved and presumably heat- and caryopsis-specific stress-responsive genes. Summarized, our data provide an important basis for further investigation of gene functions in order to aid an improved heat tolerance and reduced losses of yield in barley as a model for cereal crops.

Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling.

Sugar signalling cascades are important components of regulatory networks in cells. Compared with the situation in bacteria, yeast and animals, participants of the sugar signalling pathways in plants are poorly understood. Several genes involved in starch synthesis are known to be sugar inducible, although the signal transduction pathways remain undisclosed. We reported recently the isolation of SUSIBA2, a transcription factor involved in sugar-mediated regulation of starch synthesis. Here, we used antisense oligodeoxynucleotide (ODN) inhibition, a powerful approach in medical sciences, to block the effects of SUSIBA2 in sugar-treated barley leaves. The uptake and intracellular trafficking of an 18-mer susiba2 antisense ODN in leaves were followed by confocal microscopy. Administration of the antisense ODN to the leaves impeded susiba2 expression by RNase H activation. This dramatically diminished the ectopic expression of the iso1 and sbeIIb genes and resulted in altered starch synthesis. This study illustrates the successful exploitation of the antisense ODN technology in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and identifies SUSIBA2 as a transcriptional activator in plant sugar signalling. Based on our findings, we propose a model for sugar-signalling control of starch synthesis.