Differential Expression Profiling of Microspores During the Early Stages of Isolated Microspore Culture Using the Responsive Barley Cultivar Gobernadora

Abstract

In barley, it is possible to induce embryogenesis in the haploid and uninucleate microspore to obtain a diploid plant that is perfectly homozygous. To change developmental fates in this fashion, microspores need to engage in cellular de-differentiation, interrupting the pollen formation, and restore totipotency prior to engaging in embryogenesis. In this work, we used the barley cultivar Gobernadora to characterize the transcriptome of microspores prior to (day 0) and immediately after (days 2 and 5) the application of a stress pretreatment. A deep RNA-seq analysis revealed that microspores at these three time points exhibit a transcriptome of ∼14k genes, ∼90% of which were shared. An expression analysis identified a total of 3,382 differentially expressed genes (DEGs); of these, 2,155 and 2,281 DEGs were respectively identified when contrasting expression at days 0 and 2 and at days 2 and 5. These define 8 expression profiles in which DEGs share a common up- or down-regulation at these time points. Up-regulation of numerous glutathione S-transferase and heat shock protein genes as well as down-regulation of ribosomal subunit protein genes was observed between days 0 and 2. The transition from microspores to developing embryos (days 2 vs. 5) was marked by the induction of transcription factor genes known to play important roles in early embryogenesis, numerous genes involved in hormone biosynthesis and plant hormonal signal transduction in addition to genes involved in secondary metabolism. This work sheds light on transcriptional changes accompanying an important developmental shift and provides candidate biomarkers for embryogenesis in barley.

Keywords

• barley
• transcriptome analysis
• isolated microspore culture