Isolation of tissues and preservation of RNA from intact, germinated barley grain

Abstract

Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19 611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24 h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination.

Temporal tissue‐specific regulation of transcriptomes during barley (Hordeum vulgare) seed germination

“…Major carbohydrate metabolism (starch synthesis and degradation), ABA metabolism and signal transduction, abiotic stress and late embryogenesis abundant encoding protein functions were significantly enriched in downregulated DEGs of at least two organs. These observations are consistent with the commencement of cell activity and growth, the repression of ABA signaling that permits germination, and with the known dynamics of transcripts encoding starch metabolism enzymes (An and Lin, ; Betts et al , ).…”

“…This comprised 3598 DEGs in plumule, 5524 in radicle and 6209 in scutellum, reflecting the fact that some genes are shared between organs. The number of DEGs detected in scutellum is comparable with that detected in a hand‐dissected scutellum samples (Betts et al , ). However, hand dissection is limited to sample sizes that the researcher may physically handle so, for example, the previous study was able to isolate whole embryos but not separate the plumule and radicle.…”

“…Transcription factors have an important role in regulating gene expression spatially and temporally during germination (Bassel et al , ; Narsai et al , ; Sánchez‐Montesino et al , ). However, the details of which transcription factors act as regulators when and where are poorly understood, particularly in crop species (Betts et al , ).…”

“…Tight coordination of activities between seed organs and cells over time is required for a seed to germinate successfully, and this is achieved in part through the action of each cell type’s genome regulatory program. How the functions of distinct seed cell types are controlled by differential regulation of the genome has not yet been fully resolved due to the technical challenge of obtaining tissue‐specific transcript abundance data at appropriately small scales (Penfield et al , ; Endo et al , ; Dekkers et al , ; Betts et al , ).…”

Sprouted Barley Flour as a Nutritious and Functional Ingredient

“…The impact of germination time on β-glucan levels was stronger at lower temperatures. It has been reported that germination causes an increase of β-glucanases activity, which are the primary enzymes responsible for breaking down cereal endosperm cell walls during germination, thus reducing β-glucan levels [28,29]. Our results agree with those reported by Rimsten et al [30], who showed slight losses in β-glucan levels in barley grains germinated for 96 h after steeping at 48 • C. On the contrary, larger reductions of β-glucan content (50% after 48-120 h of germination) were found in germinated barley [31].…”