Regulation of cell cycle activity in the embryo of barley seeds during germination as related to grain hydration

Various studies indicate that cell division is a post-germination phenomenon, with radicle protrusion occurring by cell elongation, while others demonstrate that induction of the cell cycle occurs in osmo-conditioned seeds prior to radicle growth. The aim of the present work was to investigate the occurrence of the cell cycle during germination as related to grain hydration, using: (i) a flow cytometry technique to estimate the percentage of cell nuclei in G₁ and G₂ phases of the cell cycle; and (ii) reverse transcription-PCR (RT-PCR) in order to characterize the expression of the genes encoding cyclin-dependent kinases (CDKA1, CDKB1, and CDKD1) and cyclins (CYCA3, CYCB1, and CYCD4), the main genes involved in the cell cycle and its regulation. Radicle tips of embryos were isolated from seeds placed for various times on water at 30 °C and from grains partially hydrated at moisture contents ranging from 11% to 51% fresh weight (FW), which prevent radicle elongation. Abscisic acid (ABA) contents of the embryos during seed germination at 30 °C and after 48 h of partial hydration were also measured. In dry embryos, cells are mostly arrested in the G₁ phase of the cell cycle (82%), the remaining cells being in the G₂ phase, and the ABA content of the embryo was 432.7 ng g⁻¹ dry weight (DW). Seed imbibition was associated with a sharp decrease in ABA content as early as 5 h, while the cell cycle reactivation was a late process taking place ~4-6 h prior to radicle protrusion. Hydration of seeds resulted in a decrease in embryo ABA content, but it remained at a high level (207-273 ng g⁻¹ DW) even after 48 h at 0.41-0.51 g H₂O g⁻¹ FW. The cell population of the radicle tips in the G₂ phase of the cell cycle, i.e. 4C nuclei, increased from 9% up to 34% at a moisture content of 51% FW. In dry seeds, CDKA1 and CDKD1 mRNAs were present at low levels, but transcripts of CDKB1, CYCA3, CYCB1, and CYCD4 were not detected. Radicle protrusion was associated with a higher expression of CDKA1, CDKB1, CYCA3, and CYCB1. Blockage of germination of partially hydrated grains resulted in a reduction in the expression of CDKA1 and CDKB1, and of CYCA3 and CYCB1, and in a reinforcement of that of CDKD1 and CYCD4. Patterns of gene expression show differential sensitivity of the genes studied to hydration of the grain. They will be discussed with regard to embryo ABA content and embryo sensitivity to ABA.