The transition from seed to seedling represents a critical developmental step in the life cycle of higher plants, dramatically affecting plant ontogenesis and stress tolerance. The release from dormancy to acquiring germination ability is defined by a balance of phytohormones, with the substantial contribution of abscisic acid (ABA), which inhibits germination. We studied the embryonic axis of Pisum sativum L. before and after radicle protrusion. Our previous work compared RNA sequencing-based transcriptomics in the embryonic axis isolated before and after radicle protrusion. The current study aims to analyze ABA-dependent gene regulation during the transition of the embryonic axis from the germination to post-germination stages. First, we determined the levels of abscisates (ABA, phaseic acid, dihydrophaseic acid, and neo-phaseic acid) using ultra-high-performance liquid chromatography-tandem mass spectrometry. Second, we made a detailed annotation of ABA-associated genes using RNA sequencing-based transcriptome profiling. Finally, we analyzed the DNA methylation patterns in the promoters of the PsABI3, PsABI4, and PsABI5 genes. We showed that changes in the abscisate profile are characterized by the accumulation of ABA catabolites, and the ABA-related gene profile is accompanied by the upregulation of genes controlling seedling development and the downregulation of genes controlling water deprivation. The expression of ABI3, ABI4, and ABI5, which encode crucial transcription factors during late maturation, was downregulated by more than 20-fold, and their promoters exhibited high levels of methylation already at the late germination stage. Thus, although ABA remains important, other regulators seems to be involved in the transition from seed to seedling.

• Keywords
• ABA-associated genes, DNA methylation, Pisum sativum L., abscisic acid, embryonic axis, seed-to-seedling transition,
• Publication type
• Journal Article MeSH

Barley is one of the most important cereals, which is used for breweries, animal and human feeds. Genetic manipulation of plant hormone cytokinins may influence several physiological processes, besides others stress tolerance, root formation and crop yield. In planta, endogenous cytokinin status is finely regulated by the enzyme cytokinin dehydrogenase (EC 1.5.99.12; CKX), that irreversible degrades the side chain of adenine-derived isoprenoid cytokinins. Increasing grain yield by mean of manipulation of endogenous cytokinin content was assayed by the silencing of the HvCKX1 gene. Moreover, to elucidate the putative role of HvCKX1 gene on grain production, knocked-out Hvckx1 mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced HvCKX1 gene and azygous knock-out Hvckx1 mutants have been selected and analyzed. Both reduced expression of HvCKX1 gene and CKX activity were measured in different stages of barley grain development. Phenotyping of the transgenic lines revealed reduced root growth, however, plants produced more tillers and grains than azygous wild-type controls and the total yield was increased up to 15 per cent. Although plant productivity was increased, total grain biomass was decreased to 80% of WT grains. RNA-seq analysis of knock-down transgenic lines revealed that several important macronutrient transporters were downregulated in the stage of massive starch accumulation. It suggests that local accumulation of cytokinins negatively affected nutrients flow resulting in reduced grain biomass. Obtained results confirmed the key role of HvCKX1 for regulation of cytokinin content in barley.

• Keywords
• CRISPR-Cas9, barley, cytokinin, silencing, yield,
• Publication type
• Journal Article MeSH