Table_1_Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches.pdf

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.

大麦是全球最重要的谷类作物之一，广泛应用于酿酒工业以及畜禽与人类饲料生产。植物激素细胞分裂素（cytokinin）的遗传调控可影响诸多生理进程，涵盖胁迫耐受性、根系建成与作物产量等方面。在植物体内，内源细胞分裂素的稳态由细胞分裂素脱氢酶（cytokinin dehydrogenase，EC 1.5.99.12；CKX）精细调控，该酶可不可逆降解腺嘌呤源异戊二烯类细胞分裂素的侧链。本研究通过沉默HvCKX1基因，验证了通过调控内源细胞分裂素含量提升籽粒产量的可行性。为阐明HvCKX1基因在籽粒生产中的潜在功能，研究人员借助RNA引导的Cas9系统创制了Hvckx1基因敲除突变体植株。研究人员筛选获得携带沉默HvCKX1基因的纯合转基因植株，以及无外源转基因整合的Hvckx1敲除突变体，并对两类材料展开系统分析。在大麦籽粒发育的不同阶段，均检测到HvCKX1基因表达水平与CKX酶活性的显著下降。对转基因株系的表型鉴定结果显示，其根系生长受到抑制，但相较于非转基因野生型对照，转基因植株的分蘖数与籽粒产量均显著提升，总产量最高可增加15%。尽管植株整体生产力有所提升，但籽粒总生物量降至野生型植株的80%。对HvCKX1基因沉默转基因株系的RNA测序（RNA-seq）分析表明，在淀粉大规模积累阶段，多种关键的大量营养元素转运蛋白编码基因均呈现表达下调。该结果提示，局部细胞分裂素的积累会对养分运输产生负面影响，进而导致籽粒生物量下降。本研究所得结果证实了HvCKX1在调控大麦细胞分裂素稳态中的关键作用。