Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches

Abstract

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.

Silencing ofTaCKX1mediates expression of otherTaCKXgenes to increase grain yield in wheat

“…A similar relationship between reduced expression of selected CKX family genes and cytokinin accumulation in reproductive organs has been observed in other species including A . thaliana (Bartrina et al, 2011), rice (Ashikari et al, 2005) and barley (Holubova et al, 2018), but detailed data are not comparable to our research in wheat.…”

“…Interestingly, the authors observed no peaks and no differences in CKX activity at the particular stages of spike development, which is in agreement with homeostasis of the pool of isozymes in 7 DAP spikes of wheat, as suggested by us, which is independent of the level of silencing of TaCKX1 but is rather a consequence of co-regulation of expression of other TaCKX genes. A similar effect of increased grain yield, which was a consequence of higher spike and grain number, was obtained in barley with silenced by RNAi HvCKX1 , an orthologue of TaCKX1 (Zalewski et al, 2010; Zalewski et al, 2014; Holubova et al, 2018). The three CKs measured, cZ, tZ and iP, were differently regulated in earlier or later, but not precisely defined, stages of spike development (Holubova et al, 2018); therefore the results of CK contents are not comparable.…”

“…Knock-out mutation or silencing by RNAi of OsCKX2 in rice significantly increased grain number (Ashikari et al, 2005). The same effect of elevated grain number, spike number and yield was reported for RNAi-silenced HvCKX1 in barley (Zalewski et al, 2010; Zalewski et al, 2012; Zalewski et al, 2014) and repeated for the same gene under field conditions (Holubova et al, 2018). Moreover, significantly increased grain number per spike was found as the effect of the TaCKX2.4 gene silenced by RNAi (Li et al, 2018).…”

“…In the first report, by Gasparis et al (2019), knock-out ckx1 mutants showed significantly decreased CKX enzyme activity in young spikes and 10-day old roots, which corresponded to greater root length, increased surface area, and greater numbers of root hairs. In the second report, by Holubova et al (2018), root growth in Hvckx1 mutant plants was reduced, which was similar to knock-out mutants of ckx3 showing smaller roots (Gasparis et al, 2019). The role of other TaCKX genes in wheat was analysed based on natural TaCKX variation.…”

“…Moreover, significantly increased grain number per spike was found as the effect of the TaCKX2.4 gene silenced by RNAi (Li et al, 2018). Knock-out mutation of HvCKX1 by CRISPR/Cas9 editing had a limited effect on yield productivity (Gasparis et al, 2019) or no yield data were supplied (Holubova et al, 2018). However, the results were contradictory for root growth.…”