Abstract
Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields.
Highlights
In recent years, efforts taken in cereal breeding programs have focused mainly on increasing resistance to disease and abiotic stress, which is motivated by climate change and decreasing biodiversity.little progress has been made toward increasing yield potential
Knockout mutations of the HvCKX1 and HvCKX3 genes were induced by an RNA-guided Cas9 nuclease
The target sequences of both genes were selected at the 50 proximal region of the first exon to ensure that no other Cytokinin oxidase/dehydrogenase enzymes (CKX) genes would be affected by the sgRNA constructs
Summary
Efforts taken in cereal breeding programs have focused mainly on increasing resistance to disease and abiotic stress, which is motivated by climate change and decreasing biodiversity.little progress has been made toward increasing yield potential. One of the reasons for this limitation is the genetic nature of yield-related traits, which are usually determined by hundreds of quantitative trait loci, and conventional breeding methods are not sufficient for increasing yields to expected levels Modern biotechnology tools such as genetic transformation, marker-assisted selection, genome editing, and next-generation sequencing can be used as supporting methods in the conventional breeding of crop plants. Similar to that of the other most important cereal crop species, the progress of increasing the yield potential of barley is unsatisfactory, given the still increasing demand for food production. This problem may be addressed by the identification and utilization of new genes controlling grain size and grain number—the two main components of yield in cereals. This system can be utilized for the introduction of defined mutations (gene repair), for gene stacking, or for the upregulation or downregulation of gene expression [2]
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