Are TaNAC Transcription Factors Involved in Promoting Wheat Yield by cis-Regulation of TaCKX Gene Family?

NAC transcription factors (TFs) are one of the largest TF families in plants, and TaNACs have been known to participate in the regulation of the transcription of many yield-regulating genes in bread wheat. The TaCKX gene family members (GFMs) have already been shown to regulate yield-related traits, including grain mass and number, leaf senescence, and root growth. The genes encode cytokinin (CK) degrading enzymes (CKXs) and are specifically expressed in different parts of developing wheat plants. The aim of the study was to identify and characterize TaNACs involved in the cis-regulation of TaCKX GFMs. After analysis of the initial transcription factor data in 1.5 Kb cis-regulatory sequences of a total of 35 homologues of TaCKX GFMs, we selected five of them, namely TaCKX1-3A, TaCKX22.1-3B, TaCKX5-3D, TaCKX9-1B, and TaCKX10, and identified five TaNAC genes: TaNACJ-1, TaNAC13a, TaNAC94, TaNACBr-1, and TaNAC6D, which are potentially involved in the cis-regulation of selected TaCKX genes, respectively. Protein feature analysis revealed that all of the selected TaNACs have a conserved NAC domain and showed a stable tertiary structure model. The expression profile of the selected TaNACs was studied in 5 day-old seedling roots, 5-6 cm inflorescences, 0, 4, 7, and 14 days-after-pollination (DAP) spikes, and the accompanying flag leaves. The expression pattern showed that all of the selected TaNACs were preferentially expressed in seedling roots, 7 and 14 DAP spikes, and flag leaves compared to 5-6 cm inflorescence and 0 and 4 DAP spikes and flag leaves in Kontesa and Ostka spring wheat cultivars (cvs.). In conclusion, the results of this study highlight the potential role of the selected TaNACs in the regulation of grain productivity, leaf senescence, root growth, and response to various stresses.

Inhibition of the Glycogen Synthase Kinase 3 Family by the Bikinin Alleviates the Long-Term Effects of Salinity in Barley.

Crops grown under stress conditions show restricted growth and, eventually, reduced yield. Among others, brassinosteroids (BRs) mitigate the effects of stress and improve plant growth. We used two barley cultivars with differing sensitivities to BRs, as determined by the lamina joint inclination test. Barley plants with the 2nd unfolded leaf were sprayed with a diluted series of bikinin, an inhibitor of the Glycogen Synthase Kinase 3 (GSK3) family, which controls the BR signaling pathway. Barley was grown under salt stress conditions up to the start of the 5th leaf growth stage. The phenotypical, molecular, and physiological changes were determined. Our results indicate that the salt tolerance of barley depends on its sensitivity to BRs. We confirmed that barley treatment with bikinin reduced the level of the phosphorylated form of HvBZR1, the activity of which is regulated by GSK3. The use of two barley varieties with different responses to salinity led to the identification of the role of BR signaling in photosynthesis activity. These results suggest that salinity reduces the expression of the genes controlling the BR signaling pathway. Moreover, the results also suggest that the functional analysis of the GSK3 family in stress responses can be a tool for plant breeding in order to improve crops' resistance to salinity or to other stresses.

Genome-Wide Identification of Barley Long Noncoding RNAs and Analysis of Their Regulatory Interactions during Shoot and Grain Development.

Long noncoding RNAs (lncRNAs) are a class of RNA molecules with gene regulatory functions in plant development and the stress response. Although the number of lncRNAs identified in plants is rapidly increasing, very little is known about their role in barley development. In this study, we performed global identification of barley lncRNAs based on 53 RNAseq libraries derived from nine different barley tissues and organs. In total, 17,250 lncRNAs derived from 10,883 loci were identified, including 8954 novel lncRNAs. Differential expression of lncRNAs was observed in the developing shoot apices and grains, the two organs that have a direct influence on the final yield. The regulatory interaction of differentially expressed lncRNAs with the potential target genes was evaluated. We identified 176 cis-acting lncRNAs in shoot apices and 424 in grains, while the number of trans-acting lncRNAs in these organs was 1736 and 540, respectively. The potential target protein-coding genes were identified, and their biological function was annotated using MapMan ontology. This is the first insight into the roles of lncRNAs in barley development on the genome-wide scale, and our results provide a solid background for future functional studies.

TaCKX2.2 Genes Coordinate Expression of Other TaCKX Family Members, Regulate Phytohormone Content and Yield-Related Traits of Wheat.

TaCKX gene family members (GFMs) play essential roles in the regulation of cytokinin during wheat development and significantly influence yield-related traits. However, detailed function of most of them is not known. To characterize the role of TaCKX2.2 genes we silenced all homoeologous copies of both TaCKX2.2.1 and TaCKX2.2.2 by RNAi technology and observed the effect of silencing in 7 DAP spikes of T1 and T2 generations. The levels of gene silencing of these developmentally regulated genes were different in both generations, which variously determined particular phenotypes. High silencing of TaCKX2.2.2 in T2 was accompanied by slight down-regulation of TaCKX2.2.1 and strong up-regulation of TaCKX5 and TaCKX11, and expression of TaCKX1, TaCKX2.1, and TaCKX9 was comparable to the non-silenced control. Co-ordinated expression of TaCKX2.2.2 with other TaCKX GFMs influenced phytohormonal homeostasis. Contents of isoprenoid, active cytokinins, their conjugates, and auxin in seven DAP spikes of silenced T2 plants increased from 1.27 to 2.51 times. However, benzyladenine (BA) and abscisic acid (ABA) contents were significantly reduced and GA3 was not detected. We documented a significant role of TaCKX2.2.2 in the regulation of thousand grain weight (TGW), grain number, and chlorophyll content, and demonstrated the formation of a homeostatic feedback loop between the transcription of tested genes and phytohormones. We also discuss the mechanism of regulation of yield-related traits.

Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology.

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.

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (Hordeum vulgare L.).

BACKGROUND: Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley. RESULTS: We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T0 plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T1 lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T1 plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T1 plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T0 plants. CONCLUSIONS: We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of Pinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.