TaCKX, Triticum aestivum (cytokinin oxidase/dehydrogenase) family genes influence the development of wheat plants by the specific regulation of cytokinin content in different organs. However, their detailed role is not known. The TaCKX1, highly and specifically expressed in developing spikes and in seedling roots, was silenced by RNAi-mediated gene silencing via Agrobacterium tumefaciens and the effect of silencing was investigated in 7 DAP (days after pollination) spikes of T₁ and T₂ generations. Various levels of TaCKX1 silencing in both generations influence different models of co-expression with other TaCKX genes and parameters of yield-related traits. Only a high level of silencing in T₂ resulted in strong down-regulation of TaCKX11 (3), up-regulation of TaCKX2.1, 2.2, 5, and 9 (10), and a high yielding phenotype. This phenotype is characterized by a higher spike number, grain number, and grain yield, but lower thousand grain weight (TGW). The content of most of cytokinin forms in 7 DAP spikes of silenced T₂ lines increased from 23% to 76% compared to the non-silenced control. The CKs cross talk with other phytohormones. Each of the tested yield-related traits is regulated by various up- or down-regulated TaCKX genes and phytohormones. The coordinated effect of TaCKX1 silencing on the expression of other TaCKX genes, phytohormone levels in 7 DAP spikes, and yield-related traits in silenced T₂ lines is presented.

Multigene families of CKX genes encode cytokinin oxidase/dehydrogenase proteins (CKX), which regulate cytokinin content in organs of developing plants. It has already been documented that some of them play important roles in plant productivity. The presented research is the first step of comprehensive characterization of the bread wheat TaCKX gene family with the goal to select genes determining yield-related traits. The specificity of expression patterns of fifteen formerly annotated members of the TaCKX family was tested in different organs during wheat development. Based on this, the genes were assigned to four groups: TaCKX10, TaCKX5 and TaCKX4, highly specific to leaves; TaCKX3, TaCKX6 and TaCKX11, expressed in various levels through all the organs tested; TaCKX1, TaCKX2.3, TaCKX2.2, TaCKX2.1, TaCKX2.4 and TaCKX2.5 specific to developing spikes and inflorescences; TaCKX9, TaCKX8 and TaCKX7, highly specific to roots. Amplification products of tested genes were mapped to the chromosomes of the A, B or D genome using T. aestivum Ensembl Plants. Based on analysis of TaCKX transcripts as well as encoded amino acids in T. aestivum and Hordeum vulgare the number of CKX genes in wheat was limited to 11 and new numbering of selected TaCKX genes was proposed. Moreover, we found that there were developmental differences in expression of TaCKX in the first and the second spike and expression of some of the genes was daily time dependent. A very high and significant correlation was found between expression levels of TaCKX7 and TaCKX9, genes specific to seedling roots, TaCKX1, TaCKX2.1 and TaCKX2.2, specific to developing spikes, and the group of TaCKX3, 4, 5, 6, 10 and 11, highly expressed in leaves and other organs. The genes also co-operated among organs and were included in two groups representing younger or maturating stages of developing plants. The first group was represented by seedling roots, leaves from 4-week old plants, inflorescences and 0 DAP spikes; the second by developing spikes, 0 DAP, 7 DAP and 14 DAP. The key genes which might determine yield-related traits are indicated and their possible roles in breeding strategies are discussed.

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.

TaCKXfamily genes influence development of wheat plants by specific regulation of cytokinin content in different organs. However, their detailed role is not known. TheTaCKX1, highly and specifically expressed in developing spikes and in seedling roots, was silenced by RNAi-mediated gene silencing viaAgrobacteriumand the effect of silencing was investigated in 7 DAP spikes of T₁and T₂generations. Various levels ofTaCKX1silencing in both generations influence different models of co-expression with otherTaCKXgenes and parameters of yield-related traits. Only a high level of silencing in T₂resulted in strong down-regulation ofTaCKX11 (3), up-regulation ofTaCKX2.1,2.2,5and9(10), and a high yielding phenotype. This phenotype is characterised by higher spike number, grain number and grain yield, as well as slightly higher mass of seedling roots, but lower thousand grain weight (TGW) and slightly lower spike length. Content of most of cytokinin forms in 7 DAP spikes of silenced T₂lines increased from 40 to 76% compared to the non-silenced control. The CKs cross talk with other phytohormones.Each of the tested yield-related traits is regulated by various up- or down-regulatedTaCKXgenes and phytohormones. Unexpectedly, increased expression ofTaCKX2.1in silent forTaCKX1T₂plants up-regulated trans- and cis-zeatin and trans-zeatin glucosides, determining lower TGW and chlorophyll content in flag leaves but higher grain yield. The coordinated effect ofTaCKX1silencing on expression of otherTaCKXgenes, phytohormone levels in 7 DAP spikes and yield-related traits in silenced T₂lines is presented.One-sentence summary: Different levels ofTaCKX1silencing influence various models of coordinated expression ofTaCKXgenes and phytohormone levels in 7 DAP spikes, as well as yield parameters.

The influence of silenced TaCKX1 and TaCKX2 on coexpression of other TaCKX gene family members (GFMs), phytohormone regulation and yield-related traits was tested in awned-spike cultivar. We documented a strong feedback mechanism of regulation of TaCKX GFM expression in which silencing of TaCKX1 upregulated expression of TaCKX2 genes and vice versa. Additionally, downregulation of TaCKX2 highly upregulated the expression of TaCKX5 and TaNAC2-5A. In contrast, expression of these genes in silenced TaCKX1 was downregulated. Silenced TaCKX1 T₂ lines with expression decreased by 47% had significantly higher thousand grain weight (TGW) and seedling root mass. Silenced TaCKX2 T₂ lines with expression of TaCKX2.2.1 and TaCKX2.2.2 decreased by 33% and 30%, respectively, had significantly higher chlorophyll content in flag leaves. TaCKX GFM expression, phytohormone metabolism and phenotype were additionally modified by Agrobacterium-mediated transformation. Two novel phytohormones, phenylacetic acid (PAA) and topolins, lack of gibberellic acid (GA) and changed phytohormone contents in the 7 days after pollination (DAP) spikes of the awned-spike cultivar compared to a previously tested, awnless one, were detected. We documented that major mechanisms of coregulation of the expression of TaCKX GFMs were similar in different spring wheat cultivars, but, depending on content and composition of phytohormones, regulation of yield-related traits was variously impacted.