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.

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.

Members of the TaCKX gene family (GFMs) encode the cytokinin oxygenase/dehydrogenase enzyme (CKX), which irreversibly degrades cytokinins in the organs of wheat plants; therefore, these genes perform a key role in the regulation of yield-related traits. The purpose of the investigation was to determine how expression patterns of these genes, together with the transcription factor-encoding gene TaNAC2-5A, and yield-related traits are inherited to apply this knowledge to speed up breeding processes. The traits were tested in 7 days after pollination (DAP) spikes and seedling roots of maternal and paternal parents and their F₂ progeny. The expression levels of most of them and the yield were inherited in F₂ from the paternal parent. Some pairs or groups of genes cooperated, and some showed opposite functions. Models of up- or down-regulation of TaCKX GFMs and TaNAC2-5A in low-yielding maternal plants crossed with higher-yielding paternal plants and their high-yielding F₂ progeny reproduced gene expression and yield of the paternal parent. The correlation coefficients between TaCKX GFMs, TaNAC2-5A, and yield-related traits in high-yielding F₂ progeny indicated which of these genes were specifically correlated with individual yield-related traits. The most common was expressed in 7 DAP spikes TaCKX2.1, which positively correlated with grain number, grain yield, spike number, and spike length, and seedling root mass. The expression levels of TaCKX1 or TaNAC2-5A in the seedling roots were negatively correlated with these traits. In contrast, the thousand grain weight (TGW) was negatively regulated by TaCKX2.2.2, TaCKX2.1, and TaCKX10 in 7 DAP spikes but positively correlated with TaCKX10 and TaNAC2-5A in seedling roots. Transmission of TaCKX GFMs and TaNAC2-5A expression patterns and yield-related traits from parents to the F₂ generation indicate their paternal imprinting. These newly shown data of nonmendelian epigenetic inheritance shed new light on crossing strategies to obtain a high-yielding F₂ generation.

Members of the TaCKX gene family (GFM) encode oxidase/dehydrogenase cytokinin degrading enzymes (CKX), which play an important role in the homeostasis of phytohormones, affecting wheat development and productivity. Therefore, the objective of this investigation was to test how the expression patterns of the yield-related TaCKX genes and TaNAC2-5A (NAC2) measured in 7 days after pollination (DAP) spikes and the seedling roots of parents are inherited to apply this knowledge in the breeding process. The expression patterns of these genes were compared between parents and their F₂ progeny in crosses of one mother with different paterns of awnless cultivars and reciprocal crosses of awned and awnless lines. We showed that most of the genes tested in the 7 DAP spikes and seedling roots of the F₂ progeny showed paternal expression patterns in crosses of awnless cultivars as well as reciprocal crosses of awned and awnless lines. Consequently, the values of grain yield in the F₂ progeny were similar to the pater; however, the values of seedling root mass were similar to the mother or both parents. The correlation analysis of TaCKX GFMs and NAC2 in spikes and spikes per seedling roots reveals that the genes correlate with each other specifically with the pater and the F₂ progeny or the mother and the F₂ progeny, which shape phenotypic traits. The numbers of spikes and semi-empty spikes are mainly correlated with the specific coexpression of the TaCKX and NAC2 genes expressed in spikes or spikes per roots of the pater and F₂ progeny. Variable regression analysis of grain yield and root mass with TaCKX GFMs and NAC2 expressed in the tested tissues of five crosses revealed a significant dependency of these parameters on the mother and F₂ and/or the pater and F₂ progeny. We showed that the inheritance of yield-related traits depends on the specific cooperative expression of some TaCKX GFMs, in some crosses coupled with NAC2, and is strongly dependent on the genotypes used for the crosses. Indications for parental selection in the breeding of high-yielding lines are discussed.