Silencing of TaCKX1 Mediates Expression of Other TaCKX Genes to Increase Yield Parameters in Wheat

TaCKX, Triticumaestivum (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.


Introduction
Wheat (Triticum aestivum L.) is the third most economically important crop in the world after corn and rice, and probably the most important in moderate climates. It provides approximately 20% of human calories and protein [1]. The large genome of this high-yielding species, composed of three (AABBDD) genomes, has been very challenging for improving traits [2]. However, it might be a great reservoir to sustain a further increase of grain productivity [3]. The continuous increase of wheat production is necessary to feed the rapidly growing world population [4]. Biotechnological tools implemented in the process of increasing wheat productivity are expected to be beneficial.
Cytokinins (CKs) are important regulators of plant growth and development, influencing many agriculturally important processes [5]. This regulation might occur at the posttranscriptional and/or posttranslational level [6,7], or by the modulation of context-dependent chromatin accessibility [8]. all of them is to apply RNAi-mediated gene silencing, which allows silencing of all the homologues. Moreover, this tool made it possible to obtain a number of lines with different levels of silencing, which in the case of genes coding proteins of key importance for life gave a possibility to regenerate plants for analysis [33]. The introduction of a silencing cassette by stable transformation results in a stable, and inherited to T 4 , effect of silencing [21,34]. The applicability of Agrobacterium-mediated transformation compared to a biolistic one for gene silencing of the developmentally regulated gene HvCKX10 (2) was proved to be reliable [24].
We present the first report on the role of TaCKX1 in the co-regulation of expression of other TaCKX genes, phytohormone content, and their joint participation in the regulation of yield-related traits in wheat. Various levels of gene silencing in T 1 and T 2 have been related to different patterns of other TaCKX expression, strongly influencing yield-related traits. Models of regulation of phytohormone levels and phenotypic traits in non-silenced and highly silenced T 2 plants by the coordinated expression of TaCKX genes are proposed.

Expression Levels of Silenced TaCKX1 in Segregating T 1 and T 2 Plants
Expression levels of TaCKX1 were measured in 44 segregating T 1 plants from 8 T 0 PCR+ lines. In 14 T 1 plants relative expression (related to the control = 1.00) ranged from 0.39 to 0.88 with the mean of 0.67 (±0.14). In 30 T 1 plants, relative expression ranged from 0.90 to 1.52 with the mean of 1.16 (±0.18) (Figure 1). The proportion of silenced to non-silenced plants changed in the T 2 generation. There were 42 silenced from 0.24 to 0.88 plants with the mean of 0.54 (±0.14) and 20 non-silenced plants. Eight of them, with low relative expression ranging from 0.24 to 0.40 (mean 0.33 ±0.14) and representing different T 1 lines, were selected for further analysis. homologues. Moreover, this tool made it possible to obtain a number of lines with different levels of silencing, which in the case of genes coding proteins of key importance for life gave a possibility to regenerate plants for analysis [33]. The introduction of a silencing cassette by stable transformation results in a stable, and inherited to T4, effect of silencing [21,34]. The applicability of Agrobacteriummediated transformation compared to a biolistic one for gene silencing of the developmentally regulated gene HvCKX10 (2) was proved to be reliable [24]. We present the first report on the role of TaCKX1 in the co-regulation of expression of other TaCKX genes, phytohormone content, and their joint participation in the regulation of yield-related traits in wheat. Various levels of gene silencing in T1 and T2 have been related to different patterns of other TaCKX expression, strongly influencing yield-related traits. Models of regulation of phytohormone levels and phenotypic traits in non-silenced and highly silenced T2 plants by the coordinated expression of TaCKX genes are proposed.

Expression Levels of Silenced TaCKX1 in Segregating T1 and T2 Plants
Expression levels of TaCKX1 were measured in 44 segregating T1 plants from 8 T0 PCR+ lines. In 14 T1 plants relative expression (related to the control = 1.00) ranged from 0.39 to 0.88 with the mean of 0.67 (±0.14). In 30 T1 plants, relative expression ranged from 0.90 to 1.52 with the mean of 1.16 (±0.18) (Figure 1). The proportion of silenced to non-silenced plants changed in the T2 generation. There were 42 silenced from 0.24 to 0.88 plants with the mean of 0.54 (±0.14) and 20 non-silenced plants. Eight of them, with low relative expression ranging from 0.24 to 0.40 (mean 0.33 ±0.14) and representing different T1 lines, were selected for further analysis.

Co-Expression of Silenced TaCKX1 with Other TaCKX Genes in T 1 and T 2 and CKX Enzyme Activity
Mean relative expression of TaCKX1 in the selected 8 lines was 0.67 in T 1 and was decreased to 0.33 in T 2 ( Figure 2). Similarly, in the case of TaCKX11 (3) related gene expression was 0.81 in T 1 and was decreased to 0.34 in T 2 . Relative expression levels of TaCKX2.2 and TaCKX9 (10) were decreased in T 1 to 0.51 and 0.39 and increased in T 2 slightly above the control level, to 1.08 and to 1.10 respectively. Mean relative values for TaCKX2.1 were similar to control in T 1 (1.05) and slightly increased in T 2 (1.17). Relative expression of TaCKX5, which was in T 1 below the control level (0.84), was significantly increased to 1.82 in T 2 . The relative values of CKX enzyme activity in both generations were around the control, 1.00.
Mean relative expression of TaCKX1 in the selected 8 lines was 0.67 in T1 and was decreased to 0.33 in T2 (Figure 2). Similarly, in the case of TaCKX11 (3) related gene expression was 0.81 in T1 and was decreased to 0.34 in T2. Relative expression levels of TaCKX2.2 and TaCKX9 (10) were decreased in T1 to 0.51 and 0.39 and increased in T2 slightly above the control level, to 1.08 and to 1.10 respectively. Mean relative values for TaCKX2.1 were similar to control in T1 (1.05) and slightly increased in T2 (1.17). Relative expression of TaCKX5, which was in T1 below the control level (0.84), was significantly increased to 1.82 in T2. The relative values of CKX enzyme activity in both generations were around the control, 1.00.
The effect of TaCKX1 silencing on the levels of expression of selected TaCKX genes is presented by the expression ratio indicator (Table 1), which is a quotient of the mean relative value in silent per mean relative value in non-silent, control plants. In the case of TaCKX1 and TaCKX11 (3), the ratio indicator, significantly decreased in T1, was strongly decreased in T2. The value of the ratio indicator for TaCKX2.2 was not changed in T1 compared to the control and was only slightly decreased in T2. The expression ratio indicator of TaCKX9 (10), strongly decreased to 0.59 in T1, rose above the control level (1.15) in T2. Already high in T1, the expression ratio indicator for TaCKX2.1 (1.22) increased to 1.32 in T2. The phenotype ratio indicator for CKX enzyme activity was 1.01 in T1 and 0.99 in T2.

Figure 2.
Comparison of means of relative CKX enzyme activity and selected gene expression levels in T1 (bars) and T2 (line) generation of silenced lines. *-significant at p < 0.05; **-significant at p < 0.01.  Figure 2. Comparison of means of relative CKX enzyme activity and selected gene expression levels in T 1 (bars) and T 2 (line) generation of silenced lines. *-significant at p < 0.05; **-significant at p < 0.01.
The effect of TaCKX1 silencing on the levels of expression of selected TaCKX genes is presented by the expression ratio indicator (Table 1), which is a quotient of the mean relative value in silent per mean relative value in non-silent, control plants. In the case of TaCKX1 and TaCKX11 (3), the ratio indicator, significantly decreased in T 1 , was strongly decreased in T 2 . The value of the ratio indicator for TaCKX2.2 was not changed in T 1 compared to the control and was only slightly decreased in T 2 . The expression ratio indicator of TaCKX9 (10), strongly decreased to 0.59 in T 1 , rose above the control level (1.15) in T 2 . Already high in T 1 , the expression ratio indicator for TaCKX2.1 (1.22) increased to 1.32 in T 2 . The phenotype ratio indicator for CKX enzyme activity was 1.01 in T 1 and 0.99 in T 2 . In T 1 segregating plants, CKX enzyme activity significantly correlated with spike length (0.51; n = 16) and grain weight (0.50; n = 16), but in T 2 these correlations were not significant.

Influence of TaCKX1 Silencing on Phenotypic Traits and Chlorophyll Content in Flag Leaves of T 1 and T 2 Plants
The values of phenotypic traits in T 1 plants with slightly decreased relative expression of TaCKX1 (0.67 ± 0.14) compared to control plants (1.00) were on the same level in the case of plant height and lower for number of spikes, spike length, grain number, and grain yield (Supplementary Table S2). Higher values were obtained for TGW. Data for chlorophyll content measured by SPAD in the flag leaves of first spikes and the next spikes were similar. All these differences were not significant. Opposite results were obtained for some traits in T 2 plants with highly silent TaCKX1 (0.33 ± 0.06) compared to the control (1.00) (Supplementary Table S3). Silent T 2 plants were substantially smaller, had a higher number of spikes, number of grains, grain yield, seedling root weight, and lower SPAD values for the flag leaves of first spikes. TGW and spike length were significantly lower than in control plants.
These differences between the slightly silenced T 1 and highly silent T 2 generation are expressed by comparison of ratio indicators of phenotypic traits in both generations ( Figure 3). There were no changes in plant height, TGW or spike length in T 1 plants compared to the control. However, these values were respectively 7%, 10%, and 25% lower in T 2 plants. Opposite phenotype ratio indicators for number of spikes per plant and number of grains per plant were about 21% and 30% lower in T 1 and 57% and 29% higher in T 2 . These differences for spike number, grain number, and TGW were significant.

Influence of TaCKX1 Silencing on Phenotypic Traits and Chlorophyll Content in Flag Leaves of T1 and T2 Plants
The values of phenotypic traits in T1 plants with slightly decreased relative expression of TaCKX1 (0.67 ± 0.14) compared to control plants (1.00) were on the same level in the case of plant height and lower for number of spikes, spike length, grain number, and grain yield (Supplementary Table S2). Higher values were obtained for TGW. Data for chlorophyll content measured by SPAD in the flag leaves of first spikes and the next spikes were similar. All these differences were not significant. Opposite results were obtained for some traits in T2 plants with highly silent TaCKX1 (0.33 ± 0.06) compared to the control (1.00) (Supplementary Table S3). Silent T2 plants were substantially smaller, had a higher number of spikes, number of grains, grain yield, seedling root weight, and lower SPAD values for the flag leaves of first spikes. TGW and spike length were significantly lower than in control plants.
These differences between the slightly silenced T1 and highly silent T2 generation are expressed by comparison of ratio indicators of phenotypic traits in both generations ( Figure 3). There were no changes in plant height, TGW or spike length in T1 plants compared to the control. However, these values were respectively 7%, 10%, and 25% lower in T2 plants. Opposite phenotype ratio indicators for number of spikes per plant and number of grains per plant were about 21% and 30% lower in T1 and 57% and 29% higher in T2. These differences for spike number, grain number, and TGW were significant. Correlation coefficients among the expression of all tested TaCKX genes and enzyme activity, and phenotypic traits in non-silent and highly silent T2 are included in Supplementary Table S4a,b. All these correlations are graphically presented in Figures and described in Section 2.6.

Phytohormone Content in 7 DAP Spikes of T2.
tZGs, which were mainly composed of tZ9G, tZ7G, tZOG and tZ9GOG, were the most abundant cytokinin group in 7 DAP spikes ( Figure 4a). Their mean content in control plants was 6.97 ng/g biomass and in silent T2 was 6.24 ng/g biomass respectively. The second most abundant was tZ with the level of 3.74 ng/g biomass in the control and 4.59 ng/g biomass in silent T2. The content of cZ was Correlation coefficients among the expression of all tested TaCKX genes and enzyme activity, and phenotypic traits in non-silent and highly silent T 2 are included in Supplementary Table S4A,B. All these correlations are graphically presented in Figures and described in Section 2.6.

Phytohormone Content in 7 DAP Spikes of T 2
tZGs, which were mainly composed of tZ9G, tZ7G, tZOG and tZ9GOG, were the most abundant cytokinin group in 7 DAP spikes ( Figure 4a). Their mean content in control plants was 6.97 ng/g biomass and in silent T 2 was 6.24 ng/g biomass respectively. The second most abundant was tZ with the level of 3.74 ng/g biomass in the control and 4.59 ng/g biomass in silent T 2 . The content of cZ was slightly lower to tZ (2.90 ng/g biomass) in control but higher (5.10 ng/g biomass) in silent plants. cZOG was more abundant in the control than the groups of silent plants, and the content was 1.27 and 0.57 ng/g biomass respectively. The concentration of DZGs (sum of DZ7G, DZOG, DZ9G and DZOGR) was higher in silent (1.61 ng/g biomass) than in control plants (1.11 ng/g biomass). Low concentrations (below 0.5 ng/g biomass) were measured for iP and BA. The concentration of IAA was also low and on a comparable level in control and in silent plants (0.23 and 0.24 ng/g biomass respectively). In the case of ABA, the concentration in the control was slightly decreased in silent plants (2.61 and 2.29 ng/g biomass respectively). The concentration of GA was increased from 0.28 ng/g biomass in the control to 2.93 ng/g biomass in silent plants, which was more than a 10-fold increase.
Int. J. Mol. Sci. 2020, 21, 4809 6 of 20 slightly lower to tZ (2.90 ng/g biomass) in control but higher (5.10 ng/g biomass) in silent plants. cZOG was more abundant in the control than the groups of silent plants, and the content was 1.27 and 0.57 ng/g biomass respectively. The concentration of DZGs (sum of DZ7G, DZOG, DZ9G and DZOGR) was higher in silent (1.61 ng/g biomass) than in control plants (1.11 ng/g biomass). Low concentrations (below 0.5 ng/g biomass) were measured for iP and BA. The concentration of IAA was also low and on a comparable level in control and in silent plants (0.23 and 0.24 ng/g biomass respectively). In the case of ABA, the concentration in the control was slightly decreased in silent plants (2.61 and 2.29 ng/g biomass respectively). The concentration of GA was increased from 0.28 ng/g biomass in the control to 2.93 ng/g biomass in silent plants, which was more than a 10-fold increase.
Most of the phytohormone ratio indicators in the group of six silent T2 plants ( Figure 4b) were much higher than in control plants. There were the following cytokinins: tZ (1.

Coordinated Effect of TaCKX1 Silencing on Expression of Other TaCKX Genes and Phytohormone Level in 7 DAP Spikes as Well as Phenotype in T 2
A graphic presentation of the coordinated effect of TaCKX1 silencing on expression of other TaCKX genes and phytohormone levels in 7 DAP spikes as well as the phenotype of T 2 plants is presented in Figure 5. The significant decrease of expression of TaCKX1 was coordinated with the significant decrease of TaCKX11 (3), which presumably resulted in a significant increase of most CKs: tZ, tZGs, cZ, DZGs, iP, as well as GA. The increased phytohormone level in the first 7 DAP spikes positively influenced traits such as spike number and grain number, reaching the ratio indicators 1.57 and 1.29, respectively, and negatively influenced TGW (0.78), spike length (0.86), plant height (0.93), and flag leaf senescence (0.95). Opposing data were obtained for TaCKX2.1 and TaCKX9 (10), which showed increased expression in silenced 7 DAP spikes (1.32 and 1.15 respectively). This might have influenced the decreased ratio indicators for phytohormones-cZOG (0.45), BA (0.27), and ABA content (0.88), and slightly increased ratio indicators for yield-related traits: root weight and grain yield (1.07 and 1.03 respectively). Expression ratio indicators for TaCKX5 and TaCKX2.2 were both close to 1.00, but their expression significantly increased compared to T 1 and positively correlated with the expression of TaCKX2.1 and TaCKX9 (10) respectively.

Coordinated Effect of TaCKX1 Silencing on Expression of Other TaCKX Genes and Phytohormone Level in 7 DAP Spikes as Well as Phenotype in T2
A graphic presentation of the coordinated effect of TaCKX1 silencing on expression of other TaCKX genes and phytohormone levels in 7 DAP spikes as well as the phenotype of T2 plants is presented in Figure 5. The significant decrease of expression of TaCKX1 was coordinated with the significant decrease of TaCKX11 (3), which presumably resulted in a significant increase of most CKs: tZ, tZGs, cZ, DZGs, iP, as well as GA. The increased phytohormone level in the first 7 DAP spikes positively influenced traits such as spike number and grain number, reaching the ratio indicators 1.57 and 1.29, respectively, and negatively influenced TGW (0.78), spike length (0.86), plant height (0.93), and flag leaf senescence (0.95). Opposing data were obtained for TaCKX2.1 and TaCKX9 (10), which showed increased expression in silenced 7 DAP spikes (1.32 and 1.15 respectively). This might have influenced the decreased ratio indicators for phytohormones-cZOG (0.45), BA (0.27), and ABA content (0.88), and slightly increased ratio indicators for yield-related traits: root weight and grain yield (1.07 and 1.03 respectively). Expression ratio indicators for TaCKX5 and TaCKX2.2 were both close to 1.00, but their expression significantly increased compared to T1 and positively correlated with the expression of TaCKX2.1 and TaCKX9 (10) respectively.

Models of Co-Regulation of Phytohormone Levels and Phenotype Traits by Coordinated Expression of TaCKX Genes in Non-Silenced and Silenced T2 Plants
Two different models of co-regulation of TaCKX expression, phytohormone levels and phenotypic traits in non-silenced and silenced plants of the T2 generation are proposed (Figure 6ah) based on correlation coefficients (Table S4A,B).
Plant height (Figure 6a). There was no correlation between plant height and expression values of any TaCKX expressed in 7 DAP spikes of non-silent as well as silent plants. In the first group of plants this trait negatively correlated with BA and positively with IAA and GA content. By contrast,  expression of TaCKX2.1 was strongly positively correlated with growing values of tZ, tZGs, cZ, and DZGs as well as GA in silent plants. A strong negative correlation was observed between the gene expression and chlorophyll content, which means that increasing expression of TaCKX2.1 in 7 DAP spikes results in lower chlorophyll content in silent plants.

Discussion
First, 7 DAP spike was chosen as a research objective in wheat since decreased HvCKX1 expression at this stage in barley resulted in higher yield due to the higher spike and grain number [20,21]. The TaCKX1 gene is an orthologue of HvCKX1 and both genes are specifically expressed in developing spikes [23], indicating their possibly important role in the regulation of yield-related  In the case of grain number, the main player positively correlated with the trait is TaCKX5, increased expression of which was correlated with slightly higher IAA content, which resulted in higher grain number. Spike number is also positively regulated by TaCKX5 co-expressed with TaCKX2.1, and both genes were positively correlated with growing CKs, DZGs and iP as well as GA, determining higher spike number. Both traits are highly correlated (0.88) with each other.
Seedling root weight (Figure 6g). There was strong, positive correlation between TaCKX9 (10) expression in 7 DAP spikes and seedling root weight in non-silenced plants. Moreover, CKX activity negatively correlated with tZ (in spikes) and the trait, which finally resulted in lower root weight. The decreasing expression of TaCKX11 (3) in the case of silent plants was positively correlated with decreasing content of cZOG and strongly positively correlated with the trait. Increasing expression levels of TaCKX9 (10) plus TaCKX2.2 negatively correlated with decreasing content of cZOG and root weight.
Chlorophyll content measured by SPAD in flag leaves of first spikes (Figure 6h). There was no correlation between expression level of any TaCKX measured in 7 DAP spikes of non-silent plants and the trait. The only correlations were between phytohormone content and the trait, positive for cZ and negative for GA, which resulted in higher SPAD values (chlorophyll content). Increasing expression of TaCKX2.1 was strongly positively correlated with growing values of tZ, tZGs, cZ, and DZGs as well as GA in silent plants. A strong negative correlation was observed between the gene expression and chlorophyll content, which means that increasing expression of TaCKX2.1 in 7 DAP spikes results in lower chlorophyll content in silent plants.

Discussion
First, 7 DAP spike was chosen as a research objective in wheat since decreased HvCKX1 expression at this stage in barley resulted in higher yield due to the higher spike and grain number [20,21]. The TaCKX1 gene is an orthologue of HvCKX1 and both genes are specifically expressed in developing spikes [23], indicating their possibly important role in the regulation of yield-related traits. The samples were taken from the middle part of the spikes, when anthesis starts, in order to ensure a similar developmental stage of spikelets for research. The 7 DAP spikes of wheat represent the middle of cell division/cell expansion stage [35,36].

Various Levels of TaCKX1 Silencing Influence Different Models of Co-Expression with Other TaCKX Genes and Parameters of Yield-Related Traits
Various levels of silencing of TaCKX1 in T 1 and T 2 generate different results of co-expression with other TaCKX genes and plant phenotype. For example, the expression of TaCKX9 (10) was highly and significantly correlated with TaCKX1 only in T 1 . However, a new and strong positive correlation between TaCKX9 (10) and TaCKX2.2 in highly silenced T 2 was observed. Slightly decreased co-expression of silenced TaCKX1 together with TaCKX11 (3) in T 1 was much stronger in T 2 , indicating their positive co-regulation. It should be underlined that there is no homology between the sequence of TaCKX1 used for silencing and sequences of other TaCKX genes tested. Therefore, the process of RNAi silencing was specifically addressed to TaCKX1 silencing. It indicates that the level of silencing of the modified gene affected variable levels of expression of the other TaCKX genes in a co-operative process maintaining homeostasis of CKX enzyme in the research object. The models of co-regulation of other CKX by highly silenced TaCKX1 and knock-out HvCKX1 [27] differ between these species.
The differences in the levels of expression of TaCKX1 and various co-expression of other TaCKX genes in T 1 and T 2 resulted in opposite phenotypic effects. Since spike number, grain number, and grain yield were reduced in T 1 , the same yield-related traits were significantly higher in highly silenced T 2 plants. High-yielding phenotype occurred when highly silenced TaCKX1 co-operated with down-regulated TaCKX11 (3) but up-regulated TaCKX5, TaCKX2.2, TaCKX2.1, and TaCKX9 (10). These differences showed that both levels of silencing might be helpful to better understand the function of developmentally regulated genes. Unexpectedly, changes in the expression levels of co-working TaCKX did not result in different enzyme activity, even in highly silenced T 2 plants. This might be explained by the fact that down-regulation of TaCKX1 and TaCKX11 (3) is compensated for by the up-regulation of TaCKX2.2, TaCKX5, and TaCKX9 (10), and therefore the contribution of isozymes encoded by the genes in the general pool of CKX enzyme activity is the same. Since CKX enzymes indicate different specificities for the particular cytokinin hormone [37], the cytokinin contribution and phenotypic traits of modified plants were changed accordingly, with consequent differences in the active pool of CKs influencing phenotype.

Co-Operating Effect of TaCKX on the Level of Active CKs in Silenced Plants
Since CKX isozymes specifically degrade CKs, the highly decreased expression of TaCKX1 and TaCKX11 (3) in 7 DAP spikes is expected to result in the observed increase of most major forms of CKs: tZGs, tZ, and cZ in silenced plants. We documented that both tZ and cZ, which are isomers of zeatin, together with their derivatives are a major group of isoprenoid CKs in 7 DAP spikes. It has already been shown that trans-zeatin is the predominant form after anthesis [36,38], but comprehensive analysis of cytokinins during spike, spikelet, ovule and grain development has not yet been reported for wheat using LC-MS/MS [22]. The content of DZGs increased by 40% in silent compared to non-silent wheat plants, suggesting that this less known isoprenoid form of CKs might also play an important role in plant productivity. Interestingly, isoprenoid iP was represented in 7 DAP spikes of non-silent plants at very low quantities, but its content in 7 DAP spikes of silent plants was increased by 32%. A similar relationship between the reduced expression of selected CKX family genes and cytokinin accumulation in reproductive organs has been observed in other species including A. thaliana [39], rice [19], and barley [25], but detailed data are not comparable to our research in wheat.
The physiological significance of these isoprenoid forms is still not very well known. tZ and iP, which are susceptible to CKX, were found the most abundant and bioactive CKs in maize, whereas cZ, which shows low affinity to CKX was reported to have a weak biological impact and unknown biological role [40,41]. However, the cZ concentrations changed significantly during development in maize grain, as well as in shoot and root tissues [42,43]. High levels of cZ at the first developmental stage of barley spike observed by Powell et al. [44] might indicate an important role of this form in early barley embryo development, what is also documented in our results (discussed further below).
The BA is represented in 7-DAP spikes of wheat at trace amounts but their content was significantly decreased in silent plants. However, their correlations with the TaCKX genes as well as yield-related traits of non-silenced plants indicate their importance (discussed in more detail below). Interestingly, BA was found to participate in posttranscriptional and/or posttranslational regulation of protein abundance in Arabidopsis, showing high specificity to shoots and roots, and affected differential regulation of hormonal homeostasis [45].

Cross Talk of CKs with Other Phytohormones
Negative correlations between ABA content and TaCKX2.2 and TaCKX9 (10) expression, and positive with TaCKX11 (3), were associated with a slight decrease of ABA content in 7 DAP spikes of silenced plants. Moreover, ABA was strongly positively correlated with BA. The main auxin, IAA, remained at the same level. A ten-fold increase of GA content in silenced comparing to non-silenced plants was observed. Such cross regulation of CKs and other plant hormones is documented in other species. In maize kernels the CKX1 gene is up-regulated by cytokinin and ABA, and abiotic stress [18]. In tobacco altered cytokinin metabolism affected cytokinin, auxin, and ABA contents in leaves and chloroplasts [46], which host the highest proportion of CK-regulated proteins [47]. Moreover, auxin, ABA and cytokinin are involved in the hormonal control of nitrogen acquisition and signalling [48], which often limits plant growth and development. All four phytohormones, CKs, GA, IAA, and ABA, were found to be involved in the regulation of grain development in drought conditions [49]. Moreover, in shoots, BA up-regulated the abundance of proteins involved in ABA biosynthesis and the ABA response, whereas in the roots, BA strongly up-regulated the majority of proteins in the ethylene biosynthetic pathway [45]. We proved that IAA, GA, and ABA contents are also co-regulated by CKs in non-silenced and silenced 7 DAP spikes. Up-regulation of major CKs and down-regulation of some minor ones in silent plants influence GA, ABA, and IAA content in a similar manner as in abiotic stress conditions.

Coordinated Effect of TaCKX Gene Expression on the Content of CKs, Other Phytohormones and Yield-Related Traits
Plant height in non-silenced plants is down-regulated by BA and up-regulated by IAA and GA content in the first 7 DAP spikes, resulting in taller plants. Oppositely, increased content of tZ and tZGs negatively correlated with the trait in silent plants, stimulated plant height. As it was already showed [50,51] and similarly to our results, plant height and root weight are regulated by CKs and IAA in opposite ways. This may be dependent on basipetal auxin flow in the stem, which suppresses axillary bud outgrowth, and similarly as in pea, auxin derived from a shoot apex suppresses the local level of CKs in the nodal stem through the regulation of CKX or IPT genes [52].
The main role in spike length seemed to be played by cZ and its glucoside. Increased content of cZOG in non-silenced plants negatively correlated with ABA, resulting in longer spikes. In silent plants the trait is positively regulated by TaCKX2.2 together with TaCKX5, and the latter is a positive regulator of enzyme activity and negative of cZ content. Consequently, a higher content of cZ in 7 DAP spikes led to shorter spikes. cZOG, found as a positive regulator of longer spikes, is a sugar conjugate of cZ-0-glucoside, which is the inactivated form of cZ, showing metabolic stability against CKX activity [53]. Moreover, 0-glucosylation of cZ is catalysed by a specific 0-glucosyltransferase, cisZOG1, discovered in maize [54], and this form mainly functions in the early stages of seed development. Knowledge of function of cZ degradation pathways via the CKX enzyme is limited. Interestingly, two Arabidopsis genes, CKX1 and CKX7, expressed in stages of active growth, were shown to have high preference for cZ [37]. In our case the TaCKX5 positively regulated CKX activity and negatively cZ content.
None of the tested individual TaCKX genes was involved in high TGW in non-silenced plants, but a negative correlation with cZ and positive with GA was found. Otherwise a significant negative correlation of TaCKX2.1 and a positive correlation of TaCKX11 (3) in determining low TGW were observed in silenced plants. Unexpectedly increased expression of the first one positively influenced tZ, cZ, and iP content and negatively GA content, and the opposite was true for the second gene, resulting in lower TGW. Therefore both TaCKX2.1 and TaCKX11 (3), acting in an opposite manner, maintain homeostasis of CKX enzyme activity and co-regulate TGW in silenced plants. A greater concentration of CKs, especially tZ, was observed during the grain filling stage of high-yielding cultivars [44]. We might suppose that the observed higher concentrations of tZ and other CKs at the 7 DAP stage, which originally was a consequence of TaCKX1 silencing, might accelerate germination of the grains, which resulted in smaller grains/lower TGW than in non-silenced plants. The silenced TaCKX1 co-work with down-regulated TaCKX11 (3) in increasing CK content as well as up-regulating TaCKX2.1, with seems to play a regulatory role. The involvement of GA in TGW and other traits demonstrated by us might be the effect of co-regulation of CKX and other gibberellin-responsive genes regulating yield-related traits as well [55,56]. Fahy et al. [57] suggested that final grain weight might be largely determined by developmental processes prior to grain filling. This is in agreement with our observations, in which yield-related traits are differently regulated in two groups of plants, non-silent and silent. Therefore, we might suppose that the coordinated co-regulation of expression of TaCKX genes and related CKs takes place during whole plant and spike development and small seeds in silenced plants are determined at earlier stages.
Grain yield, which is very strongly correlated with grain and spike number in non-silent plants but with TGW in silent plants, is a more complex feature. Two groups of genes up-regulating or down-regulating grain yield in non-silent plants have been found. The first one includes TaCKX1, 2.2, and 5 positively regulating iP content but negatively BA. The second comprises TaCKX11 (3) acting in down-regulation of tZGs. Both groups might determine lower grain yield. It is worth to mention that TaCKX5, which is highly expressed in inflorescences and leaves might be a main player of this trait. Higher grain yield was positively regulated by enzyme activity and both, down-regulated TaCKX11 (3) as well as up-regulated TaCKX2.1 in silenced plants. Again, the TaCKX2.1 positively regulated tZGs and cZ content just like for TGW, which is rather untypical for a gene encoding a CKX enzyme degrading CKs. Therefore, the positive regulation of the main CK content by TaCKX2.1 observed by us supports its role in regulation of expression of other genes rather than encoding the CKX isozyme.
As observed in barley cultivars, changes in cytokinin form and concentration in developing kernels correspond with variation in yield [44]. Interestingly, the authors observed no peaks and no differences in CKX activity at the particular stages of spike development. This is in agreement with the 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 [20,21,25]. In this research, CKX activity was decreased, however according to Zalewski et al. [20], it was measured not in 7 DAP spikes, but in 0 DAP spikes and seedling roots. Therefore this inconsistency might be result of measurements in various organs/developmental stages. Another explanation is that these two cereal species varied three times in ploidy level, what might influence differences in action of both orthologues. The TaCKX homologues located on A, B and D chromosomes might significantly affect homeostasis of pooled CKX isozymes in wheat. Incomparable to the results obtained for RNAi silenced TaCKX1 and HvCKX1, no changes in yield parameters were observed in mutant lines with knock-out of HvCKX1 (Gasparis et al., 2019). These essential phenotypic differences between RNAi-silenced TaCKX1 and HvCKX1 or knocked out by CRISPR-Cas9 HvCKX1 might be the result of different processes involved in inactivation of the gene. The first one is regulated at the posttranscriptional and the second at the transcriptional level. Since CKs might regulate various developmental and physiological processes at the posttranscriptional level [6,7] or by modulation of context-dependent chromatin accessibility [8], the way of deactivating TaCKX function seemed to be important.
Spike number and grain number are highly correlated in both non-silent and silent plants and are regulated by the same groups of TaCKX genes as well as phytohormones. The first group includes TaCKX1, 2.2 and 5 positively regulating iP but negatively BA. The second comprises TaCKX11 (3) and 2.1 acting in the opposite way, and homeostasis of these hormones in non-silenced plants maintains a lower spike number. The main role in controlling higher spike and grain number in silent plants seemed to be played by TaCKX5, highly expressed in seedling roots, leaves, inflorescences and 0 DAP spikes. These correlations are not significant because they were measured in a stage of plant development in which the number of spikes and seed number have already been set. As reported, the higher spike number was the consequence of a higher tiller number, which was positively correlated with the content of endogenous zeatin in the field-grown wheat after exogenous hormonal application [58]. Shoot branching might also be dependent on the acropetal transport of cytokinin [52].
Root weight was positively correlated with lower expression of TaCKX9 (10) in 7 DAP spikes of non-silent plants and, negatively with increased expression of this gene in silenced plants. Therefore the gene might determine lower root weight in the first group of plants, but higher in the second. Increased expression of TaCKX9 (10) down-regulated cZOG. The same cZOG was up-regulated by TaCKX11 (3), but expression of this gene in 7 DAP spikes of silent plants is strongly decreased. Both cZ and cZOG are involved in spike length regulation as well as TGW and grain yield in the group of silenced plants.
Although both tested organs are in different developmental stages, correlations between TaCKX9 (10) and TaCKX11 (3) expression in 7 DAP spikes and weight of seedling roots are reasonable. The TaCKX9 (10) is mainly expressed in younger organs from seedling roots to 0 DAP spikes and highly expressed in leaves. The TaCKX11 (3) is expressed in all organs tested [23] and both seemed to regulate seedling roots as well, although in the opposite manner. Therefore, we should take into consideration the possible action of cytokinin transport and signalling genes as well as other phytohormones which take part in hormonal crosstalk to control the regulation of root growth [59]. Accordingly, cZ type CKs found as the major forms in phloem are translocated from shoots to roots [60,61]. Some CKX genes might be induced by transcription factors [62,63], what is also observed in our unpublished yet data.
The lower plant height and higher root weight observed in the group of silenced plants of wheat is in agreement with opposed regulation of these traits by CKs and IAA mentioned above [64,65]. Up-regulated content of active cZ in 7 DAP spikes, might influence down-regulation of this CK in roots. It has been documented that such suppressing cZ levels mediated by overexpression of AtCKX7 affected root development in Arabidopsis [66]. A higher weight of seedling root was also obtained by silencing via RNAi or knock-out via CRISPR/Cas9 of HvCKX1 in barley plants, as in wheat, and the trait corresponded with decreased activity of CKX enzyme measured in roots (Zalewski et  It was previously demonstrated that level of chlorophyll content in flag leaves is associated with the senescence process, in which CKs suppress inhibition of senescence [67]. During this processes, proteins are degraded and nutrients are re-mobilised from senescing leaves especially to the developing grains [68]. We might suppose that slower spike ripening in non-silent plants, which is dependent on lower CK content in the 7 DAP spike, causes a slower flow of micronutrients as well as CKs from flag leaf to spike. Therefore, prolonged chlorophyll content in the flag leaf of the first spike negatively correlated with TGW but positively with plant height. Opposite data were obtained for flag leaves of silent plants, in which higher content of CKs in 7 DAP spikes might be the result of faster flow accelerating leaf senescence. The reduced chlorophyll content in flag leaves of the first spike of silent plants positively correlated with grain yield. The important role of tZ and less active cZ in the suppression of senescence was proven in maize leaves [69] and in an oat-leaf assay [37]. It was also documented that delayed senescence of wheat stay-green mutant, tasg1, at the late filling stage was related to high cytokinin and nitrogen contents [70].

Vector Construction
The hpRNA type of silencing cassette was constructed in pBract207 (https://www.jic.ac. uk/technologies/crop-transformation-bract/). It contains the Hpt selection gene under the 35S promoter and cloning sites for the cloning silencing cassette under the Ubi promoter. The vector is compatible with the gateway cloning system. For cloning purposes a coding sequence of TaCKX1 (NCBI JN128583) 378 codons long was used. In the first step, the cassette was amplified using: EAC11-F: 5 -TTGAATTCGACTTCGACCGCGGCGTTTT-3 and EAC12-R: 5 -TTGAATTC ATGTCTTGGCCAGGGGAGAG-3 and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). In the next step, the cassette was cloned to the destination Bract7 vector in the gateway reaction. The presence of the silencing cassette in the vector was verified by restriction analysis and sequencing. The vector was electroporated into the AGL1 strain of Agrobacterium tumefaciens and used for transformation.

Plant Material, Agrobacterium-Mediated Transformation and In-Vitro Culture
The spring cultivar of common wheat (Triticum aestivum L.) Kontesa was used as a donor plant for transformation experiments as well as transgenic plants. Seeds were germinated into Petri dishes for one day at 4 • C and then five days at room temperature in the dark. Six out of ten seedlings from each Petri dish were replanted into pots with soil. The plants were grown in a growth chamber under controlled environmental conditions with 20 • C/18 • C day/night temperatures and a 16 h light/8 h dark photoperiod. The light intensity was 350 µmol·s −1 ·m −2 .
Agrobacterium-mediated transformation experiments were performed according to our previously described protocols for wheat [71,72]. Putative transgenic plants were regenerated and selected on modified MS media containing 25 mg·L −1 of hygromycin as a selectable agent. First, 7 days after pollination, (DAP) spikes from T 1 , T 2 , and control plants were collected for RT-qPCR and phytohormone quantification. Only 1 in 3 of the middle part of each spike was used for experiments (upper and lower parts were removed).

PCR Analysis
Genomic DNA was isolated from well-developed leaves of 14-day plants according to the modified CTAB procedure [73] or by using the KAPA3G Plant PCR Kit (Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA, USA). The PCR for genomic DNA isolated by CTAB was carried out in a 25 mL reaction mixture using Platinum Taq DNA Polymerase (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and 120 ng of template DNA. The reaction was run using the following program: initial denaturation step at 94 • C for 2 min, 35 cycles of amplification at 94 • C for 30 s, 65 • C for 30 s, 72 • C for 30 s with a final extension step at 72 • C for 5 min. The PCR for genomic DNA isolated by KAPA3G was carried out in a 50 µL reaction mixture using 1 U of KAPA3G Plant DNA Polymerase and a 0.5 × 0.5 mm leaf fragment. The reaction was run using the following program: initial denaturation step at 95 • C for 3 min, 40 cycles of amplification at 95 • C for 20 s, 68 • C for 30 s, 72 • C for 30s with a final extension step at 72 • C for 2 min.
Putative transgenic T 0 and T 1 plants were tested with two pairs of specific primers amplifying a fragment of the hpt selection gene. The sequences of the primers for the first pair were: hygF1 5 -ATGACGCACAATCCCACTATCCT-3 and hygR1 5 -AGTTCGGTTTCAGGCAGGTCTT-3 , and the amplified fragment was 405 bp. The sequences of the primers for the second pair were: hygF2 5 -GACGGCAATTTCGATGATG-3 and hygR2 5 -CCGGTCGGCATCTACTCTAT-3 , and the amplified fragment was 205 bp.
Non-transgenic null segregants were used as a control.

RNA Extraction and cDNA Synthesis
Total RNA from 7 DAP spikes was extracted using TRI Reagent (Sigma-Aldrich, Hamburg, Germany) and 1-bromo-3-chloropropane (BCP) (AppliChem GmbH, Darmstadt, Germany) according to the manufacturer's protocol. The purity and concentration of the isolated RNA were determined using a NanoDrop spectrophotometer (NanoDrop ND-1000) and the integrity was checked by electrophoresis on 1.5% (w/v) agarose gels. To remove the residual DNA the RNA samples were treated with DNase I, RNase-free (Thermo Fisher Scientific, Waltham, MA, USA). Each time 1 µg of good quality RNA was used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer's instructions. The obtained cDNA was diluted 20 times before use in RT-qPCR assays.

Quantitative RT-qPCR
RT-qPCR assays were performed for 6 target genes: TaCKX1  Relative expression/silencing of TaCKX1 was related to mean expression of the gene in non-silenced control plants set as 1.00. Relative expression of other TaCKX genes was related to each tested gene set as 1.00 in non-silenced plants.
Statistical analysis was performed using Statistica v13.3 software (StatSoft, Kraków, Poland). The normality of data distribution was tested using the Shapiro-Wilk test. To determine whether the means of two sets of data of expression levels, phytohormone concentrations, and yield-related traits between non-silenced and silenced lines are significantly different from each other (for p value less than p < 0.05), either the Student's t-test or the Mann-Whitney test was applied. Correlation coefficients were determined using parametric correlation matrices (Pearson's test) or a nonparametric correlation (Spearman's test).
For the measurement of phytohormones, 200 mg of plant powders were placed into the 2-mL Eppendorf tubes, suspended in 1 mL of (v/v) 50% ACN, and homogenized in a bead mill (50 Hz, 5 min) using two 5-mm tungsten balls. Then, samples were homogenized using the ultrasound processor VCX 130 (max. power 130 W, max. frequency 20 kHz, 5 min) equipped with titanium probe and mixed in laboratory shaker (90 rpm, dark, 5 • C, 30 min). Samples were centrifuged (9000× g, 5 min) and collected in a glass tube. For the quantification of ABA, AXs, CKs, and GA 3  Prepared extracts were purged using a Waters SPE Oasis HLB cartridge (Waters Corporation, Milford, MA, USA), previously activated and equilibrated using 1 mL of 100% MeOH, 1 mL water, and 1 mL of (v/v) 50% ACN [74]. Then, extracts were loaded and collected to the Eppendorf tubes and eluted with 1 mL of 30% ACN (v/v). Samples were evaporated to dryness by centrifugal vacuum concentrator, dissolved in 50 µL of (v/v) 30% ACN and transferred into the insert vials. Detection of analyzed phytohormones was performed using an Agilent 1260 Infinity series HPLC system (Agilent Technologies, Santa Clara, CA, USA) including a Q-ToF LC/MS mass spectrometer with Dual AJS ESI source; 10 µL of each sample was injected on the Waters XSelect C 18 column (250 mm × 3.0 mm, 5 µm), heated up to 50 • C. Mobile phase A was 0.01% (v/v) FA in ACN and phase B 0.01% (v/v) FA in water; flow was 0.5 mL min −1 . Separation of above hormones was done in ESI-positive mode with the following gradient: 0-8 min flowing increased linearly from 5 to 30% A, 8-25 min 80% A, 25-28 min 100% A, 28-30 min 5% A.
For the optimization of MS/MS conditions, the chemical standards of analyzed phytohormones were directly injected to the MS in positive ([M + H] + ) ion scan modes, then areas of detected standard peaks were calculated. [M + H] + was chosen because of its significantly better signal-to-noise ratios compared to the negative ion scan modes.
Chlorophyll content was measured using an SPAD chlorophyll meter.

Conclusions
Based on the 7 DAP spike as a research object, we have documented that silencing of TaCKX1 by RNAi strongly influenced up-or down-regulation of other TaCKX genes, as well as phytohormone levels and consequently phenotype. This co-regulation is dependent on the level of silencing of the gene and is independent of cross-silencing of other TaCKX genes. Detailed analysis revealed that each tested yield-related trait is regulated by various up-or down-regulated TaCKX genes and phytohormones. Key genes involved in the regulation of grain yield, TGW, or root weight in highly silenced plants are TaCKX2.1 and TaCKX11 (3) acting antagonistically, and increased expression of the first one determines growth of tZ, tZ derivatives, and cZ, whereas decreased expression of the second down-regulates content of cZOG. A key role in determination of the high-yielding phenotype seemed to be played by the growing content of tZ in 7 DAP spikes, which might accelerate maturation of immature grains by speeding up nutrient flow from flag leaves. This finally led to reduction of TGW but enhancement of grain number and yield. The latter traits are the result of a higher spike number, which is determined in the early stages of plant development.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/13/4809/ s1. Table S1: Primer sequences designed for reference gene and each of 6 tested TaCKX genes and amplicon length. Table S2: Phenotypic traits and ratio indicator in silent T 1 and not silent, control plants. Table S3: Phenotypic traits and ratio indicator in silent T 2 and not silent, control plants. Table S4