*3.3. Regulation of Yield-Related Traits by TaCKX GFMs and TaNAC2-5A in the F<sup>2</sup> Generation*

The grain number, grain yield, spike number, and TGW were strongly positively correlated with *TaCKX2.1* and *TaCKX2.2.2* independent of the parent; however, only in the crosses resulted in decreased yield. In contrast, negative correlations were observed between *TaCKX2.1*, *TaCKX2.2.2,* and TGW in a reciprocal cross of C7/C8 and *TaCKX2.2.2* in a one-way cross (C4), in which the F<sup>2</sup> progeny had a higher yield. All these correlations

prove our earlier observations [6,7]. Modified wheat lines with 60% decreased expression of *TaCKX2.2.2,* and a slight decrease in the *TaCKX2.2.1* and *2.1* genes exhibit a significantly higher TGW and slightly increased yield [7]. Interestingly, this result was observed in cultivars and breeding lines that represent awnless spikes. In the owned-spike cultivar, silencing of the *TaCKX2* genes co-expressed with other *TaCKX* resulted in decreased yield; however, TGW was at the same level as in non-silent plants [5]. Furthermore, a strong feedback mechanism for regulation of the expression of *TaCKX2* and *TaCKX1* genes was observed in both awnless and owned-spike cultivars [5–7]. Silencing of *TaCKX2* genes upregulated the expression of *TaCKX1* and vice versa. This feedback mechanism could explain the observed positive correlations of the *TaCKX2* genes with yield-related traits in low-yielding F<sup>2</sup> progeny and negative correlations in high-yielding F<sup>2</sup> progeny. A similar mechanism is visible when we analyze individual traits in high-yielding F2, such as grain number, grain yield, and spike number. These traits are promoted by up-regulated in 7 DAP spikes *TaCKX2.1* and down-regulated *TaCKX1*. Silencing of *HvCKX1* in barley, which is an ortholog of *TaCKX1*, decreased CKX enzyme activity and led to increased seedling root mass and higher plant productivity [55]; however, knock-out of this gene caused a significant decrease in CKX enzyme activity but no changes in grain yield were observed [56]. These differences might be explained by differences in the level of decreased gene expression, which variously coordinate the expression of other genes, regulate phytohormone levels, and determine particular phenotypes, as was already documented in wheat [5,7].

The association of *TaCKX2* genes with yield-related traits has also been reported in different wheat cultivars or genotypes. Zhang et al. [20] showed that *TaCKX6* (renamed by Chen et al. [11] *TaCKX2.2.1-3D*), which is an ortholog of rice *OsCKX2* associated with grain number [57], is related to grain weight. Another allele of *CKX2*, *TaCKX6a02* [21], annotated as *TACKX2.1* [58], significantly correlated with grain size, weight, and grain filling rate. Wheat plants with silenced by RNAi expression of *TaCKX2.2.1-3A* (originally *TaCKX2.4*) showed a strong correlation with the number of grains per spike implied by more filled florets [59]. Since *TaCKX2.2.1-3D* was associated with grain weight, these differences in functions between *TaCKX2.2.1-3A* and *TaCKX2.2.1-3D* were interpreted as subgenome-dependent.

Grain number was also negatively correlated with *TaCKX1* and *TaCKX5*, and grain yield was negatively correlated with *TaCKX1*, predominantly for M and F2, which were characterized by decreased grain number and lower yield. This observation is also in agreement with previous research. The silencing of *TaCKX1* caused an increase in spike number and grain number but a decrease in TGW because this trait is opposite to grain number [6]. The low-yield progeny of F<sup>2</sup> showed positive correlations between the expression of *TaCKX11*, *3*, *5*, and *8*, and *NAC2* in the seedling roots and the grain number, the spike number and the grain yield; however, the higher-yield progeny of F<sup>2</sup> displayed a negative correlation between *TaCKX1* and these yield-related traits in the seedling roots of some crosses. In summary, high-yielding F<sup>2</sup> was the result of upregulation of *TaCKX2.1* in spikes and downregulation of *TaCKX1* in seedling roots. As documented earlier, *TaCKX11*, *5*, *8* and *TaNAC2-5A* are expressed in all organs, and their expression is correlated with the expression of spike-specific *TaCKX2* and *TaCKX1* [8,58].

Rice *OsCKX11* is an orthologue of wheat *TaCKX11* and is highly expressed in the roots, leaves, and panicles. The gene was shown to coordinate the simultaneous regulation of leaf senescence and grain number by the relationship of source and sink [60]. Since *TaCKX11* is expressed in seedling roots and highly expressed in leaves, inflorescences, and 0, 7, and 14 DAP spikes, it could perform a similar function. This is partly proven by silencing of the *TaCKX2* genes in awnless spikes of cv. Kontesa, which resulted in significant upregulation of *TaCKX11* and growth of TGW, and chlorophyll content in flag leaves [7]. In contrast, *TaCKX11* is significantly negatively regulated by *TaCKX1*, resulting in a higher spike number and grain number [6]. Its orthologue in rice, *OsCKX11,* was found to regulate leaf senescence and grain number by the coordinated source and sink relationship [60].

Based on a summary of the regulation of yield-related traits in high-yielding F2, it is possible to identify singular genes or groups of genes that are up- or down-regulated in 7 DAP spike or seedling roots and specifically regulate yield-related traits. Upregulated in spikes *TaCKX2.1* and downregulated in seedling roots *TaCKX1* were found to determine grain number, grain yield, spike number, and spike length. Furthermore, upregulated in 7 DAP spikes *TaCKX10* and downregulated *TaNAC2-5A,* together with others, depending on cross, control spike length, semi-empty spikes, root mass, and increased grain yield. As discussed above, high TGW is in contrary to high grain number and partly grain yield and was strongly determined by downregulated *TaCKX2.2.2* together with *TaCKX2.1* in 7 DAP spikes and upregulated *TaCKX10* and *NAC2* in seedling roots. The upregulated in seedling roots *TaNAC2-5A* participates in the determination of TGW and plant height, and the downregulation of *TaNAC2-5A* in seedling roots controls the development of semi-empty spikes and root mass.

In previous research, *TaNAC2-5A* has been documented as a gene encoding a nitrateinducible wheat transcription factor. Overexpression of the gene improved root growth, grain yield, and grain nitrate concentration [25]. This is in agreement with our observations of growth of TGW but not enhanced roots. The increase was argued to be the consequence of regulation of nitrate concentration and its remobilization in developing grains by direct binding of the *TaNAC2-5A* protein to the promoter of the nitrate transporter, *TaNRT2.5-3A* and positive regulation of its expression [61]. The expression of *TaNAC2-5A* is coregulated by expressed in 7 DAP spikes *TaCKX2* genes and expressed in 7 DAP spikes and seedling roots *TaCKX1* gene [5,7]. Independent of awnless or awned-spike genotype, downregulation of *TaCKX2* genes by RNAi significantly increased *TaNAC2-5A* expression, resulting in higher chlorophyll content in flag leaves and delayed leaf senescence. As discussed above, the strong feedback mechanism between the *TaCKX2* and *TaCKX1* genes implies that downregulation of *TaCKX1* resulted in opposite results. Similar to our observations in wheat, an ortholog of *TaNAC2-5A* in rice, *OsNAC2,* was described as a negative regulator of crown root number and root length [62]. Its expression was positively correlated with cytokinin synthesis genes, *OsIPT3*, *5*, the gene determining the formation of active cytokinins, *OsLOG3*, and negatively correlated with *OsCKX4* and *5*. The authors concluded that *OsNAC2* stimulated cytokinin accumulation by suppressing *CKX* expression and stimulating *IPT* expression by binding the OsNAC protein to the promoters of these genes. Therefore, *OsNAC2* functions as an integrator of cytokinin and auxin signals that regulate root growth. In our experiments, orthologous to *OsCKX4*, *TaCKX4* was not tested due to its weak expression in roots. However, the up-regulated expression of highly specific in seedling roots *TaCKX3* and *TaCKX8* [8,58] was antagonistically regulated by *TaNAC2-5A* in these organs, positively influencing seedling growth. Furthermore, our in silico analysis of *TaNACs* with *TaIPTs* and *TaCKXs* showed that the same NAC proteins might join promotor sites of cytokinin synthesis and cytokinin degradation genes in wheat (Iqbal et al., not published yet).

#### **4. Materials and Methods**

#### *4.1. Plant Material*

Five common wheat breeding lines and cultivars (*Triticum aestivum* L.), named S12B, S6C, D16, KOH7, and D19, which showed differences in the expression levels of *TaCKX* GFMs and *TaNAC2-5A* (*NAC2*) in 7 DAP spikes, seedling roots, and yield-related traits were selected for the study. They were used in four reciprocal crosses: (1) S12B × S6C and S6C × S12B (C1 and C2, respectively); (2) D16 × KOH7 and KOH7 × D16 (C3 and C4, respectively); (3) D19 × D16 and D16 × D19 (C5 and C6, respectively); and (4) D19 × KOH7 and KOH7 × D19 (C7 and C8, respectively) to obtain the F<sup>1</sup> and F<sup>2</sup> generations. The experimental tissue samples were collected from the parental lines and their F<sup>2</sup> progeny growing in a growth chamber during the same period.

Ten seeds of each genotype germinated in Petri dishes for five days at room temperature in the dark. Six out of ten seedlings from each Petri dish were replanted in pots

with soil. The plants were grown in a growth chamber under controlled environmental conditions with 20 ◦C day/18 ◦C night temperatures and a 16 h light/8 h dark photoperiod. The light intensity was 350 µmol s−<sup>1</sup> ·m−<sup>2</sup> . The plants were irrigated three times a week and fertilized once a week with Florovit according to the manufacturer's instructions.

The following tissue samples in three biological replicates were collected: 5-day-old seedling roots, which were cut 0.5 cm from the root base before replanting in the pots, and first 7 DAP) spikes from the same plants grown in the growth chamber. All of these samples were collected at 9:00 a.m. The collected material was frozen in liquid nitrogen and kept at −80 ◦C until use.

### *4.2. Cross-Breeding*

The maternal plant was deprived of its own anthers so that it would not self-fertilize, then pollinated by transferring three anthers from the paternal plant for each ovary of the maternal parent plant and placed in an isolator. The seeds were harvested.

#### *4.3. RNA Extraction and cDNA Synthesis*

Total RNA from 7 DAP spikes and roots from 5-day-old seedlings was extracted using TRI Reagent (Invitrogen, Lithuana) according to the manufacturer's protocol. The concentration and purity of the isolated RNA were determined using a NanoDrop spectrophotometer (NanoDrop ND-1000, Thermi Fisher Scientific, Wilmington, DE, USA), and the integrity was checked on 1.5% (*w/v*) agarose gels. To remove residual DNA, RNA samples were treated with DNase I (Thermo Fisher Scientific, Lithuana). Each time, 1 µg of good quality RNA was used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Lithuana) following the manufacturer's instructions. The cDNA was diluted 20 times prior to use in the RT-qPCR assays.

#### *4.4. Quantitative RT-qPCR*

RT-qPCR assays were performed for 10 genes: *TaCKX1*, *TaCKX2.1*, *TaCKX2.2.2*, *TaCKX3*, *TaCKX5*, *TaCKX8*, *TaCKX9*, *TaCKX10*, *TaCKX11*, and *TaNAC2-5A*. The sequences of the primers for each gene are shown in Table S2. All real-time reactions were performed on a Rotor-Gene Q (QIAGEN Hilden, Germany) thermal cycler using 1× HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne, Estonia), 0.2 µM of each primer and 4 µL of cDNA in a total volume of 10 µL. Each reaction was carried out in three biological and three technical replicates in the following temperature profile: initial denaturation and polymerase activation of 95 ◦C–12 min (95 ◦C–25 s, 62 ◦C–25 s, 72 ◦C–25 s) × 45 cycles, 72 ◦C–5 min, with melting curve at 72–99 ◦C 5 s per step. The expression of *TaCKX* genes was calculated according to the two standard curve method using *ADP-ribosylation factor* (*Ref 2*) as a normalizer. The relative expression for each *TaCKX* GFM and *TaNAC2-5A* was calculated in relation to the control female parents, set as 1.00.

### *4.5. Analysis of CKX Activity*

CKX enzyme activity was performed in the same samples subjected to *TaCKX* gene expression analysis according to the procedure developed by Frebort et al. [63] and optimized for wheat tissues. The plant material was powdered with liquid nitrogen using a hand mortar and extracted with a 3-fold excess (*v*/*w*) of 0.2 M Tris–HCl buffer, pH 8.0, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.3% Triton X-100 ((St. Louis, MO, USA). Plant samples were incubated in a reaction mixture consisting of 100 mM McIlvaine buffer, 0.25 mM of the electron acceptor dichlorophenolindophenol and 0.1 mM of substrate (N6 isopentenyl adenine). The volume of the enzyme sample used for the assay was adjusted based on the enzyme activity. The incubation temperature was 37 ◦C for 1–16 h. After incubation, the reaction was stopped by adding 0.3 mL of 40% trichloroacetic acid (TCA) and 0.2 mL of 2% 4-aminophenol (PAF). The product concentration was determined by scanning the absorption spectrum from 230 nm to 550 nm. The total protein concentration

was estimated based on the standard curve of bovine serum albumin (BSA) according to the Bradford procedure [64].

#### *4.6. Measurement of Yield-Related Traits*

Morphometric measurement of yield-related traits of selected genotypes was performed. The described traits were plant height, spike number, semi-empty spike number, tiller number, spike length, grain yield, grain number, TGW, and 5-day seedling root weight.

#### *4.7. Statistical Analysis*

Statistical analysis was performed using Statistica 13 software (StatSoft). The normality of the data distribution was tested using the Shapiro–Wilk test. The significance of the changes was analyzed using ANOVA variance analysis and post hoc tests. The correlation coefficients were determined using parametric correlation matrices (Pearson's test) or a nonparametric correlation (Spearman's test).

#### **5. Conclusions**

We indicate, for the first time, that the pattern of expression of selected *TaCKX* GFMs and *TaNAC2-5A*, and grain yield in wheat, is paternally inherited by the F<sup>2</sup> generation. Pater-origin transmission of gene expression levels sheds new light on the method of parent selection and crossing to obtain high-yielding phenotypes. We also showed which genes cooperate together by upregulation or downregulation and which function in the opposite manner in establishing yield-related traits. This knowledge can be applied to select the desirable phenotype in F2. For example, a high-yielding paternal parent with downregulated, compared to the maternal parent, expression of *TaCKX2.1* and *TaCKX11* in 7 DAP spikes and upregulated expression of *TaCKX3* and *TaCKX8* and downregulated *TaNAC2-5A* in seedling roots is expected to transmit this pattern of expression to F2, which will result in a high yield. The main problem is the antagonistic expression patterns of genes for some important yield-related traits, such as grain number, grain yield, and spike number, to TGW, which is the result of the feedback mechanism of the regulation of expression between *TaCKX1* and *TaCKX2* genes and others. The expression analysis of *TaNAC2-5A* and the in silico analysis of *TaNAC* GFMs revealed that the encoded proteins participate in the regulation of transcription of selected *TaCKX* genes responsible for cytokinin degradation and *TaIPT* genes responsible for cytokinin biosynthesis. Therefore, *TaNACs* are important additional regulators of yield-related traits in wheat, which should be taken into consideration in wheat breeding.

### **6. Patents**

Nadolska-Orczyk A., Szala K., Dmochowska-Boguta M., Orczyk W. Wzory ekspresji genów jako nowe markery molekularne produktywno´sci zbóz oraz spos ˙ ób przekazywania wysokiej produktywno´sci I strategia selekcji wysokoplonuj ˛acych odmian zbóz. (Patterns of ˙ gene expression as new molecular markers of cereal productivity and a way of transfer of high yield and the strategy for selecting high-yielding cereal varieties). Patent application filed with the Polish Patent Office (UP RP) 23 January 2023, nr P.443557.

**Supplementary Materials:** The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms24098196/s1.

**Author Contributions:** Conceptualization, A.N.-O., K.S. and W.O.; methodology, K.S. and M.D.-B.; software, K.S. and M.D.-B.; validation, W.O.; formal analysis, K.S., M.D.-B. and A.N.-O.; investigation, K.S. and J.B.; data curation, K.S., M.D.-B. and J.B.; writing—original draft preparation, A.N.-O.; writing—review and editing, A.N.-O. and W.O.; visualization, K.S., M.D.-B., J.B. and A.N.-O.; supervision, A.N.-O.; project administration, A.N.-O.; funding acquisition, A.N.-O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Ministry of Agriculture and Rural Development, grant No. 5 PBwPR 4-1-01-4-02, and the Statutory Project of PBAI-NRI. The funding body did not perform a role in the design of the study; the collection, analysis, and interpretation of data; or the writing of the manuscript.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analysed during this study are included in this published article [and its Supplementary Materials files].

**Acknowledgments:** We thank Malgorzata Wojciechowska, Izabela Skuza and Agnieszka Glowacka for excellent technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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