Winter wheat (Triticum aestivum L.) is one of the most important grain crops worldwide. In many countries, including Argentina, Australia, Pakistan, and the United States, winter wheat is cultivated for dual purposes, both as winter forage and as a summer grain, with high yields and good quality in both roles (Redmon et al., 1995; Arzadun et al., 2006; Mackown et al., 2011; Kim and Anderson, 2015). However, few wheat varieties in China are cultivated for forage production or grazing, which corresponds to the substantial lack of fresh pastures during winter in northern China (Tian et al., 2012). Dual-purpose cultivation of winter wheat has the potential to improve land use efficiency and provide green vegetation protection, compensating for the inability of warm-season forage crops to grow in this region because of the freezing weather conditions during winter (Kim and Anderson, 2015). Compared with other small grain species, winter wheat is the best suited for use as a multipurpose crop because it provides better and more consistent production of pasture, silage, hay, and grain from winter to summer (Royo et al., 1993; Juskiw et al., 2000; Moyer and Coffey, 2000). Therefore, we propose that breeding multipurpose wheat varieties can offer significant economic advantages and fill the need for green winter forage for livestock in northern China.

The ability of wheat seedlings to undergo compensatory regeneration after mowing or grazing is a key factor determining suitability for multipurpose use, as it affects whether the plant can accumulate enough carbohydrates at low winter temperatures to ensure tillering and elongation during the following spring. Studies have shown that excessive grazing and mowing of wheat seedlings for forage can limit grain yield during the following grain harvest season; therefore, it is important to select reliable cultivars and feasible mowing or grazing methods (Arzadun et al., 2006; Tian et al., 2012; Kim and Anderson, 2015). Although there have been many studies on the effects of cultivation conditions and soil nutrients on the multipurpose potential of forage wheat, few have focused on the molecular mechanisms of regeneration after mowing or grazing (Arzadun et al., 2006; Tian et al., 2012). This has greatly limited progress in the genetic improvement of multipurpose wheat varieties.

Carbohydrate metabolism and mobilization provide energy and carbon skeletons for plant growth and development (Hill, 1998; Müller et al., 1999). Plant photosynthesis produces large amounts of carbohydrates and stores them in leaves and stems (Hill, 1998; Fernie et al., 2020). When plants are exposed to various biotic and abiotic stresses, including insect feeding, mechanical damage, drought, and chilling, they cope with the stressful environment by degrading carbohydrates to provide energy and metabolic intermediates (Pommerrenig et al., 2018). Starch and other polysaccharides are degraded into fructose and glucose, which then undergo glycolysis, providing carbon skeletons for other pathways such as amino acid biosynthesis (Pommerrenig et al., 2018). The dynamic changes of carbohydrate are also closely related to plant morphogenesis (Fernandez et al., 2017). Ryegrass and forage wheat must be mowed at the correct height (Arzadun et al., 2006; Turner et al., 2007), as leaving a higher stubble after mowing maintains more carbohydrates, which aids in post-mowing regeneration (Turner et al., 2007). In addition, forage regeneration is a process of leaf cell division and expansion, and leaf growth is controlled by sugar signaling, possibly via the auxin-regulated gene involved in organ size (ARGOS) pathway, which promotes cell division via DNA-binding proteins (Wang and Ruan, 2013). Plant growth is accompanied by cell wall expansion that is a highly sophisticated process, in which carbohydrates are important skeletons and substrates (Verbancic et al., 2018). Furthermore, under stress conditions, plants synthesize various monosaccharides and disaccharides, such as glucose, fructose, galactose, sucrose, and trehalose, which provide a protective environment enabling plant cells to face stress (Pommerrenig et al., 2018). At the same time, the cutting for the forage wheat is also mechanical injury, which can induce changes in plant carbohydrate metabolism (Lukaszuk et al., 2017). In addition, previous study showed that long-chain non-coding RNAs (lncRNAs) might participate in the carbohydrate metabolism. Shi et al. (2020) reported that some lncRNAs is related to the expression regulation of key genes in starch and sucrose metabolisms, which participate in seedling development in rice and Arabidopsis. Although previous studies have shown that carbohydrates play an important role in post-mowing regeneration of wheat, the expression patterns of key genes and the changes in carbohydrate metabolism during this process remain unclear.

Leaf morphogenesis is an intriguing process resulting from the complex interplay of a multitude of regulatory pathways. Phytohormones, including auxins and cytokinins, play a key role in this process (George et al., 2008; Staden et al., 2008; Li et al., 2010; Su et al., 2011). Leaf development originates from the shoot apical meristem (SAM). Cells in the central zone (CZ) of the SAM divide at a relatively low rate and remain undifferentiated, while cells in the peripheral zone (PZ) divide faster and differentiate into various organs such as leaves and axillary nodes (Braybrook and Kuhlemeier, 2010). The slow but continuous division of stem cells in the CZ causes cells to become displaced away from the quiescent center. At a certain position, they lose their stem cell potential and become actively dividing cells. This transition is controlled by the interplay of a regulatory loop involving the homeodomain transcription factor WUSCHEL (WUS) in the PZ and the products of the CLAVATA genes (CLV1, CLV2, and CLV3) in the CZ (Kalve et al., 2014). Cytokinin (CK) regulates WUS expression via CLV-dependent and CLV-independent mechanisms to promote SAM growth and maintenance (Gordon et al., 2009). In addition, WUS represses the transcription of type A Arabidopsis response regulators (A-ARRs), which are negative regulators of CK signaling (Argueso et al., 2010; Kieber and Schaller, 2018). Once progenitor cells are outside the stem cell niche, auxin will decide whether they will contribute to the main axis or differentiate into lateral appendices such as leaf primordia. High concentrations of auxin are accumulated at the beginning of leaf development through regulation by AUX1 and PIN1, which inhibit the activity of KNOX1, limit CK biosynthesis, and promote the formation of leaf primordia in SAM (Su et al., 2011; Guenot et al., 2012; Bar and Ori, 2014). Subsequently, the transformation of the leaf primordium to a mature leaf is controlled by at least six distinct processes: cytoplasmic growth, cell division, endoreduplication, transition between division and expansion, cell expansion, and cell differentiation (Shweta et al., 2014). Most of these processes are tightly controlled by different signaling molecules, including phytohormones. A-type cyclin dependent kinase (CDKA) and D-type cyclin (CYCD) are central to the phase transition of the cell cycle, in which the cell activates DNA duplication (Gaamouche et al., 2010). Plant hormones such as auxin, CK, and gibberellin increase the level of CYCD, thereby activating CDKA (Inzé and De Veylder, 2006; Perrot-Rechenmann, 2010). It has been observed that the leaf cells of Arabidopsis go from mitosis to endoreduplication as the auxin concentration decreases (Ishida et al., 2010). Auxin plays an important role in the transition between cell division and expansion, as it induces the expression of the ARGOS gene (Hu et al., 2003). However, auxin does not always promote cell expansion, as its concentration has also been observed to decrease during leaf expansion (Braun et al., 2008), indicating a more complex dose–response relationship. The function of CK is likewise complicated. During cell division, CK is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis, while during the cell expansion phase, cytokinin stimulates cell expansion and differentiation (Skalák et al., 2019). During the post-mowing regeneration of winter wheat, the reconstruction of leaf morphology is dramatic, and phytohormones play a crucial role in this process. Application of exogenous cytokinins has been reported to promote post-mowing regeneration of barley during maturity (Christiansen et al., 1995). Further, lncRNAs play an important role in the growth and development of plants (Yamada, 2017; Matsui and Seki, 2019). Zubko and Meyer (2007) identified a natural antisense lncRNA that plays an important role in regulating the biosynthesis of cytokinins. Zhu et al. (2015) reported that lncRNAs participate in the regulation of auxin concentrations during the development phase of fruit ripening (2015). Therefore, studying changes in the expression of genes and lncRNAs related to phytohormone biosynthesis and signal transduction will help to elucidate the role of phytohormones in the post-mowing regeneration of forage winter wheat.

RNA plays an important role in plant growth and development, whether as mRNA or as a regulator such as microRNA, lncRNA, and so on. mRNA is a direct product of gene transcription, and plays an important role in the post-mowing regeneration of forage wheat. Study has shown that microRNA is involved in the post-mowing regeneration of wheat and plays an important role in the regulation of gene expression (Cui et al., 2020). LncRNAs have also been reported to be involved in the development of shoot meristems in Populus (Liu et al., 2019) and photomorphogenesis in Arabidopsis (Wang et al., 2014). Therefore, we infer that mRNAs and lncRNAs likely have important functions in the post-mowing regeneration of forage winter wheat. However, few studies have examined the role of lncRNAs in the post-mowing regeneration of forage wheat. Additionally, no previous studies have sequenced mRNAs and lncRNAs in an attempt to reveal the molecular mechanism of compensatory regeneration in winter wheat seedlings after mowing. To reveal the molecular responses of mRNAs and lncRNAs during post-mowing regeneration of winter wheat, wheat seedlings were mowed, and the stubble (including leaf sheath and young leaves in it) was harvested for transcriptional and physiological assays within 72 h after mowing. The functions of differentially expressed mRNAs and lncRNAs were annotated using orthologous alignment, and their interactions were predicted. This study showed the responses of mRNAs and lncRNAs during post-mowing regeneration of winter wheat, and suggested that carbohydrate and hormones are involved in the post-mowing regeneration of wheat.

Dual-purpose use of winter wheat for grain and forage production improves land use efficiency and plays an important role in providing winter forage worldwide (Arzadun et al., 2006; Tian et al., 2012; Giunta et al., 2015; Kim and Anderson, 2015). Compensatory regeneration of wheat seedlings after mowing or grazing is a key factor for dual-purpose use, and excessive grazing or mowing of forage wheat seedlings will limit grain yield in the following harvest season (Arzadun et al., 2006). Studying the molecular mechanism of wheat regeneration post-mowing will aid the development of dual-purpose wheat varieties with a high post-mowing regeneration capacity. Therefore, this study identified mRNAs and lncRNAs related to post-mowing regeneration of wheat seedlings. The results showed that some key mRNAs and lncRNAs participated in carbohydrate degradation and plant hormone signal transduction, which might play an important role in post-mowing regeneration of wheat seedlings.

Post-mowing regeneration of wheat is a process of morphogenesis of young leaves, and long-chain coding and non-coding RNAs play an important role in plant development and morphogenesis (Yamada, 2017). In this study, a total of 5882 mRNAs and 672 lncRNAs were identified as differentially expressed in comparison groups (Supplementary Tables 2, 3), suggesting their possible role in the post-mowing regeneration of winter wheat. Furthermore, the number of DE mRNAs and lncRNAs at different regeneration stages differed (Figures 1, 3). In all comparison scenarios, the number of DE mRNAs in the T1/T0 group was the lowest, which was consistent with the lack of visible regeneration of post-mowing wheat seedlings, whereas the number of DE lncRNAs in the T1/T0 group ranked third (Figures 1, 3). Such lncRNAs influence the transcription of downstream genes by interacting with promoters, cis-acting elements, or other regulatory activities (Zhang et al., 2017). This result suggested a potential regulatory effect of lncRNAs on the expression of mRNAs in early post-mowing regeneration of wheat seedlings. As mowed wheat seedlings regenerated, the number of DE mRNAs and DE lncRNAs both increased, which was consistent with the visible growth of mowed wheat leaves (Figures 1, 3), indicating that mRNAs and lncRNAs are involved in the late post-mowing regeneration of wheat seedlings.

Carbohydrates are the metabolic source of the materials and energy needed for plant growth; thus, they play an important role in plant morphogenesis (Hill, 1998; Turner et al., 2007). The post-mowing regeneration of wheat seedlings involves the morphogenesis of young leaves and requires large amounts of energy and materials to maintain the division and expansion of leaf cells. Cutting height is one of the most important factors affecting the post-mowing regeneration of wheat seedlings and subsequent grain production (Arzadun et al., 2006). According to previous studies, differences in cutting height result in differences in the total sugar content in the stubble, which ultimately affect post-mowing regeneration and grain production (Arzadun et al., 2006; Turner et al., 2007); similar results were observed in this study. During the post-mowing regeneration of wheat seedlings, the carbohydrate content are changed, as well as the expression of genes related to carbohydrate metabolism (Figures 2, 5–7). BAM and BGL have been reported as key enzymes for the degradation of starch and cellulose (Hill, 1998). The increased expression of their genes suggested that starch and cellulose were degraded to glucose within 72 h after mowing (Figures 2, 5, 6). The expression of 22 genes encoding INV, a key enzyme for sucrose degradation (Ruan, 2014), underwent tremendous changes (Figure 5), suggesting carbohydrate mobilization. SUS, TPP, and TPS are key enzymes involved in trehalose biosynthesis, and trehalose 6-phosphate is also a signal and homeostatic regulator of sucrose levels in plants (Müller et al., 1999; Ruan, 2014; Fichtner and Lunn, 2021). Their significantly increased gene expression suggested the degradation of sucrose and the synthesis of trehalose (Figures 5, 6). Additionally, the expression of genes encoding key enzymes related to the degradation of stachyose, raffinose, galactose, and melibiose, including GALA, GOLS, and RFS (Stick, 2001), was significantly increased during the post-mowing regeneration of wheat seedlings (Figure 6). Changes in the gene expression and carbohydrate concentration in these pathways suggested that carbohydrates in the wheat stubble had been mobilized. The carbohydrates in the stubble were degraded into glucose and fructose, which were used for the regeneration of young leaves by the mowed wheat seedlings. This was consistent with a physiological experiment (Figure 2) and a previous report (Arzadun et al., 2006; Turner et al., 2007). One most important physiological function of glycolysis is to provide energy and carbon skeletons for plant growth and development (Sung et al., 1988; Plaxton, 1996). HXK, PFK, ALDO, PDC, PGK, PPDK, PEPC, and MAE are important enzymes in glycolysis and the Krebs cycle (Plaxton, 1996), and their gene expression in stubble was increased during post-mowing regeneration. The increased expression of these genes may result in enhanced glycolytic activity, ultimately providing more ATP and carbon skeletons for the Krebs cycle and the biosynthesis of amino acids during the post-mowing regeneration of wheat seedlings (Figure 7). All these changes results in carbohydrate mobilization and may be helpful for the post-mowing regeneration of wheat. The result is consistent with a previous study (Turner et al., 2007).

The Calvin cycle is an important component of photosynthesis, and enhanced Calvin cycle activity can fix more CO₂ to produce more carbohydrates during post-mowing regeneration of wheat seedlings. In this study, significantly increased expression of genes involved in the Calvin cycle might enhance the fixation of CO₂ (Figure 8); such genes included RuBP1–18, GAPA1–4, and ALDO1–4, which encode key enzymes for CO₂ fixation (Lu et al., 2012). The increased gene expression of these enzymes in this study suggested possible increased fixation of CO₂ and the provision of more carbon for the regeneration of mowed wheat seedlings (Figure 8). This possible increased carbon storage appeared to be the material basis for carbohydrate synthesis and the regeneration of mowed young leaves in winter wheat. It may be another favorable factor for the post-mowing regeneration of seedlings.

Plant hormones are key factors regulating plant morphogenesis (Du et al., 2018; Ali et al., 2020) and may play an important role during post-mowing regeneration of wheat seedlings. The interaction of endogenous auxins and cytokinins affects leaf morphogenesis and involves the complex interplay of a multitude of regulatory pathways (Gonzalez et al., 2012; Bar and Ori, 2014; Skalák et al., 2019). Before mowing, high concentrations of auxins accumulate in the base of the wheat cauloid, which inhibits the activity of KNOX1 and limits CK biosynthesis (Su et al., 2011; Guenot et al., 2012). This is consistent with our observations of auxin and CK content (Figure 2). Wu et al. (2017) reported that shade significantly increased auxin content and resulted in a smaller leaf. This partially confirms that high concentrations of auxin inhibit growth, thus inhibiting the post-mowing regeneration of wheat. After mowing, the CK content increased significantly, and the ratio of CK to auxin also increased (Figure 2). Increased concentrations of CK promote the growth and maintenance of the SAM (Gordon et al., 2009), aiding the regeneration of mowed wheat seedlings; this is similar to the induction of explant buds by cytokinins. Regeneration is a violent process of cell division, and CDKA and CYCD are central to the phase transition of the cell cycle (Gaamouche et al., 2010). Increased CK content can increase the level of CYCD, thereby activating CDKA and accelerating the cell cycle (Inzé and De Veylder, 2006; Perrot-Rechenmann, 2010). In addition, CK controls the transition from cell division to cell expansion and stimulates cell expansion and differentiation during the cell expansion phase (Skalák et al., 2019). This is consistent with our observation of an increased number of regenerated wheat leaves (Supplementary Video 1). The increased CK concentration and high CK/auxin ratio may lead to the fast regeneration of mowed wheat leaves. Therefore, auxins, cytokinins, and their interplay potentially play an important role in the morphogenesis of leaves in mowed wheat seedlings. In addition, the crosstalk between hormone and sugar signaling may participate in post-mowing regeneration of wheat. Mounting evidence suggests interplay between hexose and auxin signaling in mediating cell proliferation, cell expansion, and seed development (Wang and Ruan, 2013). The degradation of starch produces abundant hexoses, such as glucose, which inhibits auxin signaling and facilitate cell expansion (Mishra et al., 2009). In this study, sugar and auxin content were changed, which is similar to previous studies and may be a factor of the regeneration of mowed wheat. In addition, high concentrations of CK can stimulate sugar and energy metabolism and increase the expression of FPB, GPI, MDH, and BGL, result in increased soluble sugar, increase turgor pressure and cell wall expansion as shown in previous studies (Cosgrove, 2000; Skalák et al., 2019). This may be another factor to promote the post-mowing regeneration of wheat seedlings.

In plants, lncRNAs have been reported to regulate gene expression at multiple levels by a number of complex mechanisms, including transcriptional regulation, post-transcriptional regulation, and epigenetic modifications (Chekanova, 2015; Liu et al., 2015). This regulation of gene expression can occur either cis or trans to the lncRNA by sequence complementarity or homology with RNAs or DNA (Kung et al., 2013; Yang et al., 2014). In this study, four lncRNAs (TCONS_00173435, TCONS_00131199, TCONS_00144320, and TCONS_00252591) were predicted to cis-regulate the expression of MDH3, PP2C3, CRE, and INV5, respectively (Figure 10B). However, there was less correlation between the expression trends for these lncRNAs and mRNAs. It was difficult to judge whether lncRNAs interacted with these mRNAs and other regulatory factors that might influence the expression of mRNAs. Additionally, lncRNAs can regulate gene expression of mRNA by collinearity in a variety of mechanisms (Chekanova, 2015; Rai et al., 2018). In this study, most DE lncRNAs interacted with mRNAs in this manner. Nine lncRNAs were positively co-expressed with their target mRNAs (Figure 10A) and might regulate the expression of target mRNAs by enhancing expression of the transcription complex (Zhang et al., 2017). Most of the lncRNAs were negatively co-expressed with their target mRNAs (Figure 10A) and might regulate their target mRNAs by acting as miRNA target mimics as shown by previous study (Rai et al., 2018). In all cases, DE target genes included AUX/IAA1, PP2Cs, B-ARR2, A-ARR1, CRE, MDH3, GAPA1, RuBPs, PDH, LPD, PPCK, GLGP1, GLGB1, GLGS1, GALA, and INVs. Furthermore, expression levels of INV3, INV5, INV9, INV14, GOLS1, GLGP1, and AUX/IAA1 were higher than those of their differentially expressed homologous genes, indicating that these lncRNAs might affect their expression (Supplementary Figure 4). These results indicate that lncRNAs may be involved in the regulation of regeneration in mowed wheat seedlings. Although we discovered that sugars and phytohormones were involved in post-mowing regeneration of wheat, there are some limitations to the conclusions that can be drawn from these results. Further experiments are needed to determine how phytohormones regulate post-mowing regeneration of seedlings and to verify the interaction between lncRNAs and targeted genes.

In addition, the molecular response of post-mowing regeneration must be distinguished from stress responses induced by mechanical damage. In the experiment, parts above 3 cm of wheat seedlings were mowed, but only 2 cm above the ground was sampled. The cut wound was not sampled and this reduces the influence of the injury response on the experimental results to a certain extent. But the mowing during sampling and wound signal conduction will still affect the experimental results. Even it may be that the mowing induced the plant’s regeneration signal. Because mere mechanical damage can also induce changes in carbohydrate and hormone content (Chandra et al., 2012; Lukaszuk et al., 2017). In Furthermore, mechanical damage changes the activities of sucrose hydrolysis-related enzymes (Lukaszuk et al., 2017). However, mechanical damage induced hormonal response such as ethylene, jasmonic acid, and abscisic acid (Cheong et al., 2002; Savatin et al., 2014), while regeneration was induced by cytokinin and auxin in this study. Therefore, it can be seen that there is a difference between regeneration and injury response after mowing.

Post-mowing regeneration of seedlings is a key factor in the suitability of winter wheat for dual-purpose use. This study examined the molecular responses of long-chain coding and non-coding RNAs to illuminate the mechanism of regeneration. Function enrichment analysis demonstrated that DE mRNAs were involved in carbohydrate mobilization, photosynthesis, and phytohormone signal transduction. Physiological experiments combined with gene expression verification indicated that carbohydrate mobilization and enhanced photosynthesis provide energy and carbon skeletons for cell proliferation and expansion during post-mowing regeneration of wheat. Combining the changes in auxin and cytokinin content, and gene expression, we draw the following conjecture. The decreased auxin content may relieve the inhibition of cytokinin synthesis, enhance cytokinin biosynthesis, control the transition from cell division to cell expansion, and may stimulate cell expansion and differentiation during the cell expansion phase, and eventually accelerate post-mowing regeneration of seedlings. Furthermore, 39 DE lncRNAs might affect the expression of key genes involved in carbohydrate metabolism and phytohormone signal transduction. The main finding of this study is that carbohydrates and phytohormones are involved in post-mowing regeneration of wheat seedlings, and lncRNAs may play a role in this process. This can serve to guide the breeding of wheat varieties with higher stubble sugar content and the development of external reagents to promote forage regeneration. Our next study will focus on (i) the interaction between phytohormones and carbohydrate metabolism, and (ii) the regulatory effects of lncRNA on the expression of related genes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA638505 (SRR11973944, SRR11973945, SRR11973946, SRR11973947, SRR11973948, SRR11973949, SRR11973950, SRR11973951, SRR11973952, SRR11973953, SRR11973954, and SRR11973955).

YX and CZ designed and directed this study and drafted the manuscript. GC performed the experiments, analyzed the data, and revised the manuscript. MZ determined the expression of mRNAs by qRT-PCR. HT investigated the regeneration of wheat and completed carbohydrate determination. ZW and MM determined the levels of endogenous auxins and cytokinins. SL improved the data analysis and revised the manuscript. All authors contributed to the article and approved the submitted version.

HT was employed by Shaanxi Province Seed Industry Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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