Transcription factors play a critical role in plant growth and development and can regulate development by transmitting external environmental factors, mediating hormone signal pathways, and responding to gene regulatory networks in plants (Chen et al., 2021). TCP genes, a kind of plant-specific transcription factors, are typically involved in the plant developmental process, such as seed germination (Zhang et al., 2019a), floral organ development (Wang et al., 2015), leaf morphogenesis (Sarvepalli and Nath, 2011), axillary meristem development (Aguilar-Martínez et al., 2007; Nicolas et al., 2015; Min et al., 2021), and hormone signal transduction (Kosugi and Ohashi, 2002; González-Grandío et al., 2017). The abbreviation “TCP” comes from its earliest discovered members: Teosinte Branched1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), and the PROLIFERATING CELL FACTORS (PCF) from rice (Oryza sativa) (Luo et al., 1996; Doebley et al., 1997; Kosugi and Ohashi, 1997). They all have a conserved TCP domain, a basic helix–loop–helix (bHLH) structure, which is primarily related to DNA binding, protein interaction, and protein nuclear localization (Cubas et al., 1999). It can also be divided into Class I (also known as PCF) and Class II according to the characteristics of the conserved domain of TCP proteins. Additionally, Class II can be divided into CIN subfamily and CYC/TB1 subfamily (Martín-Trillo and Cubas, 2010). The most obvious difference between Class I and Class II is the absence of four conservative amino acids in the Class I basic TCP domain. The R domain is rich in polar amino acids such as lysine, glutamic acid, and arginine and is only found in Class II members, which is used to mediate protein interaction (Cubas et al., 1999).

So far, the majority of TCP family members have been determined to play a role in plant growth and development. Previous studies have shown that the TCP members of Class I primarily promotes leaf cell proliferation, thereby regulating plant growth and development, and plays an important role in the response to stress (Aguilar Martinez and Sinha, 2013). In Arabidopsis thaliana, TCP14 and TCP15 are involved in seed germination by acting downstream of the gibberellin and vernalization pathways (Resentini et al., 2015; Xu et al., 2020). Through binding to the homologous GCCCR elements, AtTCP20 regulates the expression of cyclin and ribosomal protein genes. Furthermore, it acts as a flexible regulator to coordinate growth and division pathways in post-embryonic plant development (Li et al., 2005). Additionally, the downregulation of the expression of OsPCF6 and OsTCP21 enhances the tolerance of rice under cold stress by changing the scavenging of reactive oxygen species (Wang et al., 2014). TCP21 is particularly important during plant growth. Its low expression makes rice more susceptible to rice ragged stunt virus (RRSV) (Zhang et al., 2016; Wang et al., 2021). Overexpression lines of TCP21 exhibited increased tiller bud length, biomass, and tiller number in rice (Wang et al., 2021). PeTCP10 is induced by drought and ABA treatment and plays a vital role in plant growth and development and response to environmental stress, which can be seen due to its effects in the evident effects on drought tolerance and the lateral root growth of transgenic lines (Liu et al., 2020). However, the TCP members of Class II primarily plays an important role in morphological construction and organ development (Manassero et al., 2013; Sarvepalli and Nath, 2018). Overexpression of AtTCP1, the homolog of CYC, directly stimulates the expression of the target gene DWF4 to actively regulate brassinosteroid (BR) biosynthesis, which affects leaf development and leaf shape regulation (Guo et al., 2010; An et al., 2011). It was found that OsTCP17 (REP1), a CYC homologous gene in rice, regulates the attributes and development of palea and controls the flower symmetry of the inner and outer lemma axis (Yuan et al., 2009). In wild rice, the OsTCP15 (TIG1) gene is specifically highly expressed on the distal side of the tiller base, which promotes cell elongation by activating the expression of downstream genes such as EXPA3, EXPB5, and SAUR39, which helps to maintain a large tillering angle (Zhang et al., 2019b).

Branching affects plant morphogenesis and growth to a great extent and determines plant architecture, yield, and ecological sustainability (Richards, 2000; Wang and Li, 2008; Wang et al., 2019). TB1, an inhibitory regulator of the development of axillary buds, realizes the transformation from lateral branch growth to apical dominance, transforming teosinte with more tiller numbers into commonly cultivated maize (Doebley et al., 1997; Wang et al., 2019). It has been found in many species due to its conservative function of negatively regulating axillary bud growth (Takeda et al., 2003; Kebrom et al., 2006; Dixon et al., 2018; Shen et al., 2019). TCP18 (BRANCHED1, BRC1) and TCP12 (BRANCHEND2, BRC2) in Arabidopsis are the two orthologous homologs of ZmTB1 in maize, which are highly expressed in axillary buds and negatively regulate the growth of axillary buds, whereas SML 6, 7, and 8 promote branching through transcriptional inhibition of BRC1 and the non-transcriptional regulation of auxin (Aguilar-Martínez et al., 2007; Wang et al., 2020). Strigolactones (SLs) are a new plant hormone that inhibits the germination of lateral branches and suppresses the growth of tiller buds in rice by regulating the transcriptional level of OsTB1 (Takeda et al., 2003; Nicolas and Cubas, 2016; Wang et al., 2018). Meanwhile, OsMADS57 and OsTB1 jointly regulate the transcription of its target genes OsWRKY94 and D14, realizing the transition between organogenesis and cold adaptation defense in rice at different temperatures (Chen et al., 2018). As wild cucumbers were domesticated, it was found that two light response elements inserted in the promoter region promoted the expression of CsBRC1, which directly restrained the auxin outflow from the lateral buds mediated by PIN3 (encoding auxin transporter) and reduced the production of branches due to the accumulation of auxin in axillary buds (Shen et al., 2019). To date, homologous genes of TB1 were subsequently identified in numerous gramineous plants such as rice (Takeda et al., 2003), wheat (Dixon et al., 2018), and sorghum (Kebrom et al., 2006). They are very conservative in function, which can cause bud dormancy, and effectively regulate the growth of axillary buds in response to hormones and the external environment which controls the tiller number (Wang et al., 2019).

Bamboo is one of the fast-growing non-timber forest resources and has significant economic, cultural, and ecological value (Zhao et al., 2017). Ma bamboo (Dendrocalamus latiflorus Munro) is the most widely cultivated clumping bamboo in Southern China. Its bamboo shoots taste good and are high in nutritional value. Mature bamboo can be used as building materials, decorations, and ornamental planting. Therefore, it is a fast-growing and environmentally friendly clump bamboo species that can be cultivated for its shoots and timber. Bamboo shoots have a longer period of emergence because they are often exposed to the soil surface, which is easily frozen in winter. When cultivating bamboo forests, seasonal shooting and the low germination rate of shoot buds directly affect the yield of bamboo shoots and timber. The formation, growth, and development of bamboo shoots involved in the morphogenesis of lateral branches are unique and affect the yield of bamboo shoots and timber. Therefore, it is important to explore the role of genes related to lateral branch formation in the outgrowth of bamboo shoots, which will help to clarify the molecular mechanism of bamboo shoot development.

Whereas, the TCP family has been characterized in many species, such as Arabidopsis (Yao et al., 2007), rice (Yao et al., 2007), sorghum (Francis et al., 2016), and Moso bamboo (Liu et al., 2018), less is known about the TCP transcription factors in Ma bamboo. Recently released genomic data about Ma bamboo, including a representative of hexaploid clumping bamboo (AABBCC, 2n = 6x = 72), allowed us to perform a genome-wide analysis of the TCP transcription factors in Ma bamboo (Zheng et al., 2022). In this study, 66 TCP transcription factor members of Ma bamboo were identified, and the phylogenetic relationship, gene structure and motif information, collinearity, tissue differential expression analysis, subcellular localization analysis, and transcriptional self-activation analysis were analyzed. Additionally, we performed transcriptome analysis of bamboo shoot buds at different developmental stages to clarify the function of the TCP family during bamboo shoot outgrowth. Through the phenotypic observation of transgenic Arabidopsis, we concluded that DlTCP12-C plays a significant role in controlling the number of branches. Our study reveals the basic information and evolutionary relationship of plant-specific TCP transcription factors in Ma bamboo, clarifies the role of candidate genes in bamboo shoot growth and development by transcriptome analysis, and preliminarily outlines on the function of candidate TCPs, all of which provides valuable insights into future investigations.

As we all know, members of gene families tend to expand through several evolutionary mechanisms, including tandem duplication, large-scale chromosome segmental duplication, and translocation, This leads to the evolution of complex phenotypes (Cannon et al., 2004; McCarthy et al., 2015). In Ma bamboo, numerous genes have undergone triple genome duplication due to whole-genome duplication events (Zheng et al., 2022). Approximately 280 gene families have undergone expansion during their evolution from paleotropical woody bamboos to allohexaploid Ma bamboo, which could be the reason for some of bamboos' unique traits, including their rapid vegetative growth and high biomass (Zheng et al., 2022). In this study, a total of 66 plant-specific TCP transcription factors were identified in Ma bamboo, and the number of genes was about three times that of rice, Arabidopsis, and Moso bamboo (Table 1). In other words, all DlTCPs possessed 3–5 homologous genes, indicating that they are preferentially retained during polyploidization. Similarly, there were three closely related homologous genes in the TCP family of hexaploid wheat. However, their cis-acting elements were quite different, which could lead to the sub-functionalization of wheat homologous genes (Zhao et al., 2018). Collinear analysis demonstrated that the main reason for the expansion of the TCP gene family is chromosome polyploidization (whole-genome duplication) and large-scale chromosome segment duplication, which primarily occurs between three subgenomes A, B, and C of D. latiflorus, and only a few chromosomes do not possess TCP transcription factors (Figure 3). The TCP family has expanded due to large-scale segment duplication events that also occurred in upland cotton, whereas 74 GhTCP genes were identified in the allotetraploid plant upland cotton genome (AADD) (Li et al., 2017). Tandem duplication events, which are important events driving the occurrence of new biological functions, have only been found one time between DlTCP5-B and DlTCP6-B. This suggests that TCP transcription factors have conservative and irreplaceable functions in Ma bamboo (Shang et al., 2012). The TCP family could have a continually increasing role in the development of the typical hexaploid bamboo species D. latiflorus. While they may have functional redundancy, the sub-functionalization of these homologous genes and gene dosage could make Ma bamboo more adaptable during growth and development (He et al., 2022).

Plants have several complex regulatory mechanisms and signal networks, which can quickly perceive the external environment and regulate gene expression to adapt to unpredictable environmental changes and resist several biotic and abiotic stresses in the long-term evolutionary process. Transcription factors can regulate the occurrence of biological processes such as plant morphology, developmental patterns, and stress responses to varying degrees. As the plant-specific transcription factors, the TCP family plays a vital role in plant growth and development. A group of functionally redundant phylogenetic-related class I TCP genes (AtTCP7, AtTCP8, AtTCP22, and AtTCP23) had similar expression patterns in young leaves, regulating leaf development by controlling cell proliferation (Aguilar Martinez and Sinha, 2013). In Arabidopsis, AtTCP14 directly activates the growth potential of the embryo during seed germination, whose expression level is highest before seed germination (Tatematsu et al., 2008). In addition, the AtTCP14 mutant was highly sensitive to abscisic acid and gibberellin synthase inhibitors, indicating that AtTCP14 regulates seed germination by regulating hormone response (Tatematsu et al., 2008; Manassero et al., 2013). The senescence phenotype of TCP19 and TCP20 double mutants was significantly enhanced in Arabidopsis, and classic genetic and molecular methods have been used to demonstrate that TCP19 and TCP20 are involved in controlling leaf senescence in Arabidopsis, despite functional redundancy (Danisman et al., 2013). OsPCF7 is primarily expressed at the tillering stage and plays an important role in the tillering and heading process of rice seedlings. It significantly affects the panicle numbers, the number of filled grains per plant, and the grain yield per plant (Li et al., 2020). CsTCP3 is induced by gibberellin, photoperiod, and temperature and directly participates in the development of axillary buds by controlling the content of auxin in axillary buds, which affects the number of lateral branches in cucumber (Wen et al., 2020). It can integrate upstream environmental factors and hormone signals and further affect the development of axillary meristem, to adapt the plant architecture to environmental conditions. Biological processes from shoot bud germination to growth is crucial for the growth and development of bamboo (Shou et al., 2020), all of which directly affect the yield of bamboo shoots and timber in Ma bamboo. Therefore, we collected the apical buds of bamboo shoots from four representative developmental stages for transcriptome analysis to find out the relevant gene sets involved in this process (Figure 7A). A total of 29 TCP transcription factors were subsequently found as differentially expressed genes from RNA-seq data. Interestingly, TCP transcription factors exhibited spatiotemporal expression specificity during the development of bamboo shoots (Figure 8A). From the dormancy stage (S1) to the rapid high growth stage (S4), the number of downregulated genes gradually decreased; in contrast, the number of upregulated genes gradually increased (Figure 8B). These results suggested that there is an alternation and switching mechanism in the function of TCP transcription factors to better adapt to the growth and development of bamboo shoots in Ma bamboo. TB1 is the key node gene for lateral bud outgrowth, which plays a conservative role in many species (Takeda et al., 2003; Dixon et al., 2018; Li et al., 2021). Whereafter, the preliminary functional verification results confirmed the critical role of DlTCP12-C in inhibiting axillary bud growth and lateral branch growth in overexpressed transgenic Arabidopsis (Figure 12). Future research will verify the biological function and regulation pathway of DlTCP12-C in shoot buds development using genetic transformation in Ma bamboo and assess whether there is functional redundancy among other members of the CYC/TB1 subfamily.

Transcription factors have the binding activity of specific DNA sequences or the characteristics of known DNA-binding domains, so they bind to cis-acting elements on the target site to ensure that the target gene is expressed at a specific intensity, in a specific time and place. In our study, a large number of cis-acting regulatory elements related to plant hormone signals, organ development, stress response, MYB transcription factors, and WRKY transcription factor-binding sites accumulated in the promoter of DlTCPs, indicating that TCP transcription factors could act as a central regulatory integrin regulated by environmental factors, hormone signals, and upstream transcription factors to affect plant growth and development. GhTCP14a/22 is involved in controlling cotton fiber growth through the gibberellin, brassinosteroids, and auxin signal transduction pathways, which play a remarkable role in the development of cotton fiber and are primarily expressed during fiber initiation and elongation (Li et al., 2017). DWARF27 (D27) is a key gene involved in strigolactone synthesis, which can sense strigolactone signaling and activate downstream TB1-like TCP transcription factors by recruiting SCF complexes to stimulate the ubiquitination and degradation of DWARF53 (D53) repressor proteins (Kerr and Beveridge, 2017). Cytokinins and sugars also inhibit the expression of TB1 (Mason et al., 2014; Patil et al., 2021). Auxin upregulates the expression of MAX3 and MAX4 through the AXR1-AFB-mediated signaling pathway, but downregulates the members of the IPT family, promoting strigolactone biosynthesis, and the inhibition of cytokinin biosynthesis, which further promotes the expression of TB1 (Nordström et al., 2004; Tanaka et al., 2006). A number of two closely related TCP transcription factors TCP14 and TCP15 affect the development of foliage and trichomes, participate in cytokinin-regulated signal pathways, and stimulate the expression of cytokinin-regulated gene RESPONSE REGULATOR 5 through interaction with SPINDLY (SPY) (Steiner et al., 2012). Jasmonic acid is a kind of plant hormone of lipids (oxylipins), which is involved in plant development, abiotic stress response, and the interaction between plants and microorganisms. AtTCP4 reportedly directly targets LIPOXYGENASE2 (LOX2), encoding a chloroplast enzyme gene involved in α-linolenic acid biosynthesis and jasmonic acid synthesis, and is involved in the regulation of jasmonic acid biosynthesis and leaf development (Vick and Zimmerman, 1983; Danisman et al., 2012). The LsAP2 transcription factor further regulates leaf morphology in lettuce by inhibiting the activity of the CIN-like TCP transcription factor (Luo et al., 2021). The TCP-mediated complicated hormone signal regulatory network further emphasizes the important role of TCP in affecting plant growth patterns and biological processes, and more in-depth research is needed to clarify the pathway of TCP transcription factors. Our transcriptome and promoter element analyses indicate that TCP transcription factors regulate the expression of target genes through transcriptional regulation and hormone signals, which affect related biological processes in plants. Recent studies have made clarified the mechanism of the direct targeting regulation of TCP transcription factors by miRNA. Some TCP members directly targeted by miR319 are widely involved in plant hormone signal transduction, leaf development, vascular formation, and response to abiotic stress (Fang et al., 2021). Comprehensive analysis of catechin metabolism profiles and TCP gene expression profiles in different plant tissues at different developmental stages indicated that the CsmiR319b/CsTCP3-4 module was not only related to shoot tip development, but also played a potential role in catechin biosynthesis in tea plants (Yu et al., 2021). Genetic and molecular analyses indicated that PtoTCP20, the direct target gene of miR319a, regulated the proliferation of vascular cambium along with PtoWOX4a and promoted the differentiation of secondary xylem by activating the transcription of PtoWND6, thereby regulating the secondary growth of Populus tomentosa stem (Hou et al., 2020). A total of five TCP genes were found to contain miR319 directly targeted binding sites in 3′ UTR (Supplementary Table S6). These miR319-targeted DlTCPs were the members of the CIN subfamily. The regulation mechanism of DlTCPs related to the bud growth of bamboo shoots requires further study.

In short, this study identified 66 plant-specific TCP transcription factors in the D. latiflorus genome using bioinformatics and analyzed their evolutionary relationship, duplication events, promoter cis-elements, tissue expression patterns, subcellular localization, and self-activating transcriptional activity. Transcriptome analysis of different developmental stages of bamboo shoot buds was used to preliminarily study the function of TCP transcription factors in Ma bamboo, providing a series of differentially expressed genes that could be involved in the growth and development of bamboo shoots. Subsequently, the conservative function of DlTCP12-C, which negatively regulates axillary bud development and lateral branch growth, was confirmed in overexpressed transgenic Arabidopsis. This comprehensive study of TCP transcription factors in Ma bamboo provides several candidate genes worthy of further analysis, including the regulatory mechanism of bamboo shoot bud growth and development.

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: National Center for Biotechnology Information (NCBI) BioProject database under Accession Number PRJNA792061.

GQ and RZ conceived this project. KJ and YW designed experiments, interpreted the results, and wrote the manuscript. JX, ZL, HF, and BH performed the experiments and analyzed the data. YW provided technical guidance for the experiment. All authors read and approved the submission of this manuscript.

This research was funded by the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding, Grant Number 2021C02070-4.

The 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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