**3. Discussion**

A number of TCP proteins had been recently identified in various plants due to completion of their whole-genome sequence, including *Arabidopsis*, rice, tomato (*Solanum lycopersicum*), and watermelon (*Citrullus lanatus*), *Orchis italica*, and *Populus euphratica* [27–31]. The allotetraploid, *G. hirsutum*, is not only the world's most important fiber crop, but is also a model polyploid crop. Despite being among the largest and most diverse gene families, the TCP gene family has not been systematically identified in the *G. hirsutum* genome. In this study, we identified 73 *TCP* genes in the sequenced genome of *G. hirsutum*. We analyzed their phylogenetic relationship, genomic distribution, conserved protein motif, and exon–intron organization. Over 80% of *GhTCP* genes were intronless, which was quite similar to the structure of *G. raimondii* and *G. arboretum TCP* genes [32,33]. Generally speaking, most *GhTCPs* within the same subclade showed similar gene structure in terms of numbers and lengths of introns and exons. Furthermore, similar to the exon–intron organization, members of the same subclade also showed similar motif composition, indicating their functional similarities. Additionally, some motifs were only present at specific subclades, such as the R domain, suggesting that they can have subclade-specific functions.

The *GhTCP* genes possessed an expanded family, with approximately three-fold size compared with *Arabidopsis*, tomato, and rice, and approximately two-fold compared with *G. arboreum* and *G. raimondii*. This suggests that although plant *TCP* genes may derive from a common ancestor, many had undergone distinct patterns of differentiation with the divergence of different lineages. Based mainly on amino acid sequence differences, especially in the basic region of the TCP domain, the TCP transcription factors are divided into three groups. There were 50 *GhTCP* genes in the PCF group, 16 in the CIN group, and seven in the CYC/TB1 group. The numbers of genes in each group were approximately twice those in *G. arboretum* and *G. raimondii*. According to a recent study, all tetraploid cotton species (AtDt) evolved from A-genome diploid, *G. arboretum,* and D-genome diploid, *G. raimondii*, at around 1–2 Mya [34]. In addition, previous studies indicated that gene duplication contributed to increasing the number of gene family members on various scales, including whole-genome duplication [35]. The expansion of regulatory genes is rarely achieved simply through single gene duplication alone, implying that genome duplication contributed to the amplification of the TCP gene family in *G. hirsutum*.

TCP transcription factors was involved in the regulation of cell growth and proliferation, which performed diverse functions in multiple aspects of plant growth and development [3]. We determined the spatial and temporal expression profiles of *G. hirsutum* TCP genes in 10 tissues, which included different developmental stages of ovule and fiber, using transcriptome analysis. The expression in different tissues varied widely among *GhTCP* genes and different organs for individual TCP genes. This implies functional divergence of *GhTCP* genes during different plant developmental processes. *GhTCP15* and *GhTCP71* were relatively highly expressed in ovules and fibers at 10 DPA. Previous study demonstrated that *GbTCP* was preferentially expressed in elongating *G. barbadense* fiber during5 to 15 DPA [36]. Overexpression of *GbTCP* enhanced root hair initiation and elongation in *Arabidopsis* and regulated branching. Both *GbTCP* in *G. barbadense* and *GhTCP71* are orthologs of *AT1G69690* in *Arabidopsis* (named *AtTCP15*), compared with which they had only one amino acid difference within the TCP domain [37]. *AtTCP15* was expressed in trichomes and rapidly dividing tissues and vascular tissue, and the protein promoted mitotic cell division, but inhibited endo-reduplication by modulating the expression of several key cell-cycle genes [15,38]. In our study, *GhTCP14* was also expressed predominantly in fiber cells, especially at the initiation and elongation stages of development as previously reported. Induced expression of *GhTCP14* can increases the density and length of root hairs and trichomes and affects gravitropism of *Arabidopsis* [39]. These results suggested that cotton fiber and *Arabidopsis* root hair elongation may have a similar regulatory mechanism for *TCP* genes.

Many *Arabidopsis* TCP genes with similar functions tended to cluster in the same clade, implying that TCP genes within the same clade may have similar functions in *G. hirsutum*. In *Arabidopsis*, some angiosperm members of the CIN-like clade involved in leaf and flower

morphogenesis are targeted by miR319–for example, *AtTCP2*, *3*, *4*, *10*, and *24* [40]. Loss of function of these genes results in enlarged leaves, due to an excess of cells that are smaller in size, while their gain of function leads to smaller leaves [41,42]. The miR319, previously known as "miR-JAW", was first described in *Arabidopsis* because its involvement in the control of leaf morphogenesis [43]. Several studies had reported the involvement of miR319 in plants in response to stress conditions via downregulation of its target genes [44]. Transgenic creeping bentgrass overexpressing a rice miR319, *Osa-miR319a*, exhibited enhanced salt and drought tolerance [45]. In this study, we observed upregulation of miR319 and downregulation of the targets in both salt and drought treatments. To understand the responses of *GhTCP* genes to stresses, the expression profiles were investigated in response to abiotic stresses, such as heat, salinity, and drought. In total, 40 *GhTCP* genes exhibited variations in expression. It is noteworthy that some genes showed instantaneous upregulation, and decreased slowly during continued stress. For example, *GhTCP6*, *14*, *35*, and *51* exhibited their highest expression at 3 h of dehydration and salinity treatment. However, no significantly upregulated expression was found at late time points. It is plausible to postulate that these genes might be the part of a stress-signaling system. The functions of these stress-responsive *GhTCP* genes in abiotic stress resistance will be further characterized in future work.

In this study, a total of 73 non-redundant TCP encoding genes were identified in *G. hirsutum*. Our results provided evidence for the relationship between structure and function in the *G. hirsutum* TCP gene family, and laid the foundation for further identification of the functions of the *GhTCP* gene family and their relationship with miR319.
