**3. Discussion**

Moso bamboo is a perennial plant characterized by rapid growth and a long vegetative stage that lasts for decades or even longer before flowering [25]. Dof proteins are a group of plant-specific TFs that are involved in diverse plant-specific biological processes [16]. In addition, recent research on *Dofs* is mainly in annual plants, and is limited in perennials. Therefore, researching the roles of *Dofs* in moso bamboo is necessary. In this study, a *Dof* gene, *PheDof12-1*, is identified from moso bamboo as a nucleus-localized transcription factor that contains typical zf-dof domains.

In recent decades, reports have indicated that Dof transcription factors are involved in stress response. In Arabidopsis, the expression level of *AtCDF3* is upregulated by cold, drought, high salinity, and ABA treatment [30], and overexpression of *35S::SlCDF1* and *35S::SlCDF3* increases Arabidopsis's tolerance to salt and drought stresses [18]. In wheat, *TaDof14* and *TaDof15* are significantly induced under drought treatment [35]. Previous research has suggested that drought or other environmental stresses are functional in the flowering stage of bamboo, and the transcription levels of *Dof* genes are upregulated in drought stress [36]. In addition, studying the tolerance of *PheDof12-1* will help to characterize moso bamboo cultivars such as salt, cold, and drought tolerance. In this study, *PheDof12-1* exhibited differential expression patterns under the conditions of drought, cold, salt, and ABA and GA<sup>3</sup> treatments. Through the drought, cold, salt, and GA<sup>3</sup> stresses, the expression pattern of *PheDof12-1* is basically upregulated in roots, stems, and leaves, indicating that it might participate in abiotic stress and hormone pathways, which is consistent with previous reports [36,37]. The results provide a better understanding of the stress tolerance of *PheDof12-1* in moso bamboo.

*Hd1*/*CO* and *Hd3a*/*FT* are conserved genetic pathways that regulate photoperiodic flowering between rice and Arabidopsis by their genomic comparison [38]. In Arabidopsis, *CDF1*–*CDF3* are suggested to participate in photoperiodic flowering [39]. *JcDof3* is a circadian clock regulated gene involved in the regulation of flowering time in *Jatropha curcas* [40]. In rice, *OsDof12* and *CDF1* belong to the same group [41], and overexpression of *OsDof12* resulted in early flowering by increasing the expression of *Hd3a* and *OsMADS14* under LD conditions [22]. *PheDof12-1* is the homologous gene of *OsDof12*, and Dof-Hd3a-MADS-flowering may play an important role in moso bamboo flowering [36]. Therefore, we researched the function of *PheDof12-1* in flowering time by ectopic expression in Arabidopsis for the first time, and the transgenic lines overexpressing *PheDof12-1* show earlier flowering than the wild-type plants under LD conditions. In addition, *FT*, *SOC1*, and *AGL24* are upregulated and *FLC* and *SVP* are downregulated in the transgenic lines. *FT* promotes flowering [42], which is activated by *CO* in the phloem [43]. SOC1 is a core regulator of flowering in Arabidopsis, which can interact with SVP and AGL24 proteins, but SVP and AGL24 have opposite effects on flowering time, acting as floral repressor and inducer, respectively [44]. *FLC* encodes a MADS domain-containing transcription factor that acts as an inhibitor of flowering [45]. This leads us to suspect that *PheDof12-1* promotes flowering time by regulating *FT*, *SOC1*, *AGL24*, *FLC*, and *SVP* directly or indirectly, suggesting that it might retain some function in the control of flowering time through similar molecular mechanisms to those observed when expressed in Arabidopsis.

Diurnal oscillation of the transcription levels of *CDFs* has been reported in Arabidopsis and other species [21,23]. In Arabidopsis, *CDF1*–*CDF3* and *CDF5* show maximum expression at the beginning of the light period, decreasing to a minimum between 16 and 20 h, then rising again during dawn [21]. In tomato, *SlCDF1* and *SlCDF3* exhibit maximum expression at the beginning of the day, while *SlCDF2*, *SlCDF4*, and *SlCDF5* exhibit maximum levels during the night [18]. In rice, *OsDof12* is strongly inhibited by dark treatment [22]. In the study, *PheDof12-1* exhibited significantly diurnal expression patterns with high mRNA levels at the beginning of the light period under LD and SD conditions, supporting the assumption that it is a true homologue of the Arabidopsis CDFs. In Arabidopsis, CDFs can bind to the *CO* promoter to repress its transcription [20], and PttCDF3 can bind directly to the *PttCO2* promoter in *Populus* [33]. In moso bamboo, the diurnal expression pattern of *PheCOL4* is consistent with *PheDof12-1*, and Y1H analysis shows that PheDof12-1 binds directly to the promoter

of *PheCOL4*. These results support the hypothesis that flowering regulator CO, a target of CDFs, is controlled precisely [21], which is similar to the situation in Arabidopsis and *Populus*.
