*2.3. Expression Patterns of PheDof12-1 under Stress Treatments*

Previous reports have shown that Dof TFs are involved in abiotic stress [30]. To determine the expression pattern of *PheDof12-1* in moso bamboo under different stresses, we performed detailed qRT-PCR with *TIP41* and *NTB* as internal reference genes. The results show that *PheDof12-1* was responded to cold, drought, and salt stresses. In drought stress, *PheDof12-1* was induced and upregulated at each time point, and levels of transcripts in leaves and stems were slightly elevated, but a sharp increase occurred after 1 h in roots, peaking at 70.9-fold. This implies that *PheDof12-1* is induced and has a positive function in response to drought stress (Figure 2A–C). In cold treatment using *NTB* as a reference gene, the expression of *PheDof12-1* rapidly increased in leaves, reaching 86.1-fold at 24 h (Figure 2I). Regarding salt treatment, the maximum increase was observed at 12 h, reaching 12.5-fold in leaves when *TIP41* was used as the reference gene (Figure 2F), but the expression level was first induced and then decreased in roots. To further investigate the functions of *PheDof12-1*, we initially analyzed the effects of gibberellin A3 (GA3) and abscisic acid (ABA) on its expression (Figure 2J,K). In GA<sup>3</sup> stress, the transcription level of *PheDof12-1* was induced and upregulated at almost every time point, peaking at 15.0-fold at 24 h. Under ABA treatment, the translation level of *PheDof12-1* initially decreased and then increased, was lowest at 6 h, dropping to undetectable levels, and reached a peak at 48 h. All of these data indicate that *PheDof12-1* takes part in the hormones and different abiotic stresses of moso bamboo.

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**Figure 2.** Relative expression of *PheDof12-1* in different tissues of moso bamboo under drought: (**A**) root, (**B**) stem, (**C**) leaf; salt: (**D**) root, (**E**) stem, (**F**) leaf; under cold: (**G**) root, (**H**) stem, (**I**) leaf; and under (**J**) gibberellin A3 (GA3) and (**K**) abscisic acid (ABA) treatments. **Figure 2.** Relative expression of *PheDof12-1* in different tissues of moso bamboo under drought: (**A**) root, (**B**) stem, (**C**) leaf; salt: (**D**) root, (**E**) stem, (**F**) leaf; under cold: (**G**) root, (**H**) stem, (**I**) leaf; and under (**J**) gibberellin A3 (GA<sup>3</sup> ) and (**K**) abscisic acid (ABA) treatments.

#### *2.4. Overexpression of PheDof12-1 Promotes Early Flowering in Arabidopsis 2.4. Overexpression of PheDof12-1 Promotes Early Flowering in Arabidopsis*

In order to verify the subcellular localization of PheDof12-1, we further amplified its coding region and fused it to the N-terminal of the eGFP vector. The subcellular localization assay indicated that PheDof12-1 was localized in the nucleus, in accordance with its function as a transcription factor (Figure 3B). To study the genetic functions of *PheDof12-1*, we transformed it in Arabidopsis. The overexpressed plants showed an early flowering phenotype under LD conditions (Figure 3A), whereas *PheDof12-1* overexpression had no effect on flowering time under SD conditions (not shown). The flowering time was about 10 days earlier than wild-type, and the number of rosette leaves of overexpressed lines was smaller than that of wild Arabidopsis (Figure 3C). We further investigated the transcription levels of *FT*, *SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1* (*SOC1)*, *AGAMOUS-LIKE 24* (*AGL24)*, *FLOWERING LOCUS C* (*FLC*), and *SHORT VEGETATIVE PHASE* (*SVP*) in the T3 generation to ascertain the downstream effects of this construct. *FT*, *SOC1*, and *AGL24* were upregulated, while *FLC* and *SVP* expression were rather low compared with wildtype (Figure 3D). These data suggest that *PheDof12-1* might regulate flowering by controlling the expression of *FT*, *SOC1*, *AGL24*, *FLC*, and *SVP*. In order to verify the subcellular localization of PheDof12-1, we further amplified its coding region and fused it to the N-terminal of the eGFP vector. The subcellular localization assay indicated that PheDof12-1 was localized in the nucleus, in accordance with its function as a transcription factor (Figure 3B). To study the genetic functions of *PheDof12-1*, we transformed it in Arabidopsis. The overexpressed plants showed an early flowering phenotype under LD conditions (Figure 3A), whereas *PheDof12-1* overexpression had no effect on flowering time under SD conditions (not shown). The flowering time was about 10 days earlier than wild-type, and the number of rosette leaves of overexpressed lines was smaller than that of wild Arabidopsis (Figure 3C). We further investigated the transcription levels of *FT*, *SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1* (*SOC1)*, *AGAMOUS-LIKE 24* (*AGL24)*, *FLOWERING LOCUS C* (*FLC*), and *SHORT VEGETATIVE PHASE* (*SVP*) in the T3 generation to ascertain the downstream effects of this construct. *FT*, *SOC1*, and *AGL24* were upregulated, while *FLC* and *SVP* expression were rather low compared with wild-type (Figure 3D). These data suggest that *PheDof12-1* might regulate flowering by controlling the expression of *FT*, *SOC1*, *AGL24*, *FLC*, and *SVP*.

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**Figure 3.** Analysis of an early flowering phenotype by overexpression of PheDof12-1 in Arabidopsis. (**A**) Phenotypes of overexpressing *PheDof12-1* transgenic lines (L3, L4) and wild-type (WT) plants as control under long-day (LD) conditions. (**B**) Subcellular localization of PheDof12-1. (**C**) Flowering time scored as number of rosette leaves at flowering of wild-type and transgenic plants under LD conditions. (**D**) Transcription levels of *FT*, *SOC1*, *AGL24*, *FLC*, and *SVP* in wild-type and transgenic plants. Arabidopsis *Actin* was used as the internal reference gene. Error bars indicate standard deviations. Asterisks indicate statistically significant difference between wild-type and transgenic plants (*p* < 0.01 by Student's t-test). **Figure 3.** Analysis of an early flowering phenotype by overexpression of PheDof12-1 in Arabidopsis. (**A**) Phenotypes of overexpressing *PheDof12-1* transgenic lines (L3, L4) and wild-type (WT) plants as control under long-day (LD) conditions. (**B**) Subcellular localization of PheDof12-1. (**C**) Flowering time scored as number of rosette leaves at flowering of wild-type and transgenic plants under LD conditions. (**D**) Transcription levels of *FT*, *SOC1*, *AGL24*, *FLC*, and *SVP* in wild-type and transgenic plants. Arabidopsis *Actin* was used as the internal reference gene. Error bars indicate standard deviations. Asterisks indicate statistically significant difference between wild-type and transgenic plants (*p* < 0.01 by Student's *t*-test).

#### *2.5. PheDof12-1 Interacts with Photoperiod-Related Regulators 2.5. PheDof12-1 Interacts with Photoperiod-Related Regulators*

In Arabidopsis, CDFs are transcriptional repressors that bind to *CO* and *FT* promoters to repress their transcription [20]. To explore whether PheDof12-1 can form heterodimers with other proteins, an interaction prediction was performed using STRING (https://stringdb.org/) based on the interaction network of rice orthologous genes. As shown in Figure 4E, PheDof12-1 interacted with 10 identified proteins. Among them, the B-box protein (PH01004196G0130), Dof transcription factor (PH01001184G0160), grain size gene (*PheSLR1*) [31], photoperiodic flowering response gene (*PH01002431G0090*) [32], and drought-induced protein (PH01000199G0750) were identified, suggesting that PheDof12-1 may be involved in growth and development, photoperiodic response, and abiotic stress. *CDF1*, *CDF2*, *CDF3*, and *CDF5* had high mRNA levels at the beginning of the light period in In Arabidopsis, CDFs are transcriptional repressors that bind to *CO* and *FT* promoters to repress their transcription [20]. To explore whether PheDof12-1 can form heterodimers with other proteins, an interaction prediction was performed using STRING (https://stringdb.org/) based on the interaction network of rice orthologous genes. As shown in Figure 4E, PheDof12-1 interacted with 10 identified proteins. Among them, the B-box protein (PH01004196G0130), Dof transcription factor (PH01001184G0160), grain size gene (*PheSLR1*) [31], photoperiodic flowering response gene (*PH01002431G0090*) [32], and drought-induced protein (PH01000199G0750) were identified, suggesting that PheDof12-1 may be involved in growth and development, photoperiodic response, and abiotic stress.

Arabidopsis [21], and CDFs displayed a similar expression pattern in *Populus* [33]. So, we detected the expression patterns under photoperiod treatments. The results show that *PheDof12-1* was similarly expressed under both LD and SD conditions. The transcription level of *PheDof12-1*  decreased with the increased light time, reaching the minimum value before dark (Figure 4A,B), with high mRNA levels at the beginning of the light period, which was consistent with the expression patterns of CDFs in Arabidopsis and *Populus*. The highly similar expression pattern of CDFs in *Populus*, Arabidopsis, and moso bamboo suggests a functional conservation. CO and CO-like (COL) proteins are members of the B-box family, playing a central role in the photoperiod response pathway by mediating between the circadian clock and the floral integrators *CDF1*, *CDF2*, *CDF3*, and *CDF5* had high mRNA levels at the beginning of the light period in Arabidopsis [21], and CDFs displayed a similar expression pattern in *Populus* [33]. So, we detected the expression patterns under photoperiod treatments. The results show that *PheDof12-1* was similarly expressed under both LD and SD conditions. The transcription level of *PheDof12-1* decreased with the increased light time, reaching the minimum value before dark (Figure 4A,B), with high mRNA levels at the beginning of the light period, which was consistent with the expression patterns of CDFs in Arabidopsis and *Populus*. The highly similar expression pattern of CDFs in *Populus*, Arabidopsis, and moso bamboo suggests a functional conservation.

[34]. CDFs are transcriptional repressors that bind to the *CO* promoter to repress its transcription [20]. PheDof12-1 interacted with B-box proteins by interaction prediction; moreover, *PheDof12-1* and *PheCOL4* had similar expression patterns under photoperiod treatments (Figure 4C,D), suggesting that PheDof12-1 may interact with PheCOL4 in moso bamboo. To examine whether the PheDof12-1 protein regulated *PheCOL4* expression by directly binding to the promoter region, the *PheCOL4* promoter sequence was investigated. We performed a targeted yeast one-hybrid (Y1H) assay using CO and CO-like (COL) proteins are members of the B-box family, playing a central role in the photoperiod response pathway by mediating between the circadian clock and the floral integrators [34]. CDFs are transcriptional repressors that bind to the *CO* promoter to repress its transcription [20]. PheDof12-1 interacted with B-box proteins by interaction prediction; moreover, *PheDof12-1* and *PheCOL4* had similar expression patterns under photoperiod treatments (Figure 4C,D), suggesting that PheDof12-1 may interact with PheCOL4 in moso bamboo. To examine whether the PheDof12-1 protein regulated *PheCOL4* expression by directly binding to the promoter region, the *PheCOL4* promoter sequence was investigated. We performed a targeted yeast one-hybrid (Y1H) assay using PheDof12-1, and PheCOL4 was inserted upstream of the reporter plasmid pHIS2

and cotransfected into the yeast cells with the AD-PheCOL12-1 effector plasmid. The binding of PheCOL12-1 and the promoter of *PheCOL4* was indicated by the growth of transfected yeast cells on a nutrient-deficient medium (synthetic dextrose (SD)/-Trp-Leu-His) plus 3-amino-1, 2, 4-triazole (3-AT) and 5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside (X-α-Gal). The results show that all transformants tested were found to grow well on the SD/-Leu/-Trp medium when transferred onto SD/-Trp/-Leu/-His/3-AT/X-α-Gal plates for 3 days; only the yeast cells of AD-PheDof12-1 + pHIS2-PheCOL4 vectors and the positive control grew strong and turned blue (Figure 4F). This result suggests that PheDof12-1 could bind to the promoter of PheCOL4 and regulate *PheCOL4* expression in moso bamboo. PheDof12-1, and PheCOL4 was inserted upstream of the reporter plasmid pHIS2 and cotransfected into the yeast cells with the AD-PheCOL12-1 effector plasmid. The binding of PheCOL12-1 and the promoter of *PheCOL4* was indicated by the growth of transfected yeast cells on a nutrient-deficient medium (synthetic dextrose (SD)/-Trp-Leu-His) plus 3-amino-1, 2, 4-triazole (3-AT) and 5-bromo-4 chloro-3-indoxyl-α-D-galactopyranoside (X-α-Gal). The results show that all transformants tested were found to grow well on the SD/-Leu/-Trp medium when transferred onto SD/-Trp/-Leu/-His/3- AT/X-α-Gal plates for 3 days; only the yeast cells of AD-PheDof12-1 + pHIS2-PheCOL4 vectors and the positive control grew strong and turned blue (Figure 4F). This result suggests that PheDof12-1 could bind to the promoter of PheCOL4 and regulate *PheCOL4* expression in moso bamboo.

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**Figure 4.** PheDof12-1 protein binds to the promoter region of *PheCOL4*. Relative expression of *PheDof12-1* under (**A**) LD and (**B**) SD conditions. Transcription level of *PheCOL4* under (**C**) LD and (**D**) SD conditions. (**E**) Interaction network of PheDof12-1 in moso bamboo. Colored balls (protein nodes) in the network were used as a visual aid to indicate different input proteins and predicted interactions. Enlarged protein nodes indicate the availability of 3D protein structure information. Gray lines connect proteins that are associated by recurring text mining evidence. (**F**) Yeast onehybrid (Y1H) assay for AD-PheDof12-1 and pHIS2-PheCOL4. The reporter pHIS2 vector carrying the corresponding fragment and the effector AD-PheDof12-1 vector were cotransfected into yeast Y187 cells. Growth of the transfected yeast cells on a 3-AT and X-α-Gal medium indicates that the PheDof12-1 protein can bind to the PheCOL4 promoter. **Figure 4.** PheDof12-1 protein binds to the promoter region of *PheCOL4*. Relative expression of *PheDof12-1* under (**A**) LD and (**B**) SD conditions. Transcription level of *PheCOL4* under (**C**) LD and (**D**) SD conditions. (**E**) Interaction network of PheDof12-1 in moso bamboo. Colored balls (protein nodes) in the network were used as a visual aid to indicate different input proteins and predicted interactions. Enlarged protein nodes indicate the availability of 3D protein structure information. Gray lines connect proteins that are associated by recurring text mining evidence. (**F**) Yeast one-hybrid (Y1H) assay for AD-PheDof12-1 and pHIS2-PheCOL4. The reporter pHIS2 vector carrying the corresponding fragment and the effector AD-PheDof12-1 vector were cotransfected into yeast Y187 cells. Growth of the transfected yeast cells on a 3-AT and X-α-Gal medium indicates that the PheDof12-1 protein can bind to the PheCOL4 promoter.
