Identiﬁcation and Characterization of PRE Genes in Moso Bamboo ( Phyllostachys edulis )

: Basic helix–loop–helix (bHLH)/HLH transcription factors are involved in various aspects of the growth and development of plants. Here, we identiﬁed four HLH genes, PePRE1 - 4 , in moso bamboo plants that are homologous to Arabidopsis PRE genes. In bamboo seedlings, PePRE1/3 were found to be highly expressed in the internode and lamina joint by using quantitative RT-PCR analysis. In the elongating internode of bamboo shoots, PePRE genes are expressed at higher levels in the basal segment than in the mature top segment. Overexpression of PePRE s ( PePRE s-OX ) in Arabidopsis showed longer petioles and hypocotyls, as well as earlier ﬂowering. PePRE1 overexpression restored the phenotype due to the deﬁciency of AtPRE genes caused by artiﬁcial micro-RNA. PePRE1-OX plants showed hypersensitivity to propiconazole treatment compared with the wild type. In addition, PePRE1/3 but not PePRE2/4 proteins accumulated as punctate structures in the cytosol, which was disrupted by the vesicle recycling inhibitor brefeldin A (BFA). PePRE genes have a positive function in the internode elongation of moso bamboo shoots, and overexpression of PePREs genes promotes ﬂowering and growth in Arabidopsis . Our ﬁndings provided new insights about the fast-growing mechanism of bamboo shoots and the application of PRE genes from bamboo.


Introduction
Moso bamboo (Phyllostachys edulis) is one of the fastest growing non-timber tree plants all over the world, with considerable ecological, economic, and cultural value [1,2]. In spring, it can grow up to 1 m in less than 24 h and reach a final height of 20 m in 45-60 days [3]. The rapid expansion of bamboo stems is driven by the cell division and elongation of internodes, which are regulated by a combination of endogenous phytohormones and environmental factors such as auxin, gibberellin acid (GA), brassinosteroids (BRs), and light [4]. Recent studies demonstrate that the BR, auxin, GA, and phytochrome pathways converge through direct interactions among their transcription factors/regulators, then pass to a tripartite module of helix-loop-helix (HLH) and basic helix-loop-helix (bHLH) factors, which is named the HHbH module [5]. The HLH/bHLH cascade regulates cell elongation downstream of various hormonal and environmental signaling pathways [6,7].
Paclobutrazol-resistant (PRE) proteins are a typical bHLH transcription factors, homologs to the human Id-1 (inhibitor of DNA binding 1) protein which lacks the basic domain required for DNA binding. PRE proteins dimerize with other bHLH factors to inhibit their DNA-binding activity [8,9]. IBH1 (ILI1 binding bHLH protein) interacts with HBI1 (homolog of BEE2 interacting with IBH1), a positive regulator of cell elongation, and inhibits its transcriptional activity, thereby promoting the hypocotyl elongation by inhibiting the DNA-binding activity of HBI1 [10,11]. PRE1 and ILI1 promote cell elongation both in Arabidopsis and rice by interacting with IBH1 and forming a pair of antagonistic bamboo PRE proteins are clustered with rice PREs but not AtPREs (Figure 1b). Protein sequence alignment of the PREs showed that all bamboo PREs proteins have highly conserved helix-loop-helix (HLH) domains but not basic domains that are critical for DNA binding (Figure 1a). The expression patterns of PePRE genes were investigated in different tissues of moso bamboo seedlings by quantitative reverse transcription PCR (qRT-PCR) (Figure 1c,d). PePRE2 and PePRE3 are found highly expressed in roots, whereas PePRE1 and PePRE4 were hardly detected ( Figure 1d). Interestingly, only the PePRE1 gene was detected in leaves, suggesting its unique function in leaf development (Figure 1d). Consistent with a previous report about the critical function of PRE genes on the lamina joint bending [12], four bamboo PRE genes were found to be expressed in the lamina joint ( Figure 1d). As all PePREs were expressed in the internode and sheath, PePRE1 and PePRE3 showed predominant expressions in the sheath and internode, respectively ( Figure 1d). Together, these results suggested that PePRE genes showed tissue-specific patterns in moso bamboo seedlings.

Expression of PePRE Genes in Elongating Bamboo Shoots
Bamboo shoot growth is attributed to cell proliferation in the intercalary meristem and subsequent cell elongation in the elongation zone of the internodes. The expression patterns of bamboo PRE genes were investigated in the elongating bamboo shoot ( Figure  2a). PePRE1 and PePRE3 transcript levels were highest in the elongating internode (EIN)

Expression of PePRE Genes in Elongating Bamboo Shoots
Bamboo shoot growth is attributed to cell proliferation in the intercalary meristem and subsequent cell elongation in the elongation zone of the internodes. The expression patterns of bamboo PRE genes were investigated in the elongating bamboo shoot (Figure 2a). PePRE1 and PePRE3 transcript levels were highest in the elongating internode (EIN) and scale leaves, whereas PePRE2 and PePRE4 transcript levels were highest in the scale leaves ( Figure 2b). All PePREs showed higher expression levels in the internodes than in the nodes (Figure 2b). To further explore the role of PePRE genes in internode growth, the elongating internode of bamboo shoots was divided into top, middle, and basal regions (Figure 2a bottom right). Generally, expression of PePRE genes accumulated in the middle and basal regions, whereas PePREs were hardly detected in the upper parts. PePRE1 and PePRE3 were predominantly expressed in the middle and basal regions (Figure 2c). There was high PePRE2 expression in scale leaves, followed by the unelongated internodes. Interestingly, PePRE4 showed the lowest expression levels in the internodes and had relatively higher expression in the upper parts. Collectively, these data suggest that PePREs have a prevalent accumulation in the elongating tissue of bamboo shoots.  (Figure 2c). There was high PePRE2 expression in scale leaves, followed by the unelongated internodes. In terestingly, PePRE4 showed the lowest expression levels in the internodes and had rela tively higher expression in the upper parts. Collectively, these data suggest that PePREs have a prevalent accumulation in the elongating tissue of bamboo shoots.

Overexpressing PePREs in Arabidopsis
To further understand the biological function of PePREs, we generated overexpres sion transgenic lines with the 35S promoter driving the PePRE cDNAs fused with green fluorescent protein (GFP) in a Col-0 background. A total of 59, 42, 64, and 14 transgenic lines for PePRE1, 2, 3, and 4 were generated, respectively, with 31 (52.5%), 34 (81%), 22 (34.4%), and 3 (21.4%) lines for each gene, respectively, showing an early flowering phe notype. Days to bolting for PePRE1-OX #19 and PePRE2-OX #21 were found to be reduced by 13.3% and 14.0%, respectively, whereas the total rosette leaf numbers of PePRE-OX plants were also reduced with exception of PePRE4-OX. Under long-day conditions PePRE1-OX transgenic plants exhibited longer petioles and had paler green leaves than control plants (Figure 3a), which were similar to the AtPRE1 overexpression lines reported in a previous study [27]. This suggested that PePREs have a conserved function similar to AtPRE1 on flowering control. A total of 45% and 82.5% of PePRE1 and PePRE3 overexpres sion transgenic lines, respectively, showed a stem-bending phenotype, which was mainly due to a broken stem with a longitudinal crack ( Figure A2). We further crossed PePRE1 OX transgenic plants with pre-amiR transgenic lines, in which four AtPREs (AtPRE1/2/5/6

Overexpressing PePREs in Arabidopsis
To further understand the biological function of PePREs, we generated overexpression transgenic lines with the 35S promoter driving the PePRE cDNAs fused with green fluorescent protein (GFP) in a Col-0 background. A total of 59, 42, 64, and 14 transgenic lines for PePRE1, 2, 3, and 4 were generated, respectively, with 31 (52.5%), 34 (81%), 22 (34.4%), and 3 (21.4%) lines for each gene, respectively, showing an early flowering phenotype. Days to bolting for PePRE1-OX #19 and PePRE2-OX #21 were found to be reduced by 13.3% and 14.0%, respectively, whereas the total rosette leaf numbers of PePRE-OX plants were also reduced with exception of PePRE4-OX. Under long-day conditions, PePRE1-OX transgenic plants exhibited longer petioles and had paler green leaves than control plants (Figure 3a), which were similar to the AtPRE1 overexpression lines reported in a previous study [27]. This suggested that PePREs have a conserved function similar to AtPRE1 on flowering control. A total of 45% and 82.5% of PePRE1 and PePRE3 overexpression transgenic lines, respectively, showed a stem-bending phenotype, which was mainly due to a broken stem with a longitudinal crack ( Figure A2). We further crossed PePRE1-OX transgenic plants with pre-amiR transgenic lines, in which four AtPREs (AtPRE1/2/5/6) were knocked-down using artificial microRNA [14]. Due to the lack of AtPREs, pre-amiR exhibited extreme dwarfism, delayed flowering, and had a reduced fertility phenotype [21]. All F1 plants showed a normal phenotype similar to the wild type ( Figure 3d). Overexpression of PePRE1 rescued the AtPRE (1/2/5/6)-deficient phenotypes of the pre-amiR mutant, including late flowering and curled leaves. To exclude AtPRE1 expression interference, the expression level of AtPRE1 was detected in all plants. Only in the AtPRE1-OX line did the AtPRE1 expression level increase; there was no difference in the Col-0 and PePRE1-OX lines (Figure 3e). These studies suggest that PePRE genes play a conserved role similar to AtPRE genes on flowering promotion and cell elongation. were knocked-down using artificial microRNA [14]. Due to the lack of AtPREs, pre-amiR exhibited extreme dwarfism, delayed flowering, and had a reduced fertility phenotype [21]. All F1 plants showed a normal phenotype similar to the wild type ( Figure 3d). Overexpression of PePRE1 rescued the AtPRE (1/2/5/6)-deficient phenotypes of the pre-amiR mutant, including late flowering and curled leaves. To exclude AtPRE1 expression interference, the expression level of AtPRE1 was detected in all plants. Only in the AtPRE1-OX line did the AtPRE1 expression level increase; there was no difference in the Col-0 and PePRE1-OX lines ( Figure 3e). These studies suggest that PePRE genes play a conserved role similar to AtPRE genes on flowering promotion and cell elongation.

Subcellular Localization of PePREs in Arabidopsis
To determine the intracellular localization of PePRE proteins, the fluorescence signals of 35S::PePRE-GFP were examined in transgenic Arabidopsis plants. PePRE-GFP signals were mainly located in the nucleus, cytosol, and plasma membranes, whereas 4 , 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was used as the indicator of nuclear area. Interestingly, PePRE1-GFP and PePRE3-GFP proteins specifically displayed small punctate structures in the cytosol (Figure 4a), which are different from the AtPRE1 protein. When treated with BFA, a vesicle trafficking inhibitor [28], aggregation of PePRE1 fusion proteins in the punctate structures was reduced. Moreover, these signals were recovered by removing BFA (Figure 4b). Taken together, PePREs are located in the nucleus, cytosol, and membrane, whereas PePRE1 and PePRE3 are distributed in the cytosol in punctate structures.

BR Regulates the Expression Levels of PePREs
The expression levels of PePRE1 and PePRE2 dramatically decreased in the aerial part, whereas BR increased the expression of PePRE1 and PePRE2 in bamboo shoot [25]. We further examined the expression profile of PRE homologous genes in various tissues of bamboo seedlings with PPZ and eBL ( Figure 5). Expression of PRE1, 3, and 4 was decreased by PPZ but recovered by subsequent BR treatment in most tissues. However,

BR Regulates the Expression Levels of PePREs
The expression levels of PePRE1 and PePRE2 dramatically decreased in the aerial part, whereas BR increased the expression of PePRE1 and PePRE2 in bamboo shoot [25]. We further examined the expression profile of PRE homologous genes in various tissues of bamboo seedlings with PPZ and eBL ( Figure 5). Expression of PRE1, 3, and 4 was decreased by PPZ but recovered by subsequent BR treatment in most tissues. However, mRNA levels of PePRE2 increased in the internode and sheath and decreased in the lamina joint and root. In addition, PePRE2 did not further respond to eBL treatment. Overexpression of the PePRE1 gene resulted in longer hypocotyl in comparison with the wild type (Figure 6a,b; Figure A1). Compared with the wild type, PePRE1 overexpressing plants have longer hypocotyls and roots (Figure 6a,b), similar to AtPRE1 overexpressing plants. Additionally, compared with AtPRE1-OX, the shoot and root parts of PePRE1-OX plants showed more sensitivity and less sensitivity to PPZ, respectively (Figures 6c,d and A3).

Discussion
Although the rapid growth of woody bamboo plants has been widely studied, is known about the molecular mechanism underlying the elongation of moso bambo this study, we investigated the unique patterns of PePRE genes, the conserved gro promoting function in Arabidopsis, and the potential important role for PePREs in the gation of moso bamboo.

PePREs Have Tissue-Specific Expression Patterns in Moso Bamboo
PePRE1 and PePRE4 are highly expressed in the shoot, whereas PePRE2 and Pe are specifically expressed in the roots of seedlings. Consistent with these tissue-sp expressions, a rice homologous gene of PePRE, PGL1 (positive regulator of grain le 1), was expressed in the floral organs, young panicle, and predominantly in the roo not the leaf [29]. The expression level of BU1 (brassinosteroid upregulated 1) is high i lamina joint in vegetative organs and the panicle at the heading stage [30]. OsILI1 is uitously expressed in rice, whereas the highest expression of OsILI1 was observed i lamina joint [12]. OsBUL1 (O. sativa brassinosteroid upregulated 1-like1), an AtPRE olog in rice, is preferentially expressed in the lamina joint where it controls cell elong and positively affects leaf angles [31,32]. FaPRE1 was proposed as a ripening-assoc gene and showed a rapid increase in expression in the receptacle during fruit enlarge [33]. In our study, all four PRE genes were highly expressed in the elongating tissue not in the mature tissues such as the nodes of the bamboo shoot (Figures 1 and 2), w is consistent with their function in promoting cell elongation. During the rapid grow

Discussion
Although the rapid growth of woody bamboo plants has been widely studied, little is known about the molecular mechanism underlying the elongation of moso bamboo. In this study, we investigated the unique patterns of PePRE genes, the conserved growthpromoting function in Arabidopsis, and the potential important role for PePREs in the elongation of moso bamboo.

PePREs Have Tissue-Specific Expression Patterns in Moso Bamboo
PePRE1 and PePRE4 are highly expressed in the shoot, whereas PePRE2 and PePRE4 are specifically expressed in the roots of seedlings. Consistent with these tissue-specific expressions, a rice homologous gene of PePRE, PGL1 (positive regulator of grain length 1), was expressed in the floral organs, young panicle, and predominantly in the root but not the leaf [29]. The expression level of BU1 (brassinosteroid upregulated 1) is high in the lamina joint in vegetative organs and the panicle at the heading stage [30]. OsILI1 is ubiquitously expressed in rice, whereas the highest expression of OsILI1 was observed in the lamina joint [12]. OsBUL1 (O. sativa brassinosteroid upregulated 1-like1), an AtPRE homolog in rice, is preferentially expressed in the lamina joint where it controls cell elongation and positively affects leaf angles [31,32]. FaPRE1 was proposed as a ripening-associated gene and showed a rapid increase in expression in the receptacle during fruit enlargement [33]. In our study, all four PRE genes were highly expressed in the elongating tissues but not in the mature tissues such as the nodes of the bamboo shoot (Figures 1 and 2), which is consistent with their function in promoting cell elongation. During the rapid growth of monocots, elongation generally occurs from top to bottom in each individual internode [34]. From our results, PePRE1 is highly expressed in the basal part of the elongating internodes, with a gradient distribution from the basal to the top parts (Figure 7), which may contribute to the fast growth of bamboo shoot. A previous report showed that GRFs (growth-regulating factors) and ARF genes were highly expressed in the basal region of the elongating internode, in which ARF6 and two ARF8s targeted by miR167 and 11 GRFs targeted by miR396 were in the top region [35]. The DsEXLA2 (Dendrocalamus sinicus expansin-like A2) gene is highly expressed in the elongating internode and accelerates the plant growth rate of Arabidopsis [36]. PeGT43s (P. edulis Glycosyltransferase 43) and lignin biosynthesis are significantly upregulated within the shoot [3]. Considering the roles for PRE in Arabidopsis, PePRE genes may work downstream of ARF or GRF genes to regulate target genes in the elongating bamboo. monocots, elongation generally occurs from top to bottom in each individual internode [34]. From our results, PePRE1 is highly expressed in the basal part of the elongating internodes, with a gradient distribution from the basal to the top parts (Figure 7), which may contribute to the fast growth of bamboo shoot. A previous report showed that GRFs (growth-regulating factors) and ARF genes were highly expressed in the basal region of the elongating internode, in which ARF6 and two ARF8s targeted by miR167 and 11 GRFs targeted by miR396 were in the top region [35]. The DsEXLA2 (Dendrocalamus sinicus expansin-like A2) gene is highly expressed in the elongating internode and accelerates the plant growth rate of Arabidopsis [36]. PeGT43s (P. edulis Glycosyltransferase 43) and lignin biosynthesis are significantly upregulated within the shoot [3]. Considering the roles for PRE in Arabidopsis, PePRE genes may work downstream of ARF or GRF genes to regulate target genes in the elongating bamboo.

Phytohormones Regulates PRE Function in Bamboo Elongation
The stem elongation was contributed to by the division and expansion of individual internodes [37]. Transcriptome analysis revealed that multiple signaling pathways, including GA, auxin, and ABA, may play a role in regulating internode elongation [38]. GA plays an antagonistic regulatory role in regulating internode stem elongation in rice [39]. OsbHLH073 encodes an atypical bHLH protein and regulates plant height, internode elongation, and panicle extension by regulating GA biosynthesis genes [40]. The content of GAs in dwarf bamboo varieties is also lower than that in normal bamboos, implying that GA plays a major role in the height of Shidu bamboo [41]. Exogenous application of GA resulted in a significant increase in internode length in bamboo seedlings [42]. Those results hint that GA may play a dual role in internode elongation between shoots and seedlings of bamboo. PREs were reported to act as hostile antagonists of the bHLH family of transcription factors, which positively regulate cell elongation via multiple signaling pathways [6,7].

Phytohormones Regulates PRE Function in Bamboo Elongation
The stem elongation was contributed to by the division and expansion of individual internodes [37]. Transcriptome analysis revealed that multiple signaling pathways, including GA, auxin, and ABA, may play a role in regulating internode elongation [38]. GA plays an antagonistic regulatory role in regulating internode stem elongation in rice [39]. OsbHLH073 encodes an atypical bHLH protein and regulates plant height, internode elongation, and panicle extension by regulating GA biosynthesis genes [40]. The content of GAs in dwarf bamboo varieties is also lower than that in normal bamboos, implying that GA plays a major role in the height of Shidu bamboo [41]. Exogenous application of GA resulted in a significant increase in internode length in bamboo seedlings [42]. Those results hint that GA may play a dual role in internode elongation between shoots and seedlings of bamboo. PREs were reported to act as hostile antagonists of the bHLH family of transcription factors, which positively regulate cell elongation via multiple signaling pathways [6,7].
Bamboo PRE proteins showed different subcellular localizations, and only PRE1 and PRE3 showed punctate structures that were disturbed by BFA treatment. BFA treatment may lead to rapid protein aggregation within the endoplasmic reticulum and collapse of the Golgi apparatus [28]. The specific localizations of PePRE1 and PePRE3 may be related to the tuning of protein stability or function. PePREs are localized in both the nucleus and cytoplasm, whereas the punctate structures in cytoplasm were only observed in PePRE1 and PePRE3 in transgenic lines (Figure 4). Considering synchrony in phenotype, expression pattern, and subcellular localization, we proposed that the difference in subcellular localization may contribute to its functional variation. Certain PePRE overexpressing plants were easily dislodged caused by a broken stem ( Figure A2). PRE1 was reported to promote cell elongation by preventing IBH1 from inhibiting HBI1, which directly activates genes encoding cell-wall-loosening enzymes (e.g., EXP, etc.) [10,43]. The accumulation of PePRE may change the stem segment structure, especially the composition of the cell wall, leading to the rupture and hollowness of the stem ( Figure A2).

Various Functions of PRE in Regulating Plant Growth through Multiple Signaling Pathways
PRE proteins are important parts of the signaling pathways involved in physiological development and reproduction [21,27,44]. Due to the limitation of the transformation of moso bamboo, the function of PePREs was checked in Arabidopsis in this work. The overexpression of PePREs was associated with early flowering and promoted growth, such as longer petioles, hypocotyls, and roots ( Figure 7). PREs are involved in regulating the growth of floral organs in Arabidopsis [27,45]. FaPRE1 antagonistically modulates the transcription of genes related to both receptacle growth and ripening [23,33]. GhPRE1 has contributed to spinnable fiber formation in cotton; overexpressing GhPRE1 leads to longer fibers with improved quality parameters, indicating that this bHLH gene is useful for improving cotton fiber quality [24]. SlPRE2 was reported to regulate fruit development via the gibberellin pathway and tomato fruit pigment accumulation in tomatoes [46]. Those results suggest that PRE affects multiple aspects of the development of plants. PRE6 is a positive regulator of shade avoidance and interacts with a number of negative growth regulators (PAR1, etc.) [17]. Transcriptional regulators (ARFs and BZR1) and post-transcriptional regulators (HFR1, etc.) were key modules of the signaling network controlling shade avoidance [47,48]. Interestingly, PePRE genes are regulated by brassinosteroid levels ( Figure 5) in the elongating bamboo, and the transgenic lines also shown an altered response to PPZ treatment ( Figure 6). Shade avoidance syndrome (SAS) allows plants that are grown in densely populated environments to maximize their sunlight access [48]. As mentioned above in the relationship between those TFs and PRE, PePREs may play a positive role in rhizome elongation underground and shoot elongation aboveground. Functional analyses of PePREs will help to elucidate the mechanism of fast growth in plants as well. Light affects the dynamic growth and development of the P. pygmaeus rhizome-root system [49]. Taken together, PePRE genes show a conserved function in controlling flowering and promoting growth by responding to multiple signaling pathways. This is similar to the PRE genes from other species.
Our analysis identified PePRE genes with specific expression patterns in the seedling and shooting stage and demonstrated that PePREs are brassinosteroid-regulated genes. Overexpression of the PePRE1 gene will promote flowering, hypocotyl elongation, and root growth. The current results indicate a key role for PePRE genes in bamboo growth and development. Our findings will provide the basis for further functional characterizations of PRE family genes and the molecular mechanism of bamboo fast growth and will benefit the application of growth-promoting gene resources from bamboo.

Plant Material and Growth Conditions
The moso bamboo shoots (approximately 1.8 m above ground height) were obtained from the Bamboo Garden of Fujian Agriculture and Forestry University, Fuzhou (coordinate 119 • 14 E, 26 • 50 N). Moso bamboo seeds were collected from Gong city, Guanxi province, China (118 • 48 E, 24 • 51 N). The seeds of moso bamboo were soaked in tap water for 24 h to induce seed germination and then sown on the soil. Seedlings were cultivated for 3 weeks in a greenhouse (long-day conditions, 22 • C).
Columbia (Col-0) wild-type seeds of Arabidopsis were germinated on half MS medium (pH = 5.8) with 1% sucrose, then transferred to a greenhouse (long-day conditions, 22 • C). PePRE-OX was crossed with pre-amiR to generate PePRE-OX pre-amiR plants.

Protein Sequence Alignment and Phylogenic Tree Construction
Alignment of the protein sequences was performed using Clustal Omega and analyzed in the GENEDOC program with the default settings. A phylogenic tree based on the sequence alignment was generated using MEGA-X by the neighbor-joining method [50].

Gene Expression Analysis
Total RNA was isolated from various tissues with the Plant Total RNA Kit (Sigma, ATRN50) and extensively treated with RNase-free DNase I (Sigma, St. Louis, MO, USA, DNASE70-1SET). cDNA was generated by RT-PCR using the PrimeScript TM RT reagent Kit (Takara, Kusatsu, Japan, RR047A). qRT-PCR analysis was performed with SYBR Premix Ex Taq II (Takara, RR820) on a QuanStudio 6 Flex instrument (Applied Biosystems, Waltham, MA, USA). For each sample, qPCR was performed with three technical replicates on three biological replicates. The bamboo PeTIP41 gene [51] was used as an internal control for the qRT-PCR. The relative expression levels were calculated as E −∆Cq and normalized to PeTIP41. The primer sequences used in this study are listed in Table A1.

Vector Construction and Transformation
The full-length PePRE1 sequence was amplified and ligated into pAGM1311. A 35Spromotor-driven PePRE C-terminal fused with a GFP tag was constructed into a pAGM4673 backbone. Through Agrobacterium tumefaciens strain GV3101, these constructions were transformed into Arabidopsis (Col-0). T1 seeds were screened by an RFP selection marker and then further confirmed by qRT-PCR and Western blot.

Hypocotyl Measurements and Statistical Analysis
For hypocotyl and root length measurements, seedlings were grown for 7 days on vertically oriented plates. Seedlings were flattened and photographed before taking quantitative measurements using ImageJ software (http://rsb.info.nih.gov/, accessed on 8 April 2018) to analyze the scanned images of the seedlings. The differences among groups were assessed by one-way ANOVA and Duncan's multiple comparisons test using SPSS 23.0. GraphPad Prism 8.0 (http://www.graphpad.com/, accessed on 26 April 2018) was used to plot figures. At least 20 seedlings were measured, and experiments were repeated more than two times.

BFA Treatment
For BFA treatment, 4-day-old seedlings of PePRE1-OX were incubated in 50 µM BFA for 90 min before viewing the seedlings; control seedlings were incubated in a solution without BFA but with DMSO at the same concentration as the BFA-treated seedlings. After BFA treatment, the seedlings were washed with half MS liquid media several times and the root tips were observed for the formation of BFA compartments using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). For recovery, BFA was removed from seedlings and supplied with half MS media for another 40 min followed by immediate observation with the confocal microscope.

Conclusions
Our study identified an atypical bHLH transcription factor (PRE homologs) in moso bamboo via gene expression analysis, heterologous overexpression, etc. We verified that PePREs function as a positive regulator in the promotion of internode elongation during the fast-growth process. Overexpressing PePRE promoted Arabidopsis growth and petiole/hypocotyl elongation. Our findings shed light on bHLH-mediated fast growth to provide preliminary knowledge for fast-growing plants.

Conflicts of Interest:
The authors declare no conflict of interest.
Appendix A   Table A1. The primers used for quantitative PCR (RT-qPCR) and amplification of full-length coding sequences of PePREs in Phyllostachys edulis.