Moso bamboo gene sequences are obtained from the BambooGDB database¹ under the accession numbers in Supplementary Table 1.

In-Fusion Cloning methods were used to generate the plasmids used in this study.² The sequences subcloned into plasmids were verified by Sanger sequencing. The human cell expression vectors used in this study were pQCMV-Flag-GFP, pQCMV-GFP, and pCMV-Myc, which were described previously (Liu et al., 2017; Chen et al., 2021). pQCMV-YFP and pQCMV-mTagRFP-T vectors were modified from pQCMV-Flag-GFP by replacing GFP with YFP or mTagRFP-T (Supplementary Figure 1). To generate plasmids expressing Flag-PheCRY1c, Flag-PheCRY1d, and Flag-PhePPKb, the coding sequences (CDS) of PheCRY1c or PheCRY1d were amplified from moso bamboo cDNA, and the purified PCR products were then subcloned into SpeI/KpnI-digested pQCMV-Flag-GFP vector through in-fusion. Myc-PheCRY1c and Myc-PheCRY1d plasmids were prepared by cloning the CDS of the genes into the BamHI site of pCMV-Myc vector. For plasmids expressing PheCRY1c-YFP and PheCRY1d-YFP, the CDS of PheCRY1c and PheCRY1d were PCR-amplified from plasmids made before, and introduced into SpeI digested pQCMV-YFP vector by in-fusion. The AtBIC1-mTagRFP and PheBIC1a-mTagRFP plasmids were prepared by cloning the CDS of the AtBIC1 or PheBIC1a genes into the SpeI-digested pQCMV-mTagRFP-T vector by in-fusion. The gene-specific primers for constructs used for expressing recombinant proteins in HEK293T cells are listed in Supplementary Table 2.

pFGFP binary vectors was used for creating overexpression transgenic lines (Chen et al., 2021). The pFGFP binary vector was modified from pCambia3301, which allows the interested genes to express under control of ACTIN 2 promoter and fuse with 2 × Flag and GFP tags. For generation of the plant binary plasmids of FGFP-PheCRYs and FGFP-PheBIC1a, the CDS of PheCRY1s and PheBIC1a were cloned into BamHI-digested pFGFP vector. The primers for constructs used for plant transformation are included in Supplementary Table 2.

The wild-type Arabidopsis in this study is rdr6-11, which suppresses gene silencing (Peragine et al., 2004). The cry1cry2rdr6 was described previously (Chen et al., 2021). Arabidopsis transgenic lines were generated via Agrobacterium tumefaciens-mediated the floral dip method (Clough, 2005). Plasmids of FGFP-PheCRY1c and FGFP-PheCRY1d were introduced into cry1cry2rdr6 background to generate FGFP-PheCRY1c and FGFP-PheCRY1d overexpressing plants. FGFP-BIC1a overexpressing Arabidopsis was prepared in rdr6-11 background. The transgenic lines were screened on nutrient soil with 25 mg/L Glufosinate-ammonium and lines with a relative high protein expression level were used for phenotype analysis.

For hypocotyl phenotype analysis, seedlings were grown on Murashige and Skoog medium (MS) plates with 1% sucrose at 20∼22°C for 5 days under different light conditions. The same representative images and quantification results of control seedlings, cry1cry2, wild-type (WT), GFP-AtCRY1, GFP-AtCRY2, were used for Figures 1, 5. For endogenous CRY1 and CRY2 degradation analysis in WT, FGFP-PheBIC1a, seedlings were grown in darkness on MS plates with 1% sucrose for 7 days, then subjected to 30 μmol m^–2 s^–1 blue light for the indicated time. For FGFP-PheCRY1c and FGFP-PheCRY1d and degradation analysis, seedlings were grown in darkness on MS plates with 1% sucrose for 6 days, then subjected to 100 μmol m^–2 s^–1 blue light for the indicated time. For flowering time measurements, seeds were sown in compound soil, stratified at 4°C in darkness for 3 days and then were transferred to long day (LD; 16 h light/8 h darkness) walk-in chambers.

These methods were described previously (Chen et al., 2021).

For photobody observation in HEK293T cells, cells were grown in 20 mm glass bottom confocal dishes (Cat. # 801001, NEST), and fixed in 4% formaldehyde for 15 min before analysis under confocal microscope. Hoechst 33342 (Cat. # 14533, Sigma-Aldrich) solution, at the concentration of 10 μg/ml, was used for nuclei staining. Leica TCS SP8 confocal microscope with a HC PL APO CS2 63x/1.40 OIL objective was used to captured the microscopic images. YFP signals were detected at 500–550 nm with 488 nm as excitation, mTagRFP was excited with 554 nm laser and detected at 566–629 nm. Hoechst 33342 was excited with 405 nm laser and detected at 420–480 nm.

This method was described previously (Chen et al., 2021).

The primary antibodies used in this study are as following: anti-AtCRY1 (1:5,000, homemade) (Lin et al., 1996), anti-AtCRY2 (1:3,000, homemade) (Lin et al., 1998), anti-HSP90 (1:20,000, cat # AbM51099-31-PU, Beijing Protein Innovation), anti-GFP (1:2,000, cat # 598, MBL), anti-Myc (1:2,000, cat # 05-724, Millipore), anti-Flag (1:1,500, cat # F3165, Sigma-Aldrich).

The secondary antibodies used in this study are as following: anti-Mouse-HRP (1:15,000, cat # 31430, Thermo Fisher Scientific), anti-Rabbit-HRP (1:15,000, cat # 31460, Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 790 (1:15,000, cat # A11357), goat anti-rabbit IgG-Alexa Fluor 790 (1:15,000, cat # A11369), goat anti-mouse IgG-Alexa Fluor 680 (1:15,000, cat # A21058), goat anti-rabbit IgG-Alexa Fluor 680 (1:15,000, cat # A21109).

To investigate possible roles that cryptochromes play in moso bamboo, we searched moso bamboo (Phyllostachys edulis) genome sequence database, and identified five moso bamboo cryptochrome-like genes (PheCRY), PheCRY1a, PheCRY1b, PheCRY1c, PheCRY1d, and PheCRY2 (Supplementary Figure 2A). PheCRY1a, PheCRY1b, PheCRY1c, and PheCRY1d show higher similarity to Arabidopsis cryptochrome 1 (AtCRY1, ∼ 72–84% identity) than that to Arabidopsis cryptochrome 2 (AtCRY2, ∼ 66–74% identity) (Supplementary Figure 2B). Similar to that found in cryptochromes of other plants, PheCRYs share more extensive sequence similarity in the N-terminal photolyase homologous chromophore-binding domains (PHR domain) than in the C-terminal domains (CCE domain) (Supplementary Figure 2C). We successfully cloned two representative PheCRY genes (PheCRY1c and PheCRY1d) for further biochemical and functional analysis.

Because the transformation technique has not been established for moso bamboo, we generated FGFP-PheCRY1c (with Flag and GFP tags fused to the N-terminus of PheCRY1c) and FGFP-PheCRY1d overexpression transgenic Arabidopsis in cry1cry2 mutant background to test whether the bamboo CRY1s may complement the Arabidopsis cry1cry2 mutant. Similar to previous studies of cryptochromes in other plants (Ninu et al., 1999; Matsumoto et al., 2003; Platten et al., 2005; Chatterjee et al., 2006; Szucs et al., 2006; Zhang et al., 2008; Xu et al., 2009; Li et al., 2013a,b; Mao et al., 2014; Zhou et al., 2018; Lyu et al., 2021), our study demonstrates that bamboo CRY1 serves similar function as the Arabidopsis CRYs in regulating cell elongation. Transgenic expression of FGFP-PheCRY1c and FGFP-PheCRY1d rescued the blue light-specific long hypocotyl phenotype of the Arabidopsis cry1cry2 mutant, resulted in hypersensitivity to blue light as the GFP-AtCRY1 and GFP-AtCRY2 transgenic plants, which showed hypocotyls obviously shorter than that of the wild-type seedlings (Figure 1). Given that light inhibition of cell elongation is likely an ancient cellular response, it is not surprising that this activity of cryptochromes seems universally conserved in different plant species.

However, bamboo CRY1 could not fully rescue the late-flowering phenotype of Arabidopsis cry1cry2 double mutant (Figure 2). At the time of flowering, FGFP-PheCRY1 transgenic Arabidopsis had less rosette leaves than that of cry1cry2 mutant plants, but had more rosette leaves than that of wild-type and GFP-AtCRY1 or GFP-AtCRY2 overexpressing plants (Figure 2). Consistently, FGFP-PheCRY1 transgenic Arabidopsis flowered at about the same time as the cry1cry2 mutant (Figure 2). One transgenic line of FGFP-PheCRY1d (line #4) seemed to rescue the late-flowering phenotype of Arabidopsis cry1cry2 mutant (Figure 2B), which is inconsistent with the flowering phenotype of other FGFP-PheCRY1d overexpressing lines and FGFP-PheCRY1c overexpressing lines. We reasoned that this inconsistence may cause by T-DNA insertion in the genomic region of a gene involving in flowering regulation, because all the transgenic lines of FGFP-PheCRY1c and FGFP-PheCRY1d showed similar physiological activity in hypocotyl inhibition. Since flowering time is a more recent evolutionary “invention” of angiosperm, the moso bamboo cryptochromes and Arabidopsis cryptochromes may have a distinct mode of action in the regulation of flowering time. It’s also possible that the flowering regulation mechanisms in moso bamboo are distinct from that of Arabidopsis, especially that bamboo has the extremely long vegetative phase and bamboo plants often take decades to flower (Numata, 1970; Lin et al., 2010). In Arabidopsis, CRY1 and CRY2 primarily mediate blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation, respectively (Ahmad and Cashmore, 1993; Mockler et al., 2003). It would also be interesting to investigate whether bamboo CRY2 may have the function in promoting floral initiation.

Having demonstrated the blue light-dependent activity of PheCRY1c and PheCRY1d in transgenic Arabidopsis plants, we next asked whether the PheCRY1 may undergo similar photochemical reactions as that of Arabidopsis cryptochromes. Photo-oligomerization is the early photoreaction necessary for photoactivation of both CRY1 and CRY2 (Sang et al., 2005; Rosenfeldt et al., 2008; Wang et al., 2016; Liu et al., 2020). To investigate if the moso bamboo PheCRYs undergo homo-oligomerization in response to blue light, we examined the PheCRY1 oligomerization by co-expressing Flag-PheCRY1 and Myc-PheCRY1 in human embryonic kidney 293T (HEK293T) cells and tested the interaction of the two differentially tagged PheCRY1 by co-immunoprecipitation (co-IP) assay. In cells with similar amounts of Flag-PheCRY1 and Myc-PheCRY1 expression, Flag-beads coprecipitated more Myc-PheCRY1 in blue light than that in the dark (Figures 3A,B), demonstrating the blue light-enhanced PheCRY1 homo-oligomerization in the absence of other bamboo proteins. These results also demonstrated that, similar to Arabidopsis CRY1 and CRY2, bamboo PheCRY1 undergo oligomerization to become active. Photoexcited AtCRY1 and AtCRY2 are known to oligomerize into photobodies (Yu et al., 2009; Bugaj et al., 2013; Ozkan-Dagliyan et al., 2013; Wang et al., 2021; Liu et al., 2022), which is another early photoreaction of cryptochromes. We further investigated the PheCRY1 photobody formation in HEK293T cells. Figures 3C,D shows that PheCRY1-YFP (fused to yellow fluorescent protein) formed photobodies in the nucleus with blue light treatment, whereas no photobodies were detected in the cells in the dark, demonstrating that photoexcited PheCRY1 is capable of homo-oligomerization into photobodies in the absence of other proteins. These observations are also consistent with a hypothesis that photo-oligomerization might be an evolutionarily conserved photoactivation mechanism of cryptochrome photoreceptors. Taken together, our results demonstrate that moso bamboo PheCRY1 undergoes blue light-dependent homo-oligomerization to become photoactivated.

Photoreceptor inactivation is an important photoregulatory mechanism for the maintenance of a sustained photosensitivity of the cells. Arabidopsis cryptochromes are inactivated by at least three mechanisms: BIC inhibition of oligomerization, dark-dependent monomerization, and blue light-dependent proteolysis (Yu et al., 2007; Wang et al., 2016; Liu et al., 2020, 2022; Chen et al., 2021). In Arabidopsis, the blue light-dependent cryptochrome oligomerization is suppressed by two closely related cryptochrome inhibitory proteins, BIC1 and BIC2. We examined whether there are BIC proteins in moso bamboo and whether the putative bamboo BIC proteins might suppress photo-oligomerization of the bamboo cryptochromes. Two highly homologous PheBIC1 genes, PheBIC1a and PheBIC1b, were identified from the moso bamboo genome sequence database (Supplementary Figure 3). PheBIC1a and PheBIC1b share over 80% sequence identity and share more extensive sequence similarity in the CID domain (CRY-interacting domain) to Arabidopsis BIC1 and BIC2 (Supplementary Figure 3C), indicating that PheBICs may also function as cryptochrome inhibitors in moso bamboo. To test this hypothesis, we first examined the interaction of PheBIC1 and PheCRY1 in HEK293T cells co-expressing epitope-tagged PheBIC1 and PheCRY1 using co-IP assays. Our results show that blue light enhances PheBIC1 and PheCRY1 interaction. Both PheCRY1c and PheCRY1d coimmunoprecipitated PheBIC1a in HEK293T cells exposed to blue light, but little PheBIC1-PheCRY1 complex was coprecipitated in the dark (Figures 4A,B). This result suggests that PheBIC1 might interact with photoactivated PheCRY1 to inhibit their activities. We next tested this possibility by examining the effects of PheBIC1 on blue light-dependent PheCRY1 photobody formation by co-expressing PheBIC1-mTagRFP (fused to red fluorescent protein) and PheCRY1-YFP in HEK293T cells. AtCRY2-YFP and AtBIC1-mTagRFP co-expression cells were included as controls. Our results shows that no PheCRY1 photobodies were detected in the cells co-expressing PheBIC1 in blue light (Figures 4C,D), confirming our hypothesis that PheBIC1 act as cryptochrome-specific inhibitors in moso bamboo.

To test whether the bamboo BIC1 and their physical interaction with the bamboo CRY1 may be physiologically relevant, we prepared FGFP-PheBIC1a overexpression transgenic Arabidopsis in the wild-type background. The PheBIC1a overexpressing lines are insensitive to blue light, showing a long hypocotyl phenotype in blue light, resembling that of the Arabidopsis cry1cry2 double mutant (Figures 5A–D). This result suggests that PheBIC1 may inhibit the function of Arabidopsis cryptochromes. We then investigated this hypothesis by comparing the phosphorylation and degradation of Arabidopsis endogenous CRY1 and CRY2 in the wild-type and PheBIC1a overexpressing plants. As expected, the blue light-dependent phosphorylation of both AtCRY1 and AtCRY2 were not detectable in PheBIC1a overexpressing Arabidopsis (Figures 5E,F), and the blue light-dependent degradation of AtCRY2 was also impaired in PheBIC1a overexpressing plants (Figure 5F). In the wild-type seedlings, obvious AtCRY1 phosphorylation was detected within 15 min of blue light exposure, while no AtCRY1 phosphorylation was detected even after 60 min of blue light exposure in the PheBIC1a overexpressing Arabidopsis (Figure 5E). The phosphorylation of AtCRY2 was detected within 10 min of blue light exposure and AtCRY2 protein was no detectable after 60 min of blue light exposure in the wild-type seedings (Figure 5F). However, neither phosphorylation nor degradation of AtCRY2 were detected in the PheBIC1a overexpressing Arabidopsis in response to blue light (Figure 5F). These results are consistent with our previously published results that overexpression of Arabidopsis BICs suppressed all known photobiochemical and photophysiological activities of AtCRY1 and AtCRY2, resulted in unrestrictive hypocotyl growth in blue light (Wang et al., 2016). Results of these experiments also indicate that PheBICs may be the PheCRYs-specific inhibitors to regulate the activity of PheCRYs in moso bamboo.

Blue light-dependent proteolysis serve as another inactivation mechanism of crytochromes by removing the activated cryptochromes in cells. Depending on the plant species, CRY1, CRY2, and other types of cryptochromes have all been shown to undergo light-dependent proteolysis (Lin et al., 1998; Imaizumi et al., 2000; Reisdorph and Small, 2004; Chatterjee et al., 2006; Hirose et al., 2006). In Arabidopsis, both AtCRY1 and AtCRY2 undergo blue light-dependent degradation in the nucleus (Yu et al., 2007; Liu et al., 2022). We next examined the protein degradation of PheCRY1 in transgenic Arabidopsis. Figure 6 showed that in etiolated seedlings exposed to blue light, the PheCRY1c and PheCRY1d protein exhibits a pronounced decrease after 12 h of blue light treatment. The rate of blue light-induced degradation of PheCRY1c and PheCRY1d were slower than that of the AtCRY2, which showed apparent degradation after 2 h of blue light treatment. We reasoned that PheCRY1c and PheCRY1d might be more closely related to Arabidopsis CRY1, which requires higher intensities of blue light and longer blue light treatment time to induce its degradation. Our results also suggest that the light-dependent proteolysis may be another evolutionary conserved mechanism for inactivation of PheCRYs in moso bamboo, and the E3 ubiquitin ligases, CUL3^LRBs and CUL4^COP1/SPAs, responsible for Arabidopsis cryptochromes degradation may also be conserved between Arabidopsis and moso bamboo. However, this hypothesis remains to be tested.

Photoexcited Arabidopsis CRY1 and CRY2 further undergo blue light-dependent phosphorylation to both enhance their activities and facilitate their ubiquitination and degradation in the nucleus (Shalitin et al., 2002, 2003; Yu et al., 2007; Weidler et al., 2012; Liu et al., 2016, 2022; Miao et al., 2021). Four closely related plant-specific PPKs (PPK1, PPK2, PPK3, and PPK4) have been shown to catalyze the phosphorylation of Arabidopsis CRY1 and CRY2 in blue light (Liu et al., 2017; Ni et al., 2017). To test whether moso bamboo PheCRYs undergo blue light-induced phosphorylation and whether PhePPKs are the protein kinases catalyzing light-induced phosphorylation of PheCRYs, we searched moso bamboo genome sequence database, and found 10 closely related PhePPKs (PhePPKa–PhePPKj) genes in moso bamboo (Supplementary Figure 4). Similar to Arabidopsis PPKs, PhePPKs have two domains, a N-terminal Ser/Thr kinase domain which is homolog to casein kinase 1 (CK1) (Liu et al., 2017), and a C-terminal domain that are highly conserved within the PPK gene families (Supplementary Figure 4C). We cloned PhePPKb and examine its catalytic characteristics in HEK293T cells. We co-expressed Arabidopsis CRY2 or moso bamboo PheCRY1 with AtPPK1 or PhePPKb in HEK293T cells to investigate the catalytic activity of PPK on CRY by electrophoretic mobility upshift immunoblot assays (Shalitin et al., 2002, 2003; Wang et al., 2016). AtCRY2, PheCRY1c, and PheCRY1d exhibit retarded migration in cells co-expressing PhePPKb or AtPPK1 treated with blue light, whereas no migration upshift was detected in cells treated with green light, red light or far-red light (Figures 7A–C and Supplementary Figures 5A–C). The blue light-specific upshift migration of CRYs disappeared after protein phosphatase treatments (Figures 7D–F and Supplementary Figures 5D–F), confirming the slow migration bands were phosphorylated PheCRY1. We also found that AtCRY2 can be phosphorylated by moso bamboo PhePPK1, and AtPPK1 can phosphorylate bamboo PheCRY1 (Figures 7A,D and Supplementary Figures 5B,C,E,F), indicating that PPK-CRY (enzyme-substrate) pairing is more likely determined by structure-based mechanisms regardless of plant species. In summary, our results demonstrate that moso bamboo CRY1s undergo blue-light dependent phosphorylation in vitro, and PhePPKs are the protein kinases that catalyze the blue-light dependent phosphorylation of moso bamboo cryptochromes, indicating that the molecular basis for light-dependent phosphorylation of cryptochrome is also evolutionary conserved between Arabidopsis and moso bamboo.