Identification and Characterization of the PEBP Family Genes in Moso Bamboo (Phyllostachys heterocycla)

Moso bamboo is one of the economically most important plants in China. Moso bamboo is a monocarpic perennial that exhibits poor and slow germination. Thus, the flowering often causes destruction of moso bamboo forestry. However, how control of flowering and seed germination are regulated in moso bamboo is largely unclear. In this study, we identified 5 members (PhFT1-5) of the phosphatidyl ethanolamine-binding proteins (PEBP) family from moso bamboo genome that regulate flowering, flower architecture and germination, and characterized the function of these PEBP family genes further in Arabidopsis. Phylogenetic analysis revealed that 3 (PhFT1, PhFT2 and PhFT3), 1 (PhFT4) and 1 (PhFT5) members belong to the TFL1-like clade, FT-like clade, and MFT-like clade, respectively. These PEBP family genes possess all structure necessary for PEBP gene function. The ectopic overexpression of PhFT4 and PhFT5 promotes flowering time in Arabidopsis, and that of PhFT1, PhFT2 and PhFT3 suppresses it. In addition, the overexpression of PhFT5 promotes seed germination rate. Interestingly, the overexpression of PhFT1 suppressed seed germination rate in Arabidopsis. The expression of PhFT1 and PhFT5 is significantly higher in seed than in tissues including leaf and shoot apical meristem, implying their function in seed germination. Taken together, our results suggested that the PEBP family genes play important roles as regulators of flowering and seed germination in moso bamboo and thereby are necessary for the sustainability of moso bamboo forest.


Results
Isolation and identification of PEBP family genes in moso bamboo. To identify PEBP proteins in moso bamboo we blast screened the entire moso bamboo genome database (http://server.ncgr.ac.cn/bamboo/ blast.php) for genes providing sequence similarity with Arabidopsis and rice PEBP proteins. We obtained 6 PEBP family candidate genes from moso bamboo genome, but PH01000020G1780 were excluded from PEBP family because of harboring an incomplete PEBP domain with a lower expectation value (E = 7.8e-8). Therefore, 5 fulllength PEBP family genes were identified and designated as PhFT1-5 (Table S1),that were consistent with previously reported sequence 1 . In order to confirm this results, we cloned the full-length coding sequence of PhFT1, PhFT2, PhFT3, PhFT4 and PhFT5 from cDNA extracted from moso bamboo seedling.
Furthermore, the multiple protein sequences alignment revealed that moso bamboo PEBP family proteins have conserved PEBP domain and DPDxP motif (Fig. 2). The key amino acid residues that are distinguishable among the MFT-like (W), TFL-like (H) and FT-like (Y) clade were present at position 85 of AtFT in each moso bamboo PEBP family proteins (Fig. 2). However, the highly conserved amino acid sequences, LGRQTVYAPGWRQN in segment B and LYN triad in segment C are less conserved in PhFT4, although these motifs are determinant of FT activity and FT/TFL1 function (Fig. 2). Notably, these motives in PhFT4 are even different from FT sequences of other bamboo species (Fig. S1). Taken together, MFT-like and TFL-like clade of moso bamboo were conserved across angiosperm species, but FT-like clade is more diversified.
The developmental stage dependent PhPEBPs expression in leaf and flowering tissue in moso bamboo. To gain insights into possible roles of these PEBP family genes in the regulation of flowering and/ or flower development in moso bamboo, the expression pattern of PEBP family genes in the different developmental stages were analyzed. We could successfully obtain the samples from the flowering bamboo forest at Nanping, Fujian, China (Fig. S2). During the flowering of moso bamboo, the leaves began to die gradually (Fig. S2). Therefore, for RNA extraction leaf blades before flowering (leaf), leaf sheath and flowering tissues at bloom (flower) and developing seeds after bloom (seed) were sampled (Fig. 3a-c) and gene expression was subsequently tested by qRT-PCR analysis (Fig. 3d-h).
The expression of genes in the TFL-like clade had highly different transcript levels during the developmental stages investigated. Expression of PhFT1 and PhFT2 followd a similar pattern over time and tissue with a higher transcriptional abundance of PhFT1 compared to PhFT2. No expression was detected in leaves prior to flowering, while during blooming PhFT1 and PhFT2 were clearly expressed in floral tissues and showed high transcript , Selaginella denticulata (Sd), Bambusa tulda (Bt), Phyllostachys edulis (Pe) and Phyllostachys meyeri (Pm) was constructed by IQ-TREE 1.6.9 84 . The unit for the scale bar displays branch lengths. levels in developing seeds (Fig. 3d,e). PhFT3 expression was also higher at blooming than in leaves before flowering. During seed development expression was comparably low with our leaves samples (Fig. 3f). By contrast, PhFT4 was expressed in leaves prior flowering, however, hardly detectable in the other two samples (Fig. 3g). The expression of PhFT5 was constant between the tested tissues and developmental stages, although PhFT5 expression was slightly lower at blooming, (Fig. 3h).

Ectopic expression of PhPEBPs in transgenic arabidopsis plants.
In order to examine the function of moso bamboo PEBP family genes, fusion constructs of PhPEBP coding sequences and yellow fluorescence protein (YFP) driven by the CaMV 35S promoter were introduced into Arabidopsis thaliana wild type. We successfully obtained multiple transgenic lines for all constructs. The microscopic observation with these lines revealed that moso bamboo PEBP proteins localize in both of nucleus and cytoplasm in Arabidopsis (Fig. S3). These results are consistent with the observation that PEBP proteins from other plant species localize in both of nucleus and cytoplasm 20 .
The PhFT5 is induced by ABA during arabidopsis seed germination. Abscisic acid (ABA) negatively regulates seed maturation and germination in many species. MFT is known to be up-regulated upon ABA treatment and negatively regulate seed germination through regulating ABA signaling in Arabidopsis 26 . Therefore, we tested the germination rate of transgenic Arabidopsis expressing PhFT5-YFP in Col-0 or ABA-hypersensitive mother of ft and tfl1-3 FT (mft-3) loss-of-function mutant. In the absence of ABA, the germination rate of tested seeds were close to 100% (Fig. S5). However, in the presence of 10 mM ABA, PhFT5-YFP overexpressors exhibited a higher germination rate compared to Col-0 (Fig. 6a). In addition, the expression of PhFT5-YFP restored the phenotype of mft-3 (Figs 6b and S6). Regardless of the background genotypes, the germination rate of PhFT5-YFP overexpressors correlated with the expression level of PhFT5-YFP (Figs 6b and S6). To further determine the possible role of PhFT5 in the regulation of seed germination in moso bamboo, we examined the expression of PhFT5 during seed germination in the presence or absence of ABA in moso bamboo. As shown in Fig. 6c, the expression of PhFT5 was slightly reduced after imbibition in the absence of exogenous ABA, where 40.3% seed germination was induced (Fig. S7). On the other hand, it was up-regulated and peaked at 2 days after imbibition in the presence of exogenous ABA (Fig. 6c), where seed germination was impaired (Fig. S7). This results indicated that PhFT5 plays a conserved function in moso bamboo similar to MFT in Arabidopsis 26 .
The expression pattern of other moso bamboo PEBP family genes was further tested in moso bamboo. Similar to the expression of PhFT5, transcript levels of PhFT1, 2 and 3 are higher in moso bamboo seeds than in leaves (Fig. S8). However, different from PhFT5, PhFT1, 2 and 3 are expressed stably irrespective of ABA condition (Fig. S9). Interestingly, Arabidopsis PhFT1 overexpressor exhibited significantly low germination rate compared to wild type Col-0 in the presence of ABA (Figs 6d and S10), whereas the overexpressors of other PEBP family genes except for PhFT5 showed wild type response (Fig. 6d). Notably, PhFT1 overexpressor did not exhibit www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ obvious phenotype in the absence of ABA (Fig. S5), suggesting the potential of PhFT1 as positive regulator of ABA mediated inhibition of seed germination.

Discussion
The function of PEBP genes in moso baboo is conserved. In this study, 5 PEBP family genes from moso bamboo database have been characterized and named as as PhFT1-5 (Table S1). The number of PEBP family genes in moso bamboo is less than rice (19) 9 , maize (25) 46 and poplar (9) 47 , and similar as Arabidopsis (6) 15,21 . The numbers of PEBP family members are different in various species. But, generally, the numbers of PEBP family members in monocots are 3-4 times higher than that in dicots 48 . For example, there are 19 members of PEBP family members in rice 9 , although Arabidopsis has only 6 PEBP family members 23 . Our blast search identified only 5 PEBP genes from moso bamboo database, regardless of the fact that both moso bamboo and rice belong to the monocotyledonous grass family and the genome size of moso bamboo is much larger than that of rice (Fig. 1). The reason why moso bamboo has much lower number of PEBP genes is still not clear. Moso bamboo might have eliminated non-functional PEBP family members to develop sophisticated system during its evolution. Similar situation is also found in other species. For example, the biological function of RCN3, one of rice TFL-like genes, has not been detected in rice 32 . Thus, moso bamboo might have eliminated non-functional PEBP family members to develop sophisticated system during its evolution.
The most of reported PEBP family members had conserved amino acid sequence 49 . Phylogenetic analysis identified PhFT1, PhFT2 and PhFT3 as TFL1-like gene, PhFT4 as FT-like gene and PhFT5 as MFT-like gene (Figs 1 and 2). The PEBP family member from moso bamboo and other species exhibited high sequence homology (Figs 1, 2 and S1). Importantly, Tyr 85 in AtFT and His 88 in AtTFL1 28 that distinguish FT-like from TFL-like are conserved (Fig. 2). These results suggest that FT-like gene and TFL1-like gene obtained share conserved protein sequences in moso bamboo. FT and TFL1 homologs play flowering induction and repression function in angiosperms, respectively 50 , such as in rice 31,32,51 , poplar 52-54 , soybean [55][56][57][58] , pea 59-61 , grapevine 10,62 , kiwifruit 63 , rose 64,65 and so on. Consistently, PhFT4 promotes flowering in Arabidopsis, but PhFT1, PhFT2 and PhFT3 inhibit it (Fig. 4b,c). In addition, PhTFL1-like genes also regulate floral organ development in Arabidopsis that is consistent with TFL1 homologs in Arabidopsis 66 , Lombardy poplar 47 , Gentiana 67 , Bambusa oldhamii 43 , cucumber 68 , Chrysanthemum morifolium 69 , but PhFT4 does not (Fig. 5). Thus, these amino acid residues are also determinant for FT-like and TFL-like functions in moso bamboo. The MFT-like PhFT5 gene also has activity to promote flowering in a heterologous system, albeit weakly (Fig. 4b,c), and promote seed germination in Arabidopsis (Fig. 6a,b) that is similar with AtMFT's function in Arabidopsis 26,70 . Thus, it is very likely that the regulation of flowering and seeds germination through PEBP family genes in moso bamboo is conserved.
It has been proposed that FT-like can form a protein complex with FD that is a transcription factor, while TFL1-like can interact with 14-3-3 protein, in SAM to promote and inhibit flowering, respectively 23,71,72 . We found three FD homologues and seven 14-3-3 homologues in moso bamboo (Tables S4 and S5). Some of these FD and 14-3-3 like proteins contain SAP motif (R/KXX-pS/TXP) or SAP-like motif (RXXSTQF) (Figs S11 and S12), which are necessary for their physical interaction [71][72][73] . Actually, key amino acid residues, which comprise the interface for their interaction, are highly conserved (Figs S11 and S12). In addition, SAP motif and 14-3-3 recognition motifs are highly conserved in FT-like and TFL1-like sequences from moso bamboo (Fig. 2). Therefore, we speculate that PhFT4, and moso bamboo TFL1-like genes also form complex with these proteins to regulate flowering, although this regulatory mechanism should be experimentally examined in moso bamboo in the future. Although the molecular mechanisms how MFT-like gene mediate seed germination are currently unknown, PhFT5 may act as co-regulator that modify the transcription factor activity since lack DNA-binding domain as is the case with FT-like and TFL1-like genes since AtMFT regulates the transcription of ABA INSENSITIVE 5, EARLY METHIONINE-LABELLED 6 and RESPONSIVE TO DESICCATION 29A to induce seed germination 26 . Thus, it is a possibility that the molecular mechanism of PEBP family genes on regulation flowering and seed germination are conserved.
Since the vegetative phase can last several decades and data from direct studies of bamboos are limited, the regulatory events leading to phase transition and induction of flowering is largely unknown. Previous reports have speculated that endogenous factor such as circadian clock has an important effect on synchronizing flowering in bamboo 74 . In addition, phytohormones also regulate flowering time 53 . It has been discovered by in vitro tissue culture that cytokinins and auxin play positive and negative role to induce flowering, respectively [75][76][77][78] . Besides, salicylic acid and gibberellin, are also slightly effective to induce flower formation 79 . On the other hand, exogenous factors such as temperature, severe stress, drought and nitrogen concentration affect bamboo flowering [80][81][82] . Whether these endogenous and exogenous signals regulate PEBP family gene expression in moso bamboo is currently unknown. However, cis-elements associated with some of these signals are found on the promoter region of PEBP family genes. For example, the PhFT4 promoter region contains cis-elements associated with drought response and a putative binding site of FLC, a critical transcription factor regulating vernalization (Table S3) 83 . The promoter regions of TFL-like genes also contains many putative elements related to responses to phytohormone, temperature stress and drought (Table S3), implying that endogenous and exogenous factors may coordinate the transcription of FT-like and TFL1-like genes to induce moso bamboo flowering. Indeed, these expression patterns are robustly changed during transition between vegetative and reproductive phase (Fig. S8).
PhFT1 plays role in seed germination. Moso bamboo TFL1-like genes were shown conserved function on flowering repression (Fig. 4), however, it is surprisingly that PhFT1 overexpressor exhibited significantly reduced seed germination rate (Fig. 6d). This is a first discovery indicating the possible involvement of TFL1-like gene in the regulation of seed germination. It should be noted here that PhFT1 and PhFT5 oppositely regulate seed germination (Fig. 6d). This is reminiscent of the relation between function of FT-like genes and TFL1-like genes, in which they compete each other to form complex with FD 72  www.nature.com/scientificreports www.nature.com/scientificreports/ genes affects seed germination (Fig. 6d), although all PhTFL1-like genes similarly affect flowering time (Fig. 4b,c), suggesting the different action mode of PhFT1 between the regulation of flowering and germination. Whether Arabidopsis TFL1-like genes also regulate seed germination and what the binding partners of TFL1-like proteins to regulate germination are need to be addressed in the future.
Higher expression of PhTFL1-like genes and PhFT5 in seeds compared to leaf tissue also partially represents the functions of PhFT1 and PhFT5 in the regulation of seed germination (Fig. S8). In Arabidopsis, AtMFT plays a role as desensitizer in the negative feedback loop of ABA-inhibited seed germination, because positively acting AtMFT expression is induced by the exogenously applied ABA 26 . PhFT5 is induced by ABA application in moso bamboo seed (Fig. 6c) and PhFT5 promoter contains ABA responsive cis-element (Table S3), suggesting that PhFT5 also constitutes a feedback loop that modifies endogenous ABA sensitivity to regulate embryo growth in moso bamboo. Different from PhFT5, PhFT1 expression is not affected by ABA (Fig. S9). However, the promoter of PhFT1 as well as that of PhFT5 contains cis-elements responsive to other hormone, circadian clock and environmental factors such as light and temperature (Table S3). Thus, PhFT1 and PhFT5 may negatively and positively fine-tune the capability of moso bamboo seed germination in the fluctuating environment in nature.
This study provides clues to understand the function of five PEBP family genes from moso bamboo in the regulation of flowering and seed germination. The findings also highlight the possible importance of PEBP family genes in the reproduction, thereby in the maintenance of moso bamboo forest. However, further studies are required to characterize these PEBP family genes by genetic approaches to understand their roles in flowering and germination in moso bamboo.

Methods
Sequence alignment and phylogenetic analysis. Full length amino acid sequences of Arabidopsis and rice PEBP family members were used as queries squences to blast against moso bamboo genome (BambooGDB, http://server.ncgr.ac.cn/bamboo/blast.php). The resulting protein sequences with expectation values ≤10 −10 were used as new queries in a second round of blast search with similar search parameters to ensure detection of all orthologs. After removing redundant sequences, the presence of a PEBP domain was confirmed using SMART (http://smart.embl-heidelberg.de/) before subsequent analysis. The phylogenetic tree was constructed using IQ-TREE 1.6.9 84 with the JTT + G4 model, the best-fit model as determined by ModelFinder 85 . Genes used in sequence alignment and phylogenetic analysis listed in Supplemental Table 6.
Seed germination assay. For Arabidopsis, after-ripened seeds were sterilized with 75% (v/v) alcohol for 10 min, and washed once with absolute ethanol for 1 min, then dried in concentrator plus (Eppendorf). For moso bamboo, after-ripened moso bamboo seeds were sterilized with chlorine gas in a vacuum container for 5 hours. After sterilization, at least 50 Arabidopsis or moso bamboo seeds were sowed on 1/2MS (0.8% agar) medium with or without ABA (Sigma-Aldrich) and kept in the dark at 4 °C for 3 days for stratification. Seeds were then transferred to 16 h light/8 h dark photoperiod and 21 °C to examine seed germination. Germination was counted when the radicle emerges.

RNA extraction and RT-qPCR assay. Total RNA was extracted with TIANGEN RNAprep Pure Plant
Kit (DP441) according to the manufacturer's instructions. 1 μg of total RNA was used for reverse transcription with PrimeScript ™ RT reagent Kit (TaKaRa, RR047A). qRT-PCR was performed with GoTaq ® qPCR Master Mix (Promega) on a QuantStudio ™ 6 Flex Real-Time PCR System. A 40-cycle two-step amplification protocol was used for all measurements. The qPCR signals were normalized to that of the reference gene PhUBQ using the ΔCT method. All experiments incorporated three technical replicates and biological replicates. The primer sequences are listed in the supplementary material (Table S2).
PhPEBPs gene cloning and vectors construction. The full-length coding sequences of PhFT1, PhFT2, PhFT3, PhFT4 and PhFT5 were amplified by PCR from cDNA of the moso bamboo seedling with gene specific primers. The amplified PCR products were cloned into pDONR207 vector (Invitrogen) by BP reaction of the Gateway technology (Invitrogen), and then transferred into pEarlyGate 101 binary vector (Invitrogen) by LR reaction (Invitrogen). Primersequences used for plasmid construction are given in Table S2. The obtained binary vectors were transformed into Arabidopsis by Agrobacterium-mediated flower dip method, and resultant transgenic Arabidopsis were screened with 1% (v/v) basta on 1/2 MS (0.8% agar) medium.
Statistical analysis. Statistically significant differences were computed based on Student's t-tests. Published: xx xx xxxx