Bamboo Flowering from the Perspective of Comparative Genomics and Transcriptomics.

Bamboos are an important member of the subfamily Bambusoideae, family Poaceae. The plant group exhibits wide variation with respect to the timing (1-120 years) and nature (sporadic vs. gregarious) of flowering among species. Usually flowering in woody bamboos is synchronous across culms growing over a large area, known as gregarious flowering. In many monocarpic bamboos this is followed by mass death and seed setting. While in sporadic flowering an isolated wild clump may flower, set little or no seed and remain alive. Such wide variation in flowering time and extent means that the plant group serves as repositories for genes and expression patterns that are unique to bamboo. Due to the dearth of available genomic and transcriptomic resources, limited studies have been undertaken to identify the potential molecular players in bamboo flowering. The public release of the first bamboo genome sequence Phyllostachys heterocycla, availability of related genomes Brachypodium distachyon and Oryza sativa provide us the opportunity to study this long-standing biological problem in a comparative and functional genomics framework. We identified bamboo genes homologous to those of Oryza and Brachypodium that are involved in established pathways such as vernalization, photoperiod, autonomous, and hormonal regulation of flowering. Additionally, we investigated triggers like stress (drought), physiological maturity and micro RNAs that may play crucial roles in flowering. We also analyzed available transcriptome datasets of different bamboo species to identify genes and their involvement in bamboo flowering. Finally, we summarize potential research hurdles that need to be addressed in future research.

Introduction

Flowering is one of the most important adaptations in the evolution of land plants. Numerous studies have been performed on annual, herbaceous model plants from dicotyledonous (Arabidopsis, Antirrhinum) and monocotyledonous (Oryza) groups to identify and characterize important floral pathway genes (Putterill et al., 2004; Colasanti and Coneva, 2009). However, the majority of commercially important plants are perennial and there remains a gap in translating knowledge gained from annual, model plants to perennial plants. Therefore, increasing research attention is being paid to perennial plants. While poplar (Jansson and Douglas, 2007) and white spurge have emerged as model perennial dicotyledonous plants (Anderson et al., 2007), research on perennialism remains elusive in monocots.

Bamboos are an important member of subfamily Bambusoideae, family Poaceae (Kellogg, 2015). Wide variations exist across bamboo species with respect to the flowering time, ranging from annual flowering to flowering after 120 years of vegetative growth (Janzen, 1976). There are even species for which the flowering time is not yet known. Variations in flowering time are not only diverse among species, but also at the population level. For instance, in the case of gregarious flowering all the individuals of a species growing over a wide geographical area bloom within a brief interval of time, and then all die after flowering (Nadgauda et al., 1997; Bhattacharya et al., 2009; Marchesini et al., 2009; Austin and Marchesini, 2012; Chaubey et al., 2013; Xie et al., 2016). In contrast, for sporadic flowering only a few culms of a population flower at a time (Ramanayake and Yakandawala, 1998; Bhattacharya et al., 2006; Xie et al., 2016). Such a wide variation in flowering time and extent indicates that the plant group serves as a repository for a wide range of genes and expression patterns that support such a life style. The ecological consequences of bamboo flowering, such as changes in dynamics of neighboring plant populations (Sertse et al., 2011), and impacts on endangered animals that depend on bamboo shoots (Reid et al., 1991; Azad-Thakur and Firake, 2014) have been topics of active research over decades. In comparison, the molecular aspects of bamboo flowering remain at a nascent stage. Studies have been conducted to characterize a limited number of flowering genes in different bamboo species such as MADS18 from Dendrocalamus latiflorus (Bo et al., 2005), FLOWERING LOCUS T (FT) from P. meyeri (Hisamoto et al., 2008), TERMINAL FLOWER 1 (TFL1) like gene from Bambusa oldhamii (Zeng et al., 2015), FRIGIDA (FRI) from P. violascens (Liu et al., 2015), MADS1 and MADS2 from P. praecox (Lin et al., 2009), 10 genes related to floral transition and meristem identity in D. latiflorus (Wang et al., 2014) and 16 MADS box genes from B. edulis (Shih et al., 2014). Such targeted approaches are being complemented by high-throughput approaches, namely, de novo transcriptome sequencing and suppression subtractive hybridization (Lin et al., 2010; Liu et al., 2012; Zhang et al., 2012; Peng et al., 2013; Gao et al., 2014; Ge et al., 2016; Wysocki et al., 2016; Zhao et al., 2016).

The main aim of this article is to consider the current status of molecular understanding of bamboo flowering from the perspective of comparative genomics and transcriptomics. We queried the only sequenced genome of a temperate bamboo, P. heterocycla syn. P. edulis, to identify marker genes in established floral pathways (e.g., photoperiodic, vernalization, hormonal, and autonomous) and the influence of additional factors such as drought stress and physiological maturity. P. edulis is a diploid, temperate bamboo with chromosome number 2n = 48 and having a genome size of 2.075 Gb (Gui et al., 2007; Peng et al., 2013). In addition, we also explored transcriptome datasets of available bamboo taxa to assess their possible role in bamboo flowering. Finally, we have identified challenges that need to be overcome to understand what triggers bamboo flowering, the genetic controls of flowering, and the effects of gregarious monocarpic flowering cycles on bamboo evolution.

Bamboo genes related to established flowring pathways

Depending on the nature of environmental or endogenous cues, flowering pathways can be broadly classified into vernalization (cold responsive), photoperiodic (day length responsive), autonomous (endogenous factors) and hormonal pathways.

Vernalization pathway

In the model monocot Oryza the important vernalization genes are VERNALIZATION 1 (VRN1), VERNALIZATION INSENSITIVE LIKE 2, and 3 (VIL 2, 3). An additional vernalization sensitive gene VRN2 was isolated from Triticum (Dubcovsky et al., 2006), while its Brachypodium homolog BdVRN2L is vernalization insensitive (Ream et al., 2014). BLAST analyses have identified multiple copies of OsVRN1, OsVIL2, and OsVIL3 homologs in P. heterocycla genome, but the homolog of VRN2 remained undetected (Table 1). In order to understand their possible involvement in bamboo flowering, all available floral transcriptomes were searched. VRN1 was detected in the shoot tissue specific EST library of B. oldhamii (Lin et al., 2010), while VIN3 was identified from the floral transcriptomes of P. heterocycla (Peng et al., 2013) and D. latiflorus (Zhang et al., 2012). Another important vernalization gene, At.FLC, performs cold-mediated suppression of the floral activator At.FT during the seasonal transition from fall to winter (Michaels and Amasino, 1999). However, during prolonged cold exposure in winter, FLC activity is gradually down-regulated by VRN1, VRN2, and VIN3 so that flowering is delayed until spring (Levy et al., 2002; Sung and Amasino, 2004). It was believed that FLC-like genes are absent in monocot plants (Choi et al., 2011), but recently two major FLC clades, namely, MADS37 and MADS51 genes, were identified in the temperate grass Brachypodium distachyon (Ruelens et al., 2013). Our BLAST analyses, however, could not detect MADS37 or MADS51 homologs in P. heterocycla at the set criterion of e−40, identity ≥50% and length coverage ≥60% of the query sequence (Table 1).

Table 1

Identification of important flowering gene homologs in the model temperate grass- .

Flowering pathways/regulator	Genes	O. sativa identifiers used as query	BLAST hits in B. distachyon	BLAST hits in P. heterocycla
Vernalization	VRN1	Os03g54160	Bradi1g08340Bradi1g59250	PH01000606G0250PH01000222G1190
	VIL2	Os12g34850	Bradi4g05950Bradi2g36237	PH01000006G3670
				PH01000674G0720
				PH01000258G0590PH01001556G0190
	VIL3	Os02g05840	Bradi3g04140	PH01000836G0140
			Bradi1g33450	PH01000114G1300PH01002795G0050
	FLC/MADS37	n.f.c	Bradi3g41297	No hit
	FLC/MADS51	Os01g69850	Bradi2g59191	No hit
		n.f.c	Bradi2g59119	No hit
Photoperiod	PHY A	Os03g51030	Bradi1g10520Bradi1g10510Bradi1g08400	PH01000222G1330PH01000606G0390
	PHY B	Os03g19590	Bradi1g64360Bradi1g08400	PH01000013G2240PH01000013G2230PH01000222G1330PH01000606G0390
	CRY 1	Os02g36380	Bradi3g46590Bradi5g11990Bradi3g49204	PH01000349G1020PH01000968G0540PH01002373G0140PH01000263G1210PH01002304G0120
	CRY2	Os02g41550	Bradi3g49204Bradi5g11990Bradi3g46590	PH01000968G0540PH01000349G1020PH01002304G0120PH01002373G0140PH01002304G0180
	CCA1	Os08g06110	Bradi3g16515	PH01001283G0510PH01000383G0300
	ELF 3	Os01g38530	Bradi2g14290	PH01000391G0450PH01000410G0960
	ELF 4	Os11g40610	Bradi4g13227Bradi1g60090	PH01002557G0050
	TOC 1	Os02g40510	Bradi3g48880	PH01003618G0130PH01000345G0790
	COP 1	Os02g53140	Bradi3g57667	PH01000928G0310PH01000311G0870
	FKF 1	Os11g34460	Bradi4g16630Bradi1g33610Bradi3g04040	PH01002958G0010PH01000114G1110PH01000836G0340PH01002213G0250PH01007024G0030
	ZTL	Os06g47890	Bradi1g33610Bradi3g04040Bradi4g16630	PH01007024G0030PH01002213G0250PH01000836G0340PH01000114G1110PH01002958G0010
	CO	Os06g16370	Bradi1g43670Bradi3g56260	PH01005551G0030
	GI	Os01g08700	Bradi2g05226	PH01002142G0290PH01001722G0270
Autonomous	FCA	Os09g03610	Bradi4g08727	PH01002230G0270
	FY	Os01g72220	Bradi2g60817	PH01001355G0380PH01002367G0110PH01002367G0090
	FLD	Os04g0560300	Bradi5g18210Bradi3g58720	PH01000272G0440
	FPA	Os09g0516300	Bradi4g35250	PH01000191G0930
	FVE	Os01g0710000	Bradi2g47940	PH01000048G0850PH01000241G0710
	LD	Os01g70810	Bradi2g59937	PH01006816G0010
	FLK	Os12g40560	Bradi4g02690Bradi1g14320	PH01000025G1210
Gibberellic acid	GA1	Os02g17780	Bradi2g33686	PH01000557G0660PH01002827G0080PH01004049G0170
	KAO	Os06g02019	Bradi1g51780Bradi1g30807Bradi5g00467Bradi4g05240	PH01000083G0900PH01003454G0070PH01000246G0620
	GA2ox1	Os05g06670	Bradi2g34837Bradi2g12440	PH01000685G0370
	GA2ox2	Os01g22910	Bradi2g12440Bradi2g34837	PH01000685G0370
	GA2ox3	Os01g55240	Bradi2g50280Bradi2g19900Bradi2g16750Bradi2g16727Bradi2g32577Bradi2g06670	PH01000018G1890PH01001124G0470PH01001567G0040PH01000273G0650PH01000274G0980
	GA3ox1	Os05g08540	Bradi2g04840Bradi4g23570	PH01002274G0400
	GA3ox2	Os01g08220	Bradi2g04840Bradi4g23570	PH01002274G0400
	GID1	Os05g33730	Bradi2g25600	PH01001316G0350PH01002734G0310
	GID2	Os02g36974	Bradi3g46950	No hit
	GAMYB	Os01g59660	Bradi2g53010	PH01000009G0060PH01000029G1950
Integrator	FT	Os06g06320/Hd3a	Bradi1g48830Bradi2g07070Bradi5g14010Bradi3g48036Bradi2g49795Bradi1g38150Bradi2g19670Bradi4g39730Bradi4g39760Bradi3g08890Bradi4g39750Bradi4g42400Bradi3g44860Bradi5g09270Bradi1g42510	PH01002288G0050PH01001134G0390PH01003363G0220PH01002570G0010
		Os06g06300/RFT1	Bradi1g48830Bradi2g07070Bradi3g48036Bradi5g14010Bradi2g49795Bradi2g19670Bradi3g08890Bradi1g38150Bradi4g39730Bradi4g39760Bradi4g42400Bradi4g39750Bradi4g35040Bradi3g44860Bradi5g09270Bradi2g27860Bradi2g01020	PH01002288G0050PH01001134G0390PH01003363G0220PH01002570G0010PH01007086G0020
	SOC1 /MADS50	Os03g03070	Bradi3g32090Bradi1g77020Bradi3g51800	PH01000759G0450PH01000059G1270PH01000107G0570PH01002152G0120
Drought	Dof12	Os03g07360	Bradi1g73710Bradi3g25670	PH01000113G0300PH01000188G0230PH01000219G0080PH01001264G0440
Physiological maturity	LFY	Os04g51000	Bradi5g20340	No hit
	TFL1	Os11g05470/RCN1	Bradi4g42400Bradi5g09270Bradi3g44860 Bradi1g48830Bradi2g07070 Bradi3g48036Bradi2g49795 Bradi5g14010Bradi2g19670Bradi3g08890Bradi2g01020Bradi1g38150 Bradi4g39730	PH01001134G0390PH01003363G0220PH01002570G0010PH01007086G0020PH01002288G0050
		Os12g05590/RCN3	Bradi4g42400Bradi5g09270Bradi3g44860Bradi1g48830Bradi2g07070Bradi3g48036Bradi2g49795Bradi5g14010Bradi2g19670Bradi3g08890Bradi2g01020Bradi1g38150 Bradi4g39730	PH01001134G0390PH01003363G0220PH01002570G0010PH01007086G0020PH01002288G0050

The criteria used were: e.

Photoperiodic pathway

In the photoperiodic pathway, the circadian rhythm of light and dark periods plays a major role in flower initiation. In Oryza a series of genes that include PHYTOCHROMES A and B (PHYA and PHYB), CRYPTOCHROMES 1 and 2 (CRY1 and CRY2), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), EARLY FLOWERING 4 (ELF4), TIMING OF CAB EXPRESSION 1 (TOC1), CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), EARLY FLOWERING 3 (ELF3), GIGANTEA (GI), FLAVIN-BINDING KELCH REPEAT F BOX 1(FKF1) and ZEITLUPE (ZTL) receive the circadian signal and transfer it to CONSTANS (CO) for further downstream regulation. Our BLAST analyses identified at least one one homologous copy of each of these genes in the queried P. heterocycla genome (Table 1). ESTs homologous to CRY1, CRY2, PHY, FKF1, COP1, ELF3, ELF4, GI, CCA1, and CO were found in the floral transcriptomes of P. edulis, B. oldhamii, and D. latiflorus, suggesting their role in bamboo flower induction (Lin et al., 2010; Zhang et al., 2012; Peng et al., 2013; Gao et al., 2014). The transcriptional expression level of CO varied across libraries. For instance, it was low in P. edulis and correlated with the presence of L1 and GYPSY transposable elements in the regulatory region of the gene (Peng et al., 2013). On the other hand, a high level of CO expression was obtained in the floral tissues of D. latiflorus (Zhang et al., 2012). CO, along with the CCAAT box binding factor (NFY), bind to the CCAAT box of FT promoter and result in flowering (Ben-Naim et al., 2006). Therefore, the co-expression of CO and FT (i.e., CO-FT regulon) plays a crucial role in the regulation of flowering time. Our BLAST analyses identified 5 FT-like and 1 CO-like homologs in P. heterocycla (Table 1). Similarly, single or multiple FT copies have been identified and characterized in D. latiflorus, P. meyeri, and P. violascens (Hisamoto and Kobayashi, 2007, 2013; Hisamoto et al., 2008; Wang et al., 2014; Guo et al., 2015). Detailed expression analysis of PmFT revealed that its expression is primarily restricted to leaves, but highest during full bloom (Hisamoto and Kobayashi, 2013). Expression of the two FT genes and their functional diversification was reported in P. violascens (Guo et al., 2015). PvFT1 is expressed in leaves and induces flowering, while PvFT2 possibly plays a role in floral organogenesis. Another important floral integrator, SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), was identified by our BLAST analyses (Table 1) and was also expressed in the floral transcriptomes of P. edulis, Guadua inermis, Otatea acuminate, Lithachne pauciflora, and P. aurea (Peng et al., 2013; Wysocki et al., 2016).

Autonomous and hormonal pathway

In addition to environmental cues, additional flower inducing factors are present within a plant itself and are called endogenous or autonomous signals. This pathway is well studied in Arabidopsis, but is less characterized in monocot plants (Lee et al., 2005; Abou-Elwafa et al., 2011). The important genes are FLOWERING LOCUS CA (FCA), FLOWERING LOCUS D (FLD), FLOWERING LOCUS KH DOMAIN (FLK), FLOWERING LOCUS PA (FPA), FLOWERING LOCUS VE (FVE), FLOWERING LOCUS Y (FY), and LUMINIDEPENDENS (LD, Simpson, 2004). These genes promote flowering by suppressing FLC expression (Simpson, 2004; Quesada et al., 2005). Our BLAST analyses identified one or more P. heterocycla homologs for the majority of these genes (Table 1), which were reported in the floral transcriptomes of B. oldhamii (Lin et al., 2010), D. latiflorus (Zhang et al., 2012), and P. heterocycla (Peng et al., 2013) and suggest possible roles in bamboo flowering.

The role of gibberellic acid (GA) in the induction of flowering is well established in Oryza (Kwon and Paek, 2016). Many important genes related to GA biosynthesis (ent-KAURENE SYNTHETASE A- GA1, ent-KAURENOIC ACID OXIDASE-KAO, GA 2-OXIDASE-GA2ox, GA3ox) and receptors (GIBBERELLIN INSENSITIVE DWARF1- GID1, GID2) have been characterized (Sakamoto et al., 2004). GID1 and GID2 are responsible for proteasome mediated DELLA degradation and promote flowering through upregulation of GAMYB (Kwon and Paek, 2016). At least one P. heterocycla homolog has been detected for the majority of these genes in our BLAST analyses (Table 1). The possible involvement of GA in bamboo flowering is supported by the identification of GA1, SLY, GID1, GID2, GAMYB ESTs in the floral transcriptome of P. heterocycla (Gao et al., 2014) and D. latiflorus (Zhang et al., 2012).

Possible physiological and genetic factors regulating bamboo flowering

Stress

Increasing evidence suggests a link between stress and bamboo flowering (Rai and Dey, 2012; Peng et al., 2013; Ge et al., 2016). Overall expression level of general stress responsive genes involved in ABA, ethylene, sugar metabolism and Ca+2 dependent signaling pathway were 11.1-fold higher than that of the flowering genes in P. heterocycla (Peng et al., 2013). Particularly, a few members of the DNA binding with one finger (Dof) transcription factor family were highly up-regulated in the floral transcriptome (Imaizumi et al., 2005). For instance, Ph.Dof12 was about 16-fold up-regulated in the flowering tissues of P. heterocycla collected from a drought affected area (Peng et al., 2013). Similarly, 28 unigenes related to Dof3, Dof4, Dof5, Dof12, and Cycling Dof Factors (CDF) were detected in the floral transcriptome of P. edulis (Gao et al., 2014). The Dof family is composed of 15 genes in Phyllostachys and a comprehensive functional characterization of these genes may provide new insights. Particularly, analyzing the enrichment of the drought-responsive cis-elements in their promoter regions could identify candidate genes that are induced under drought conditions.

Physiological maturity and micro RNAs

Scientific evidence emerging from research on various perennial plants suggests an important role of TERMINAL FLOWER 1 (TFL1) and microRNAs (miRNAs) in maintaining a long vegetative phase (Huijser and Schmid, 2011). Our BLAST analyses identified five copies of Ph.TFL1 genes in P. heterocycla (Table 1). A functional TFL1 gene was isolated from B. oldhamii and was overexpressed in Arabidopsis (Zeng et al., 2015). The overexpressed lines showed delayed flowering, suggesting that TFL1 may have a role in maintaining vegetative growth. In addition, TFL1 may have an important function in differentiation of bamboo floral organs, as indicated by higher expression of TFL1 in late floral developmental stages relative to early stages in B. oldhamii and D. latiflorus (Wang et al., 2014).

Long maintenance of the vegetative phase in the majority of bamboos can also be regulated at the post-transcriptional level, such as by miRNAs. In rice miR156 is known to repress flowering by targeting SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SBP/SPL) transcription factor (SPLs, Xiong et al., 2006). Expression of miR156 showed significant down-regulation through the transition from vegetative to flowering stages in P. edulis (Gao et al., 2015). Additional candidates that may have roles are miR164a, miR166a, miR167a, miR535a, miR159a.1, miR164a, and miR168-3-p (Gao et al., 2015; Ge et al., 2016). In contrast, some micro RNAs may play positive roles in bamboo flowering. One such candidate is miR172, which controls flowering time and the formation of floral organs through the regulation of the AP2-like transcription factor (Lee et al., 2014). miR172a showed an increase in expression level during progression from vegetative to the flowering phase in P. edulis (Gao et al., 2015). The expression of other miRNAs such as miR169b, miR395h-5p, and miR529-3p were higher in floral tissues than in vegetative tissues.

Future challenges

Appropriate tissue sampling

Identification of proper tissue stages is critical since the majority of flowering genes are transiently expressed soon before or after floral induction. Unlike Arabidopsis or Oryza, wild bamboo floral tissue stages are not easily traceable. Therefore, tissue culture methods have been tried to induce flowering and to study defined stages of induced floral transcriptomes of B. oldhamii in vitro (Lin et al., 2010). However, this study raised doubt about comparability of the transcription patterns under in vitro conditions vs. naturally occurring flowering. A large unigene set (146,395) generated from the floral transcriptomes of naturally grown D. latiflorus could not detect the important integrator gene FT, although it was detected in the transcriptome of P. edulis. This emphasizes the need to define in vivo floral stages with higher accuracy in order to make data generated by different research groups more comparable. Therefore, we studied the microscopic histology of different flowering stages of wild B. tulda plants and compared them with the external morphology of buds to identify phenotypic markers for specific growth stages (Figure 1). The external morphological features of nodal vegetative buds are indistinguishable from those of early stage inflorescence bud. However, this is one of the most crucial tissue stages with respect to the identification of genes involved in flower induction. Close observation of the early inflorescence bud revealed that it is slightly smaller in size, pale yellow in color, and bulged in the middle (Figures 1A,C). Histological analyses reveal that the shoot apical meristem of the nodal vegetative bud is dome shaped and covered with compactly arranged leaf primordia (Figure 1B). But the early staged inflorescence meristem is slightly smaller in size and triangular in shape (Figure 1D). The middle stage floral bud could be differentiated from the early stage by its elongated shape and bright green color (Figure 1E). Histological analysis revealed that it is composed of one or two floral primordia at the base of the rachis and an undifferentiated inflorescence meristem at the apex (Figure 1F). The late inflorescence bud is easily identifiable from all the other stages by its long and slender shape (Figure 1G). It is composed of three to four visible florets having differentiated anther primordia at the base of the rachis and an undifferentiated apical inflorescence meristem (Figure 1H).

Figure 1

Important vegetative and floral developmental stages External morphology of nodal vegetative bud (~0.6 × 0.7 cm in dimension); (B) Longitudinal section (L.S.) of vegetative bud. The shoot apical meristem (SAM) is dome shaped (marked with arrow); (C) External morphology of an early stage inflorescence bud (~0.3 × 0.3 cm in dimension); (D) LS of the early stage inflorescence bud having triangular inflorescence meristem (marked with arrow); (E) External morphology of middle stage inflorescence bud (~0.8 × 0.5 cm in dimension); (F) LS of middle stage inflorescence bud showing differentiated floral primordia (marked with arrow); (G) External morphology of late stage inflorescence bud (~1.2 × 0.6 cm in dimension); (H) LS of late stage inflorescence bud having differentiated anther primordia (marked with arrow).

Gene family expansion, high sequence homology and associated challenges

Bamboos are highly polyploid plants with big genomes (2075 Mb for P. heterocycla compared to 125 Mb for A. thaliana). Consequently, the majority of genes are present in multiple copies. It would be important to dissect their evolutionary origin (orthologs-functional, paralogs-old/recent vs. tandem duplicates) and deduce their functional conservation or divergence by studying detailed transcriptional expression patterns (Das et al., 2016). However, the majority of these genes are very similar in sequence, which creates challenges in maintaining specificity in gene expression analyses. Example of this are FT and TFL1 genes, which are members of the Phosphatidylethanolamine-binding protein (PEBP) family and share high sequence similarity (>60%). However, they are functionally antagonistic to each other. There are diagnostic amino acids, which are crucial to maintain either FT (Tyr-85) or TFL1 (His-88) function (Hanzawa et al., 2005). Our BLAST analyses identified five P. heterocycla homologs each for FT and TFL1 and they are completely overlapping with each other (Table 1). Follow-up analysis indicated PH01002288G0050 as the predicted FT gene, while the other four, PH01001134G0390, PH01003363G0220, PH01002570G0010, PH01007086G0020 are TFL1. Therefore, in addition to large-scale sequence analyses such as BLAST, individual gene sequences should be checked for correct gene function annotation.

Genetic tools for functional validation

With the completion of gene sequencing and expression pattern characterization, the next challenge would be to confirm gene functions using loss- or gain-of-function mutants. This is especially important for multi copy genes for which expression data is not indicative of functional differentiation among copies. Therefore, a model plant is needed in which tissue culture and genetic transformation are easy to perform. Woody bamboos are generally recalcitrant and present several challenges (Das and Pal, 2005a). Since loss-of-function mutation analyses would be challenging, other model plants could be exploited to perform genetic complementation analyses by ectopically expressing bamboo flowering genes. Rice could be useful for such purposes due to its close evolutionary relationship, related floral biology and availability of mutant lines for several genes. However, many rice genes and associated mutant phenotypes have yet to be characterized.

Development of a new model system for tropical bamboo

The majority of available research reports are on the tetraploid bamboo Phyllostachys, predominantly found in the temperate regions of China and Japan. However, enormous biodiversity is found in the tropical regions and dominated by members of the genus Bambusa. Therefore, the genome/transcriptomes of a tropical bamboo should be characterized. These have enormous economic importance, a large population size, wide genetic diversity (Das et al., 2008), molecular methods for species level identification (Das et al., 2005), a standardized micropropagation protocol (Das and Pal, 2005b), incidents of both gregarious (Mohan Ram and Harigopal, 1981) and sporadic flowering (Bhattacharya et al., 2006), which taken together makes B. tulda a good choice as a model species of tropical bamboos.

Author contributions

MD and AP collaborated in this study. PB, SC, and SD had done the bioinformatics and histological analyses. MD wrote the paper with input from all co-authors.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.