A precise regulation of flowering time is central to plant species survival. Therefore, mechanisms have evolved in plants to integrate various environmental cues to optimize flowering time. In this study, we show that the product of the wheat gene TaFT, which integrates photoperiod and vernalization signals promoting flowering, interacts with bZIP proteins TaFDL2 and TaFDL6. We also show that TaFDL2 can interact in vitro with five ACGT elements in the promoter of the meristem identity gene VRN1, suggesting that TaFDL2 is a functional homologue of Arabidopsis FD. No direct interactions between the TaFT protein and the VRN1 promoter were detected. Transgenic wheat plants over-expressing TaFT showed parallel increases in VRN1 transcripts, suggesting that TaFT provides transcriptional activation of VRN1, possibly through interactions with the TaFDL2 protein. The same transgenic plants also showed increased transcript levels of TaFT2 (a TaFT paralogue), indicating that TaFT2 acts downstream of TaFT. The fact that TaFT2 interacts with another bZIP protein TaFDL13, which lacks the ability to interact with the VRN1 promoter, suggests that TaFT and TaFT2 have different gene targets. This observation agrees with the functional divergence observed for the TaFT and TaFT2 orthologous genes in rice. The temperate cereals analyzed so far show VRN1 transcripts in the leaves, a characteristic not observed in Arabidopsis or rice. The high levels of TaFDL2 transcripts observed in wheat leaves provide a simple explanation for this difference. We present a hypothesis to explain the conservation of VRN1 expression in the leaves of temperate cereals.
The precise control of flowering is central to plant reproductive success and species survival. Optimization of flowering time to maximize grain yield is also an important target in cereal breeding programs. Plants have evolved mechanisms to integrate various environmental signals, including photoperiod and vernalization, to enable them to flower under conditions that optimize seed production. These environmental signals are perceived by separate parts of the plant, and their integration requires precise spatial and temporal coordination. Photoperiod, for example, is perceived by the leaves, whereas cold is perceived directly by the shoot apical meristem (SAM) (Bernier, 1988).
FT is the primary target of CONSTANS (CO), a B-box zinc finger CCT protein that plays a central role in the photoperiod pathway (Putterill et al., 1995; Robson et al., 2001; Wigge et al., 2005). When Arabidopsis plants are exposed to long days (LD), CO activates FT in the leaf vascular tissue (phloem) (An et al., 2004). The FT protein then moves through the phloem to the SAM, where it interacts with the bZIP transcription factor FD to activate expression of the MADS box meristem identity gene APETALA 1 (AP1) (Abe et al., 2005; Corbesier et al., 2007; Wigge et al., 2005). The FT–FD protein interaction is spatially regulated by the preferential expression of FD in the SAM, and temporally by the integration of various environmental signals that converge to regulate FT (Wigge et al., 2005). According to this model, FD provides specificity in recognition of the DNA target, and FT acts in concert with FD to transcriptionally activate AP1 (Wigge et al., 2005).
In the temperate cereals, the role of the FT homologues seems to be similar to the one described above for Arabidopsis. Over-expression of TaFT (Triticum aestivum L.) in transgenic wheat plants significantly accelerates flowering relative to non-transgenic controls, suggesting a conserved role as flowering promoter (Yan et al., 2006). In addition, both TaFT and HvFT (Hordeum vulgare L.) are up-regulated by long days, and increased transcript levels correlate with accelerated flowering times (Turner et al., 2005; Yan et al., 2006). Arabidopsis and the temperate cereals differ in the spatial transcription profile of the main targets of FT, AP1 in Arabidopsis and the homologous VRN1 in temperate cereals. In wheat and barley, VRN1 is normally expressed in the leaves at high levels (Schmitz et al., 2000; Yan et al., 2003), whereas, in Arabidopsis, AP1 transcripts are either not detected in the leaves or are present at very low levels (e.g. in the vascular tissues of cotyledons, Abe et al., 2005). Interestingly, ectopic expression of FD in 35S::FD transgenic Arabidopsis plants results in high expression of AP1 in the leaves (Wigge et al., 2005). Based on this result, we hypothesize that a wheat functional homologue of Arabidopsis FD, designated TaFD-like (TaFDL, hereafter), will be normally expressed in leaves.
Understanding of the role of TaFT in regulation of flowering in the temperate cereals is complicated by the existence of a similar (78% identical) paralogous copy in both wheat (TaFT2) and barley (HvFT2) (Faure et al., 2007; Yan et al., 2006). As the TaFT/TaFT2 duplication occurred after the divergence between the grasses and Arabidopsis, this duplication is independent of the FT/TSF (twin sister of FT) duplication in Arabidopsis.
In this study, we provide evidence that TaFT2 is regulated by TaFT and that the products of these genes interact with different TaFDL partners. We also show that only one of the TaFDL proteins that interact with TaFT was able to interact with the VRN1 promoter. The expression of this particular TaFDL gene in the leaves provides a simple explanation for the presence of VRN1 transcripts in the leaves of temperate cereals. Finally, we discuss a hypothesis for the putative role of VRN1 expression in the leaves of temperate cereals.
FD-like genes are transcribed in wheat leaves
Using cDNA samples from leaves of hexaploid wheat Chinese Spring at the five-leaf stage and primer pairs for 16 TaFDL genes (Table S1), we were able to amplify five FDL genes. The cDNA sequences of these genes and their predicted protein sequences were deposited in GenBank with the following accession numbers as indicated in parentheses: TaFDL2 (EU307112), TaFDL3 (EU307113), TaFDL6 (EU307114), TaFDL13 (EU307115) and TaFDL15 (EU307116). This result confirmed that at least some TaFDL genes are transcribed in the leaves.
A multiple sequence alignment of the five predicted wheat FD-like proteins and the Arabidopsis proteins FD and FDP is presented in Figure 1. The alignment shows a conserved region of 69 amino acids without gaps, including perfectly conserved bZIP domains (N-X7-R-X9-L-X6-L-X6-L). The regions outside this 69 amino acid region were not well conserved among the various proteins and were excluded for the tree construction (Figure 1).
The TaFT protein interacts with the bZIP proteins TaFDL2 and TaFDL6
Yeast two-hybrid assays were used to test the interactions between TaFT and the various TaFDL proteins (Table S2). We first performed auto-activation tests and confirmed that TaFT was not able to activate the reporter genes when used alone as bait or prey. The TaFT bait construct was then co-transformed into yeast with individual prey constructs for TaFDL2, TaFDL3, TaFDL6, TaFDL13 or TaFDL15.
The two proteins used as positive controls, murine p53 and SV40 large T-antigen (Iwabuchi et al., 1993; Li and Fields, 1993), showed the expected interaction on SD -Leu -Trp -His -Ade medium (Figure 2b). Under the same selective conditions, TaFDL2 and TaFDL6 also showed very strong interactions with the TaFT bait (Figure 2b). A TaFDL6 truncation construct carrying only the bZIP domain showed a weaker interaction with the TaFT bait than the full-length protein (data not shown). These interactions were validated using reverse bait/prey constructs. When used as bait, both TaFDL2 and TaFDL6 showed strong interactions with co-transformed TaFT prey construct (data not shown).
TaFT2 protein interacts with TaFDL13
We also investigated the interactions between TaFDL proteins and TaFT2. Auto-activation tests confirmed that TaFT2 was not able to activate the reporter genes when used alone as bait or prey or in combination with TaFT (Figure 3). Interestingly, co-transformation of TaFT2 bait with TaFDL2, TaFDL3, TaFDL6, TaFDL13 and TaFDL15 prey constructs showed different results from those observed with TaFT. Whereas TaFT showed strong interactions with TaFDL2 and TaFDL6, TaFT2 interacted only with TaFDL13 among these five FD-like proteins (Figure 3). This interaction was also validated using the reverse bait/prey combination, in which TaFDL13 was used as the bait construct and TaFT2 as prey (data not shown).
TaFDL2 protein binds to bZip binding sites in the VRN1 promoter in vitro
Members of the bZIP transcription factor family exhibit DNA binding specificity to DNA motifs with an ACGT core sequence, including the A-box (TACGTA), G-box (CACGTG) and C-box (GACGTC) (Foster et al., 1994; Izawa et al., 1993), and hybrid C-box/G-box motifs (Martinez-Garcia et al., 1998). In Arabidopsis, a C-box present in the AP1 promoter has been suggested as the binding site for interaction between the FT–FD protein complex and the AP1 promoter (Wigge et al., 2005). As this is a critical step in initiation of the flowering cascade, we investigated whether the same interaction can occur in wheat.
The VRN1 (the AP1 homologue) promoter region in Triticum monococcum most likely does not extend beyond 2.3 kb upstream from its start codon, as an uninterrupted 67 kb stretch of nested retro-elements was found upstream from this region (AY188331). Within this 2.3 kb region, we found a G-box and four putative hybrid boxes (Figure 4).
Four segments of the VRN1 promoter, each including one or two of the five putative binding sites (presented to scale in Figure 4), were radiolabeled as DNA probes. The purified bZIP proteins TaFDL2, TaFDL6 and TaFDL13, which showed interactions with either TaFT or TaFT2 in the yeast two-hybrid assays, were used with the radiolabeled DNA probes in an electrophoretic mobility shift assay (EMSA). Among these three bZIP proteins, only the TaFDL2 protein was able to bind to the putative bZIP binding sites in the TaVRN1 promoter, and none of them was able to bind to the CArG box located approximately 160 bp upstream from the VRN1 start codon (Figure 4). To determine the binding specificity of TaFDL2, oligonucleotides including either four copies of the wild-type bZIP binding sequence or a mutant version in which the ACGT core sequences were replaced by AATT (Table S3) were tested in binding assays and competition experiments. As shown in Figure S1A, TaFLD2 binds specifically to the wild-type bZIP sequence in a concentration-dependent manner, and is out-competed by an excess of unlabeled cold oligonucleotides. Furthermore, mutation of ACGT to AATT in the bZIP binding sequence completely abolishes the interactions (Figure S1B).
In a separate EMSA, five overlapping segments covering the complete (2.3 kb) VRN1 promoter region were used as probes in binding reactions with TaFT protein. No interactions were observed (data not shown).
Spatial and temporal localization of TaFDL2 transcripts
Our yeast two-hybrid assays and EMSA confirmed that the wheat protein TaFDL2 interacts with TaFT and with the VRN1 promoter in a similar way as described for the homologous FT and FD proteins in Arabidopsis. Therefore, to understand the spatial and temporal regulation of VRN1 in wheat, it is important to know the spatial and temporal transcription profiles of TaFDL2. For comparative purposes, we also tested TaFDL6 and TaFDL13 transcript levels.
Abundant TaFDL2 transcripts were detected by RT-PCR in RNA samples extracted from leaves of vernalized and unvernalized plants of the common winter wheat variety Jagger (Figure 5). Two RNA samples from the vegetative and early reproductive SAM (including a small portion of the crown) extracted from Chinese Spring plants also showed abundant TaFDL2 transcripts. RT-PCR analyses for the other TaFDL genes showed that TaFDL6 transcript levels were even higher than those of TaFDL2 in all these tissues, whereas TaFDL13 transcripts were more abundant in the apices than in the leaves (Figure 5).
A quantitative RT-PCR analysis of cDNA samples from leaves further confirmed the high transcript levels of TaFDL2. We used available cDNA samples from spring wheat line Chinese Spring (CS) and the CS (Hope 7B) chromosome substitution line (dominant TaFT allele), previously characterized for TaFT and VRN1 (Yan et al., 2006). We found high levels of TaFDL2 transcripts in all the samples (close to those of ACTIN). We detected no significant differences in TaFDL2 transcript levels between the two lines, or during the 6 weeks of vernalization at 4°C (Figure S2). Plants kept at room temperature showed an approximately twofold increase in TaFDL2 transcript levels during the 6 weeks, but the differences were not significant (Figure S2). In our previous study, these same samples showed significant increases in TaFT and VRN1 transcript levels during development (in both vernalized and unvernalized plants), and higher transcript levels in CS (Hope 7B) than in CS (Yan et al., 2006).
Transcript levels of TaFT and VRN1 are correlated
We have shown that TaFDL2 transcripts are abundant in both leaves and apices, and do not seem to be a limiting factor in the induction of VRN1. Therefore, if the interactions described above between TaFDL2, TaFT and the VRN1 promoter also occur in planta, modifications in the transcript levels of TaFT should be paralleled by the VRN1 transcript levels. To test this prediction, we used two independent transgenic hexaploid wheat lines Tr-1 and Tr-2, generated in a previous study using the winter variety Jagger (Yan et al., 2006). These transgenic plants exhibit increased levels of TaFT transcripts as a result of insertion of a dominant TaFT allele for spring growth habit from the variety Hope (Yan et al., 2006).
TaFT transcript levels in the leaves of unvernalized plants from both transgenic lines (LD photoperiod) were significantly higher than in the non-transgenic sister lines or Jagger, at the two developmental stages tested (Figure 6). Transcript levels at the fifth-leaf stage were approximately fivefold higher than at the first-leaf stage, and more than eightfold higher than those of ACTIN. The VRN1 transcript levels paralleled those of TaFT, with significantly higher VRN1 transcript levels in transgenic plants than in the non-transgenic controls (Figure 6), and higher transcript levels at the fifth-leaf stage than at the first-leaf stage. In this experiment, non-transgenic control plants took approximately 124 days from sowing to heading, whereas Tr-1 and Tr-2 plants headed on average 46 and 44 days, respectively, after sowing, approximately 80 days earlier than the non-transgenic control.
A 40-fold (first-leaf stage) to 500-fold (fifth-leaf-stage) increase in TaFT2 transcript levels was observed in the TaFT transgenic lines relative to the controls, suggesting that TaFT2 may be regulated directly or indirectly by TaFT. In the non-transgenic plants, the transcript levels of TaFT2 were 20–100-fold lower than those of TaFT.
bZIP transcription factors
The bZIP transcription factors are characterized by a basic region that binds DNA and a leucine zipper dimerization motif. This family is found in all eukaryotes but has more members in plants than in humans (Homo sapiens) or worm (Caenorhabditis elegans) (Riechmann et al., 2000). Approximately 75 bZIP transcription factors involved in pathogen resistance, light and stress signaling, flower development and seed maturation have been described in Arabidopsis (Jakoby et al., 2002).
The ChIP experiment showed enrichment for the C-box region in the AP1 promoter only under conditions promoting the expression of FT, suggesting that a stable FT–FD–AP1 complex was formed in Arabidopsis (Wigge et al., 2005). The presence of a similar interaction in wheat is indirectly supported by the in vitro interactions (TaFT–TaFDL2 and TaFDL2–VRN1 promoter) described above and by the spatial correlation between the expression of TaFDL2 and VRN1. High levels of TaFDL2 and VRN1 are observed in the leaves of wheat plants expressing TaFT, paralleling the induction of AP1 in the leaves of Arabidopsis when FD is expressed ectopically in this tissue (Wigge et al., 2005).
To explore further whether a similar TaFT–TaFDL2–VRN1 complex could be detected in wheat, we included both TaFT and TaFDL2 proteins in the same binding reaction with the VRN1 promoter segments (Appendix S1). Unfortunately, we failed to observe any supershift relative to the TaFDL2 protein used alone (Appendix S1). These results suggest that either there is no stable TaFT–TaFDL2–VRN1 complex in wheat, or, more likely, that the conditions used in the binding assays were not conductive to the formation of a stable complex. It is also possible that additional proteins are necessary to stabilize this complex. ChIP studies will be necessary to confirm the existence of a TaFT–TaFDL2–VRN1 complex in wheat.
Identification of the wheat FD homologue was not a trivial task due to the limited sequence conservation among bZIP transcription factors, which is generally restricted to the bZIP motif (Figure 1). Therefore, we relied on simultaneous interaction of the TaFDL candidates with TaFT in yeast two-hybrid tests and with the VRN1 promoter in an EMSA to select the best candidate. Only TaFDL2 fulfilled both selection criteria among the five TaFDL genes identified in wheat leaves, making it the best candidate for a functional homologue of Arabidopsis FD. Expression results showed that TaFDL2 transcripts accumulate in the vegetative and reproductive apices (Figure 5), suggesting that the TaFDL2 protein is present in the correct tissue and at the correct developmental stage to be involved in flowering induction once TaFT becomes available.
Duplication and functional differentiation of FT and FT2 genes
Phylogenetic and comparative mapping analyses have shown that orthologues of wheat TaFT and TaFT2 are present in barley and rice, indicating that the duplication that originated these paralogous genes pre-dated the divergence of the grasses (Faure et al., 2007; Yan et al., 2006). The same phylogenetic analyses also showed that the TaFT/TaFT2 duplication occurred after the divergence of the grass species from the dicots. Therefore, this duplication is independent of the FT/TSF (twin sister of FT) duplication in the Arabidopsis lineage (Faure et al., 2007; Yan et al., 2006). Arabidopsis tsf mutations delay flowering and enhance the phenotype of ft mutants, whereas TSF over-expression causes precocious flowering under short days (Michaels et al., 2005; Yamaguchi et al., 2005).
Unfortunately, similarities and differences between Arabidopsis FT and TSF (Michaels et al., 2005; Sung et al., 2006; Yamaguchi et al., 2005) cannot be used to infer the functions of TaFT and TaFT2 in the grasses because independently duplicated genes may have different sub-functionalization. Therefore, the specific roles of the TaFT and TaFT2 genes need to be studied directly in the grass species. Detailed micro-co-linearity studies between the barley HvFT gene region on chromosome arm 7HS and the rice region on rice chromosome arm 6S, including Hd3a (the functional orthologue of Arabidopsis FT), have confirmed that these genes are true orthologues (Yan et al., 2006). However, this relationship is complicated by a recent tandem duplication that occurred only in the rice lineage, resulting in closely linked genes Hd3a (OsFTL2) and RFT1 (OsFTL3) (Izawa et al., 2002; Kojima et al., 2002; Yan et al., 2006). Comparative mapping studies also confirmed that HvFT2, which is located on the short arm of barley chromosome 3H, is orthologous to rice OsFTL1, which is located on the co-linear region of the short arm of rice chromosome 1 (Faure et al., 2007).
In rice, a short-day plant, Hd3a transcript levels are rapidly induced under short days (SD), but this effect can be suppressed by short exposures to light in the middle of the night (night breaks). These characteristics are not observed for other rice FT-like genes (including OsFTL1 and OsFTL3), suggesting that Hd3a is the central gene in the photoperiod pathway in rice (Ishikawa et al., 2005). Recent studies have confirmed that the Hd3a protein moves from the leaves to the apices, where it induces flowering, confirming that it is the functional homologue of Arabidopsis FT (Tamaki et al., 2007).
Whereas over-expression of Hd3a results in early flowering in rice (Kojima et al., 2002), over-expression of OsFTL1 produces more complex phenotypes including elongation of internodes, loss of apical dominance, and a terminal tissue at the apical meristem (Izawa et al., 2002). This terminal tissue is composed of multiple glumes, and occasionally a terminal floret at the tip instead of a panicle, suggesting that OsFTL1 may be involved in regulation of panicle and floral development rather than in the regulation of flowering initiation, which is mainly regulated by Hd3a (Izawa et al., 2002).
The functions described above for Hd3a and OsFTL1 may also apply to the orthologous wheat TaFT and TaFT2 genes. The finding that these wheat paralogues interact with different FD-like proteins provides a putative molecular explanation for functional differentiation. TaFT interacts with the TaFDL2 and TaFDL6 proteins, whereas TaFT2 interacts with TaFDL13, which belongs to a different clade of FD-like proteins (Figure 1). We showed here that the interaction between TaFT and TaFDL2 probably confers on TaFT the ability to regulate VRN1 transcription and therefore to affect the fate of the SAM. In contrast, TaFDL13 (the TaFT2 partner) showed no interactions with the VRN1 promoter. As TaFDL13 is mainly expressed in the apical region, it may provide TaFT2 with specificity for targets expressed in this tissue.
A role for TaFT2 in processes occurring after the SAM reproductive differentiation is also supported by the transcription profiles of HvFT2. The proteins coded by TaFT2 and HvFT2 are 98% identical (excluding the seven initial amino acids), suggesting that they may also share similar structural and functional characteristics. Faure et al. (2007) found that HvFT2 is up-regulated by LD, but this up-regulation occurs 2–3 weeks after differentiation of the SAM. Our results in wheat exhibit a similar trend. The transcript levels of TaFT in the transgenic plants over-expressing TaFT are very high from the first leaf, whereas TaFT2 transcript levels were low at the first-leaf stage and reached high levels at the fifth-leaf stage (Figure 6e,f), suggesting a delayed induction compared with TaFT. In the non-transgenic control plants, the transcript levels of TaFT were 40–70-fold higher than those of TaFT2, also suggesting a more central role for TaFT. More importantly, the strong up-regulation of TaFT2 in the two TaFT over-expressing transgenic wheat lines relative to the control suggests that TaFT2 may be regulated directly or indirectly by TaFT (Figure 6e,f).
Similarities between the Arabidopsis and wheat flowering pathways
FT transcripts in Arabidopsis and wheat are rapidly up-regulated upon transfer of plants to LD (Faure et al., 2007; Imaizumi et al., 2003; Turner et al., 2005; Yan et al., 2006). In addition, over-expression of FT in transgenic plants results in precocious flowering, confirming the conserved role of this gene as a promoter of flowering (Kobayashi et al., 1999; Yan et al., 2006). The observed protein–protein interaction between TaFT and TaFDL2, and the protein–DNA interaction between TaFDL2 and the VRN1 promoter suggest that the molecular interactions downstream of FT are also conserved between wheat and Arabidopsis. These interactions are incorporated into our current model of flowering regulation in temperate cereals (Figure 7).
The model presented in Figure 7 shows the interaction between TaFDL2 and the G-box and hybrid boxes in the VRN1 promoter (Figure 4). A similar protein–DNA interaction has been previously identified in Arabidopsis, in which FD binds to a 130 bp region of the AP1 promoter including a C-box motif (Wigge et al., 2005).
In this study, we observed a close correlation between VRN1 and TaFT transcript levels in transgenic wheat plants over-expressing TaFT. Lower transcript levels of TaFT in the transgenic plant Tr-1 relative to Tr-2, and at the first-leaf stage compared with the fifth-leaf stage (Figure 6a,b) were paralleled by similar differences in VRN1 transcript levels (Figure 6c,d). More importantly, VRN1 transcript levels were 500–1000-fold higher in the transgenic wheat plants over-expressing TaFT than in the non-transgenic control plants. Additional experiments in Arabidopsis showed that addition of a strong transcriptional activation domain to FT increases its flowering promoting activity (Wigge et al., 2005). Taken together, these results suggest that TaFT is a limiting factor in the activation of VRN1.
As we detected no binding between the TaFT protein and the VRN1 promoter by EMSA (data not shown), it is unlikely that TaFT can regulate transcription of VRN1 through direct protein–DNA interaction. These results, combined with the observed yeast two-hybrid interaction between TaFDL2 and TaFT (Figure 2) and the TaFDL2 interaction with the VRN1 promoter, suggest a model in which TaFT and TaFDL2 act in concert to regulate the transcription of VRN1 (Figure 7).
Differences between the Arabidopsis and wheat flowering pathways
Although conservation of the last steps of the photoperiod pathway suggests that their origin pre-dates the divergence of the monocot and dicot lineages, the differences in the vernalization pathways between Arabidopsis and the temperate cereals suggest independent origins. This is consistent with the view that the temperate Pooideae grasses are a specialized monophyletic group that evolved from subtropical bambusoid-like species (Clayton and Renvoize, 1986; Preston and Kellogg, 2008).
In the temperate cereals, VRN2 is also down-regulated by vernalization (Yan et al., 2004b), but, in contrast to FLC, can also be down-regulated by short days (Dubcovsky et al., 2006; Trevaskis et al., 2006) (Figure 7). The position of VRN2 upstream of VRN1 and TaFT in the model is supported by results from transgenic winter wheat plants with reduced VRN2 transcript levels (Yan et al., 2004b) and isogenic lines for VRN2 (Yan et al., 2006). Plants with reduced or non-functional VRN2 transcripts showed up-regulation of TaFT and VRN1 and a significant acceleration of flowering (Yan et al., 2004b, 2006). The increase in VRN1 transcript levels is then followed by down-regulation of VRN2 (Figure 7), suggesting the existence of a regulatory feedback loop that has not been described in Arabidopsis (Loukoianov et al., 2005).
An additional characteristic that seems to be unique for the temperate cereals is the high level of expression of VRN1 in the leaves (Schmitz et al., 2000; Yan et al., 2003). In contrast, AP1 transcripts are abundant in the induced SAM and floral primordia in Arabidopsis, but are undetectable or present at much lower levels in some vegetative tissues (e.g. vascular tissues of cotyledons, Abe et al., 2005). Interestingly, transgenic 35S:FD Arabidopsis plants with ectopic expression of FD show high levels of AP1 transcripts in the leaves (Wigge et al., 2005), suggesting that the spatial differences between AP1 and VRN1 transcription profiles are the result of differences between the spatial transcription profiles of Arabidopsis FD and its wheat homologue (TaFDL2). The results presented here support this hypothesis. We first identified TaFDL2 as the functional homologue of Arabidopsis FD by its ability to interact with TaFT and the VRN1 promoter, and then showed that TaFDL2 exhibits high transcript levels in the leaves of both vernalized and non-vernalized winter wheat plants from very early developmental stages (Figure 5). The simultaneous presence of TaFDL2 and TaFT in the leaves of spring or vernalized winter wheat plants provides a simple explanation for the simultaneous presence of VRN1. The regulation of TaFT by various environmental cues provides temporal specificity to this interaction.
Although the results presented above explain the molecular mechanism by which VRN1 transcription is regulated in the leaves, they do not explain why such a complex regulation of a meristem identity gene has been conserved in the leaves of the temperate grasses analyzed so far. Up-regulation of VRN1 transcripts in the leaves of winter genotypes has been confirmed in wheat (Danyluk et al., 2003; Trevaskis et al., 2003; Yan et al., 2003), barley (Trevaskis et al., 2003), Lolium (Petersen et al., 2004) and oats (Preston and Kellogg, 2008), suggesting conservation across several tribes of temperate cereals (Triticeae, Poeae and Aveneae).
An interesting observation is that VRN1 transcript levels are negatively associated with the accumulation of COR (cold-responsive) genes and with the degree of frost tolerance (Danyluk et al., 2003). Frost tolerance increases during the acclimatization of wheat plants to cold but non-freezing temperatures, but decreases after the transition between the vegetative and reproductive apices. Near-isogenic lines for the VRN1 gene carrying the recessive vrn1 allele (winter growth habit) can tolerate 11°C lower freezing temperatures than lines carrying the dominant Vrn1 allele (spring growth habit) (Limin and Fowler, 2006). Similarly, spring lines grown under SD, which down-regulates the VRN1 transcript levels, can tolerate 8.5°C lower temperatures than the same lines under LD (Limin and Fowler, 2006). A similar result has been reported for barley. In a double-haploid population segregating for VRN-H1, lines carrying the recessive vrn-H1 allele showed higher transcript levels of CBF (C-repeat binding factors) than those carrying the dominant Vrn-H1 allele (Stockinger et al., 2007). In addition, lines grown under SD (reduced VRN-H1 levels) showed higher CBF transcript levels than lines grown under LD. CBF transcription factors are rapidly up-regulated by cold temperatures, inducing the expression of COR genes and playing a critical role in cold acclimatization and frost tolerance in temperate cereals (Francia et al., 2004; Knox et al., 2008; Miller et al., 2006; Skinner et al., 2005; Vágújfalvi et al., 2003). Based on these results, it is tempting to speculate that the temperate cereals have developed the ability to use the presence of VRN1 in the leaves as a signal to down-regulate the cold-tolerance regulatory network. As VRN1 is up-regulated upon the arrival of the spring, its presence would prevent up-regulation of the frost tolerance genes in the spring but not in the fall (autumn), when cold temperatures are an indication of future frost events.
In summary, we have identified the wheat functional homologue of Arabidopsis FD, TaFDL2 and confirmed that the protein coded by this gene interacts with TaFT and the VRN1 promoter. Transcription of TaFDL2 in the leaves provides a simple explanation for the characteristic expression of VRN1 in the leaves in temperate cereals. We also showed here that TaFT2 is partially regulated by TaFT, and that the proteins coded by the two genes interact with different bZIP proteins, providing the molecular basis for the sub-functionalization of these paralogous genes.
Wheat FD-like genes
The database used for the searches is available at the TGI website (http://compbio.dfci.harvard.edu/tgi/). Starting with the Arabidopsis FD protein sequence At4g35900, a TBLASTN search was carried out against the wheat (Triticum aestivum) gene index (release 10.0) to search for FD-like genes in wheat. The top 16 gene sequences producing high-scoring segment pairs were chosen and investigated further. Primers were designed to amplify cDNAs for each of the top 16 FD-like genes (Table S1). Restriction sites (underlined) were included in the oligos to facilitate the cloning of the PCR products into yeast vectors.
A multiple sequence alignment and a neighbor-joining phylogenetic tree were constructed using MEGA version 4 (Tamura et al., 2007). The tree was based on a conserved ungapped 69 amino acid region including the bZIP domain (Figure 1). Bootstrap confidence values for the nodes were based on 1000 iterations.
Yeast two-hybrid assays
The yeast cloning vectors pGBKT7 and pGADT7, control vectors pGADT7-T and PGBKT7-53, and the yeast strain AH109 used in the yeast two-hybrid assays were obtained from Clontech (http://www.clontech.com/). The yeast two-hybrid assays were performed according to the manufacturer’s instructions. Wheat full-length TaFT and TaFT2 cDNAs were fused to GAL4 DNA binding domain of pGBKT7 or the GAL4 activation domain of pGADT7 to generate bait and prey constructs, respectively. Full-length coding regions of TaFDL2, TaFDL6, TaFDL13 and TaFDL15, and a 375-bp cDNA fragment of TaFDL3 (including the putative bZIP binding domain) were cloned in-frame with the GAL4 activation domain into the prey vector pGADT7.
Table S2 lists the primers and restriction sites used to generate yeast bait and prey constructs. The appropriate plasmids were transformed into yeast strain AH109 using the lithium acetate method, and selected on SD medium lacking leucine (Leu) and tryptophan (Trp). After 4 days of incubation at 30°C, yeast cells were re-plated on selection plates containing SD medium lacking Leu, Trp, histidine (His) and adenine (Ade) for the interaction test.
For validation, coding regions of the three FDL proteins that showed interactions with either TaFT or TaFT2 bait constructs were switched from prey constructs to the bait vector pGBKT7 using restriction sites EcoRI and PstI for TaFDL2, BamHI and PstI for TaFDL6, and EcoRI and BamHI for TaFDL13. The new constructs were re-tested by yeast two-hybrid assays with TaFT or TaFT2 prey constructs.
Electrophoretic mobility shift assays (EMSA)
TaFDL2, TaFDL6 and TaFDL13 cDNA fragments (Table S3) were cloned in-frame with the GST coding region into the corresponding sites of pGEX-6p-1 (GE Healthcare, http://www.gelifesciences.com) to generate TaFDL2–GST, TaFDL6–GST and TaFDL13–GST fusion constructs. Four VRN1 promoter fragments containing one or two putative bZIP binding sites were generated by PCR (Table S3) and cloned into the pGEM-T Easy vector (Promega, http://www.promega.com/). Detailed EMSA protocols are described in Appendix S1.
Quantitative PCR and RT-PCR analyses
RNA samples were extracted from leaves and apices using the TRIZOL method (Invitrogen, http://www.invitrogen.com/). ACTIN was used as an endogenous control for both the RT-PCR and quantitative PCR SYBR Green® (Applied Biosystems, http://www3.appliedbiosystems.com) experiments, with primers as described previously (Dubcovsky et al., 2006). SYBR Green® systems for VRN1 and TaFT were developed in a previous study (Yan et al., 2006). The primers for the new SYBR Green® systems and RT-PCR experiments for TaFT2, TaFDL2, TaFDL6 and TaFDL13 are listed in Table S4. Quantitative PCR experiments were performed in an ABI7000. The method (Livak and Schmittgen, 2001) was used to normalize and calibrate transcript values relative to the endogenous controls. For each experiment, the same calibrator was used across replications, genotypes and environmental conditions to make units comparable. However, different calibrators were used for different experiments and therefore their units are not comparable.
RNA samples for the RT-PCR analysis of the various TaFDL transcript levels were extracted from leaves of unvernalized and vernalized (6 weeks at 4°C and LD) winter wheat variety Jagger. The apical region samples were extracted from the spring wheat variety Chinese Spring at the vegetative and early reproductive stages. Thirty plants were pooled to generate sufficient tissue. The apical region includes the SAM and a small portion of the crown.
This research was supported by the United States Department of Agriculture Cooperative State Research, Education, and Extension Services National Research Initiative (CSREES NRI) competitive grants 2007-35301-17737 and 2007-35301-18188. The authors thank Ann Blechl (USDA, Agricultural Research Service, Albany, NY, USA) for the transgenic wheat plants.
The GenBank accession numbers for the TaFDL2, TaFDL3, TaFDL6, TaFDL13 and TaFDL15 sequences are EU307112, EU307113, EU307114, EU307115 and EU307116, respectively.