Identification and mapping of FT genes in perennial ryegrass
We isolated six cDNAs encoding FTs in perennial ryegrass. Expression of recombinant protein in P. pastoris demonstrated that the prft4 gene encodes 1-SST, and the prft3 and the prft5 genes both encode 6G-FFT. High sequence similarity with perennial ryegrass cDNAs that have been functionally characterized (Chalmers et al., 2003; Lasseur et al., 2006) supports these results. For unknown reason(s), no prominent reaction occurred when we used recombinant protein obtained by expressing the prft1, prft2 or prft6 cDNAs in our system. The location of the prft1, prft2 and prft6 protein sequences within a clade comprising 6-SFT sequences from various plants in the phylogenetic analysis indicates that the prft1, prft2 and prft6 genes might encode 6-SFT.
The linkage map position of the prft1, prft3 and prft4 genes was established using a perennial ryegrass mapping population derived from a cross between inbred lines derived from the Perma and Aurora cultivars. Both prft1 and prft4 genes mapped on the distal region of LG7 in the proximity of the locus of an invertase gene (Armstead et al., 2004; Fig. 3c). Three FT genes have previously been mapped to perennial ryegrass LGs using a different mapping population derived from a pair-cross between North African6 (NA6) and Aurora6 (AU6) plants (Chalmers et al., 2005). The 1-SST gene (Lp1-SST; marker xlp1-sst) has been mapped to a distal region of LG7 of NA6. Although the alignment between these different genetic maps has not been fully established, it is very likely that the position of the prft4 gene corresponds to that for xlp1-sst, taking into account that the prft4 gene encodes 1-SST. In NA6, an FT gene marker (xlpft1), the function of which has not been characterized, has also been mapped adjacent to xlp1-sst (Chalmers et al., 2005). It is possible that the prft1 gene corresponds to xlpft1. Alternatively, it is possible that the prft1 gene is not identical to xlpft1 and that multiple FT genes are located in this chromosomal region. The 6-SFT gene in barley has been mapped to the distal region of the short arm of chromosome 1 (7H) corresponding to LG7 of perennial ryegrass (Wei et al., 2000), suggesting that the prft1 gene might encode 6-SFT. In the distal part of LG7, the prft1 and prft4 genes were closely linked with INV1:2, a gene for soluble invertase (Gallagher et al., 2004; Fig. 3c). Considering the possibility that FT genes originated from an invertase gene in plants (Sprenger et al., 1995; Vijn & Smeekens, 1999; Francki et al., 2006), it is possible that colocalization of the FT genes with the invertase gene in this chromosomal region arises from a previous duplication of the invertase gene.
The prft3 gene was mapped to the distal region of perennial ryegrass LG3. An FT gene marker (xlpft4), the function of which has not been characterized, has also been mapped to a distal region of LG3 of AU6 (Chalmers et al., 2005). Likewise, xlpft4 might be identical to the prft3 gene and encode 6G-FFT.
Very high sequence similarity between the prft1 or prft2 and prft6 genes, or between the prft3 and prft5 genes, suggests that some of these genes may be allelic. Similarly, the prft4 gene and the reported 1-SST gene (Chalmers et al., 2003), or the prft3 or prft5 genes and the reported 6G-FFT gene (Lasseur et al., 2006), may also be allelic. These possibilities remain to be examined by a more extensive analysis of the perennial ryegrass genome.
Changes in fructan content as an adaptive response to freezing temperatures and transcriptional regulation of FT genes in response to low temperature
Significant accumulation of fructans was detected in both leaf and crown tissues during cold treatment. There was little increase in plant size during cold treatment. We have measured fructan content in plants that were not cold-treated and were kept for the same period at a normal temperature (18–22°C). We found that fructan levels in plants kept at normal temperature for 14 d corresponded to 15.4 and 18.7% of those of cold-treated plants in leaf and crown tissues, respectively. Although this experiment should not be regarded as a control for the cold treatment, because plants grow considerably faster at a normal temperature, a larger increase in fructan content in cold-treated plants than in plants grown at normal temperature indicates that the observed accumulation of fructan during cold treatment involves cold-specific effects that are considerably larger than developmental effects.
Previous tests of freezing tolerance using crown tissues have shown that a similar cold treatment confers significant freezing tolerance in various perennial ryegrass accessions. For instance, the survival rate of cv. Riikka under freezing conditions at –6°C for 16 h increased from 0 to 62% following treatment (Yamashita et al., 1993). In general, the freezing tolerance of plants increases in proportion to the period of cold acclimation. More than 7 d cold treatment is required to achieve the maximum level of freezing tolerance in perennial ryegrass (Lorenzetti et al., 1971). As a consequence, changes in the fructan content during cold treatment parallel the acquisition of freezing tolerance through cold acclimation, suggesting that fructan may be closely associated with the transition to enhanced freezing tolerance.
Previous expression analyses of FT genes have shown that transcript profiles are generally consistent with both enzymatic activity measurements and levels of fructan accumulation (reviewed by Chalmers et al., 2005), suggesting that the cellular level of fructan is primarily regulated at the level of transcription of FT genes. Analysis of FT gene expression, however, has been carried out mainly during changes in the development of plants (Lüscher et al., 2000; Koroleva et al., 2001; Lidgett et al., 2002; Chalmers et al., 2003; Johnson et al., 2003; Gallagher et al., 2004; Lasseur et al., 2006), and very few analyses of expression of FT genes under cold treatment are known (see below). Moreover, there have been only a few reports that describe relationships between the expression of different FT genes (Wang et al., 2000; Lasseur et al., 2006). Our present data demonstrate for the first time in plants that the transcription of multiple FT genes is induced in a coordinated way by low temperature, and occurs in parallel with the accumulation of fructans.
Two distinct patterns of change in mRNA levels during cold treatment were detected. One pattern involves an abrupt increase in mRNA within the first 24 h of cold treatment, which is followed by a decrease after several days of cold treatment and then an increase during further cold treatment over at least 30 d. This pattern was observed for the prft3 (6G-FFT) and prft4 (1-SST) genes in leaf tissues. The induction of this gene expression in the first 24 h of cold treatment in both leaf and crown tissues probably reflects the initiation of fructan synthesis using sucrose as a substrate, and may also account for the increased fructan content of crown tissues in plants cold-treated for 4 d. This is consistent with the roles of 6G-FFT and 1-SST in the initiation of fructan synthesis (Fig. 8).
Such a two-step change in mRNA level resembles changes in the levels of carbohydrate detected in plants that undergo cold treatment (reviewed by Gaudet et al., 1999). Our electron microscope analyses have also revealed a decrease in mesophyll cell size subsequent to the initial increase during the early period of cold treatment, before it increases again during a longer period of cold treatment in perennial ryegrass (Tominaga et al., 2004). These phenomena probably reflect a response to an initial shock of cold treatment followed by a longer period of adaptation, which can be widely observed in cold-tolerant plants.
Changes in the mRNA level of putative 6-SFT genes during cold treatment in similar controlled conditions have been analysed in wheatgrass (Wei & Chatterton, 2001) and big bluegrass (Wei et al., 2002). An increase followed by a decrease in mRNA level during 15 d of cold treatment was obtained, which corresponds to the pattern found for the prft3 and prft4 genes in the present study. The wheatgrass results also indicated that the level of low molecular-weight fructan increases during the early period of cold treatment. However, because wheatgrass analyses were confined to changes in the mRNA levels of FT genes only up to 15 d of cold treatment, increases in mRNA levels during longer periods of cold treatment, as detected in the present study, were not investigated. We consider that a seasonal increase in the mRNA levels of FT genes observed in field-grown plants towards winter (Kawakami & Yoshida, 2002; Van Laere & Van den Ende, 2002) may be similar to the changes detected in the plants undergoing long-term cold treatment in the present study.
The other pattern is a gradual increase in the mRNA level over a long period of the cold treatment, which was observed for the prft1 and prft2 genes. Although we have not been able to determine the function of the prft1 and prft2 proteins by recombinant protein analysis, the expression profiles found during cold treatment suggest that the prft1 and prft2 proteins are mainly involved in long-term fructan accumulation. The gradual increase in mRNA level may be associated with the acquisition of freezing tolerance and/or storage of carbohydrates as an adaptive response to the onset of winter, as reported in wheat (Kawakami & Yoshida, 2002). Plants that accumulate fructans can store fructose units while keeping osmotic potentials within a limited range. Thus proteins encoded by the prft1 and prft2 genes are likely to be involved in the production of fructans with a large number of linear or branched polymers of fructose units. The results of the phylogenetic analysis (Fig. 2) suggest that 6-SFT may be a likely candidate for the protein encoded by the prft1 and prft2 genes as well as the homologous prft6 gene. Whether 6-SFT activity is present in Lolium species remains to be confirmed, because bifurcose, which is indicative of 6-SFT activity, has not been detected (Pavis et al., 2001b; Chalmers et al., 2005). Fructan-fructan 6-fructosyltransferase (6-FFT) may also be a likely candidate because it may also synthesize large fructan molecules, although the presence of this enzyme has only been postulated (Pavis et al., 2001b).
As a gradual increase was observed in the mRNA levels of the prft3 and prft5 (6G-FFT) genes and the prft4 (1-SST) gene following cold treatment for longer than 7 d, the prft3, prft4 and prft5 genes may also be involved in adaptation to cold conditions, possibly through supplying smaller fructan molecules, which can be used as substrates for polymerization of fructose units.
There is a high positive correlation between the levels of fructan and FT mRNA in response to cold treatment (Table S2), which is more pronounced than those reported previously for developmental changes (Gallagher et al., 2004; Lasseur et al., 2006). This suggests that relationships between fructan accumulation during cold treatment and transcriptional control of FT genes are much stronger than relationships between developmental controls of fructan level and the transcription of FT genes. It should also be noted that a marked difference in the relative amount of fructans of different size classes between leaf and crown tissues (Fig. 6) may reflect a difference in fructan synthesis and/or metabolism between source and sink organs. Because the patterns of change in the mRNA levels of the prft1–prft5 genes in leaf and crown tissues are very similar to each other (Fig. 7), a plausible interpretation of the observed differences in fructan size classes between these tissues is that fructans accumulate mainly in crown tissues as long-chain molecules suitable for carbohydrate storage, whereas in leaf tissues fructans are metabolized while being synthesized, which restricts the preferential accumulation of long-chain fructans.
Overall, these results indicate that there are at least two patterns of expression for FT genes in response to low temperature. One involves induction of FT gene expression in response to both cold shock and a long period of cold conditions. The other pattern involves the induction of FT gene expression in response to a long period of cold only. These different patterns of expression have been assigned to FT genes of different functions. This coincidence between gene expression and function of FTs suggests that the genes have acquired diversity during evolution in both function and gene expression in response to cold conditions.