Yasuharu Sanada

Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.)

Variation in the structures of plant fructans and their degree of polymerization (DP) can be explained as the result of diverse combinations of fructosyltransferases (FTs) with different properties. Although FT genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have only been functionally characterized in a few species such as wheat. A novel FT cDNA possessing 6-SFT activity has been identified and characterized from the temperate forage grass, timothy (Phleum pratense L.). The cDNA of an FT homolog, PpFT1, was isolated from cold-acclimated timothy. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT/sucrose:sucrose 1-fructosyltransferase (1-SST) activity and produced linear β(2,6)-linked levans from sucrose with higher DPs than present in graminans formed in vitro by wheat 6-SFT (Wft1). PpFT1 and Wft1 showed remarkably different acceptor substrate specificities: PpFT1 had high affinity for 6-kestotriose to produce levans and low affinity for 1-kestotriose, whereas Wft1 preferentially used 1-kestotriose as an acceptor. The affinity of the PpFT1 recombinant enzyme for sucrose as a substrate was lower than that of the Wft1 recombinant enzyme. It is also confirmed that timothy seedlings had elevated levels of PpFT1 transcripts during the accumulation of fructans under high sucrose and cold conditions. Our results suggest that PpFT1 is a novel cDNA with unique enzymatic properties that differ from those of previously cloned plant 6-SFTs, and is involved in the synthesis of highly polymerized levans in timothy.

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding.

Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.)

The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.

Ecotypic variation of water-soluble carbohydrate concentration and winter hardiness in cocksfoot (Dactylis glomerata L.)

The variation of the water-soluble carbohydrate (WSC) concentration in foliage and stubble and winter hardiness among cocksfoot (Dactylis glomerata L.) ecotypes originated from diverse countries were studied in the field in order to find a breeding material for the improvement of WSC. The total WSC concentration of foliage was found to be highest in Japanese cultivars and lowest in ecotypes from northern Europe in summer and autumn. The variation of fructan concentration in stubble among ecotypes was larger than the variation of mono and disaccharide concentrations in stubble and that of total WSC concentration in foliage in both winters over which this study was conducted. The total WSC and fructan concentrations in the stubble of ecotypes from Spain and southern Europe, derived from the Mediterranean region, were highest in summer. Ecotypes from Japan showed the highest concentration of total WSC in stubble, and were especially high in fructan concentration in autumn. Ecotypes from France, central Europe and central Asia, like Japanese cultivars, also showed a high fructan concentration. The latitude of origin was negatively correlated with WSC concentration and plant vigor in autumn. The altitude of origin was negatively correlated with plant vigor in autumn and positively correlated with fructan concentration in stubble, winter hardiness and snow endurance. Typhula snow blight resistance was related with the amount of the increase in fructan in the plants and with their growth habits in autumn. The fructan concentration of foliage and stubble in autumn was found to be closely related with winter hardiness in cocksfoot ecotypes. A breeding material which has a high WSC concentration in the foliage could not be found among the ecotypes in this study. However, some ecotypes that showed disease resistance, good winter hardiness and plant vigor would be useful as breeding materials for Hokkaido.

Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense).

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Genetic variation in water-soluble carbohydrate concentration in diverse cultivars of Dactylis glomerata L. during vegetative growth

The water-soluble carbohydrate (WSC) content of Dactylis glomerata L. (cocksfoot) during vegetative growth is lower than that of other temperate grasses. Variation in the WSC content of vegetative growth among cultivars was measured in order to assess the potential to improve forage quality. There were significant differences among cultivars for WSC content (P < 0.01) in all maturity groups. Average WSC content of the late (L) maturity group was higher than that of the early (E) and medium (M) maturity groups. Variation in the WSC content was largest among group L cultivars and smallest in group M. The WSC content of cultivars in groups M and L was consistent across 2 years (interactions between cultivars and years were not significant). Brown stripe infection was shown to reduce WSC content in susceptible cultivars. As a result of multiple regression analysis with WSC content as the dependent variable, dry matter content, heading date, thickness of stems, and resistance to brown stripe were selected as independent variables that were correlated with WSC. Therefore, variation for these traits must also be monitored in any program to increase WSC content in orchardgrass.