Dataset Information

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

ABSTRACT: 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 beta(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.

SUBMITTER: Tamura K

PROVIDER: S-EPMC2652063 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Tamura Ken-ichi K Kawakami Akira A Sanada Yasuharu Y Tase Kazuhiro K Komatsu Toshinori T Yoshida Midori M

Journal of experimental botany 20090101 3

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 gr ...[more]

PMID: 19269996

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Project description:Timothy is a perennial forage grass grown commonly in Boreal regions. This study explored the effect of vernalization and photoperiod (PP) on flowering and growth characteristics and how this related to changes in expression of three flowering related genes in accessions from different geographic origin. Large variation was found in accessions in their vernalization and PP responses. In southern accessions vernalization response or requirement was not observed, the heading date remained unchanged, and plants flowered without vernalization. On the contrary, northern types had obligatory requirement for vernalization and long PP, but the tiller elongation did not require vernalization at 16-h PP. Longer vernalization or PP treatments reduced the genotypical differences in flowering. Moreover, the vernalization saturation progressed stepwise from main tiller to lateral tillers, and this process was more synchronized in southern accessions. The expression of PpVRN1 was associated with vernalization while PpVRN3 accumulated at long PP. A crucial role for PpVRN3 in the transition to flowering was supported as in southern accession the transcript accumulated in non-vernalized plants after transfer to 16-h PP, and the apices transformed to generative stage. Differences in vernalization requirements were associated with variation in expression levels of PpVRN1 and PpVRN3, with higher expression levels in southern type. Most divergent transcript accumulation of PpMADS10 was found under different vernalization conditions. These differences between accessions can be translated into agronomic traits, such as the tiller composition of canopy, which affects the forage yield. The southern types, with minimal vernalization response, have fast re-growth ability and rapidly decreasing nutritive value, whereas northern types grow slowly and have better quality. This information can be utilized in breeding for new cultivars for longer growing seasons at high latitudes.

Project description:The sustainable production of perennial grasses in Northern Norway is at risk due to the ongoing climate change. The predicted increase in temperatures and variable weather patterns are further expected to create challenges for winter survival of timothy. Therefore, knowledge about the molecular mechanisms underlying freezing tolerance is crucial for developing robust cultivars. The current study is aimed at identifying genes involved in the freezing stress response of Timothy and studying gene expression differentiation due to field selection in contrasting environments. Four timothy cultivars, namely Engmo, Noreng, Grindstad and Snorri were field tested for three years at locations Tromsø and Vesterålen in Northern Norway. The surviving material from the field tests along with plants raised from the original seed lots were subjected to freezing tests. Following the tests, crown tissue was collected for RNA extraction. All samples from different treatment points were paired-end sequenced at 20 million reads/sample. LT50 values varied across cultivars and materials. Plant material from cold-acclimated (CA) samples was used as controls, while two pools for each cultivar were made of plant material from temperature points below the LT50, one with the higher temperatures termed Treatment 1 (T1) and one with the lower temperatures termed Treatment 2 (T2). Many genes coding for transcription factors and proteins which are known to play an important role in freezing tolerance like dehydrins, c-repeat binding factors, late embryogenesis abundant proteins are upregulated with decreasing temperatures. Moreover, genes associated with glycolysis/gluconeogenesis, TCA cycle, glutathione metabolism, proteasome, glyoxylate and dicarboxylate metabolism pathways and genes encoding heat shock proteins, autophagy-related, plasma membrane-associated proteins, sugar and amino acid transporters had elevated expression in field survivors compared to plants raised from the original material. These DEGs might be crucial for winter survival of timothy in a changing climate. Furthermore, differences in freezing stress response between northern and southern adapted cultivars, and surviving material from two field trial locations are discussed briefly

Project description:BackgroundTimothy is a long-day grass species well adapted for cultivation in northern latitudes. It produces elongating tillers not only in spring growth but also later in summer. As the quantity and quality of harvested biomass is dictated by canopy architecture and the proportion of stem-forming flowering tillers, the regulation of flowering is of great interest in forage grass production.MethodsCanopy architecture, stem morphology and freezing tolerance of vernalized timothy were investigated in greenhouse and field experiments. The molecular control of development was examined by analysing the relationship between apex development and expression of timothy homologues of the floral inducer VRN1 and repressor VRN2.Key resultsTrue stem formation and lignification of the sclerenchyma ring occur in both vernalized and regrowing stems irrespective of the developmental stage of the apex. The stems had, however, divergent morphology. Vernalization enhanced flowering, and the expression of the VRN1 homologue was elevated when the apex had passed into the reproductive stage. High VRN1 homologue expression was not associated with reduction in freezing tolerance and the expression coincided with increased levels of the floral repressor VRN2 homologue. Field experiments supported the observed linkage between the upregulation of the VRN1 homologue and the transition to the reproductive stage in vernalized tillers. The upregulation of putative VRN1 or VRN2 genes was restricted to vernalized tillers in the spring yield and, thus, not detected in non-vernalized tillers of the second yield; so-called regrowth.ConclusionsThe formation of a lignified sclerenchyma ring that efficiently reduces the digestibility of the stem was not related to apex development but rather to a requirement for mechanical support. The observed good freezing tolerance of reproductive timothy tillers could be one important adaptation mechanism ensuring high yields in northern conditions. Both VRN1 and VRN2 homologues required a vernalization signal for expression so the development of yield-forming tillers in regrowth was regulated independently of the studied genes.

Dataset Information

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

Publications

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

Similar Datasets

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