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Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties

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Abstract

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3–40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.

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Abbreviations

DP:

Degree of polymerization

DTT:

Dithiothreitol

FEH:

Fructan exohydrolase

1-FFT:

Fructan:fructan 1-fructosyltransferase

HPAEC-PAD:

High-performance anion-exchange chromatography with pulsed amperometric detection

HPLC:

High-performance liquid chromatography

LE:

Ligand exchange

RI:

Refractive index

SE:

Size exclusion

1-SST:

Sucrose:sucrose 1-fructosyltransferase

6-SFT:

Sucrose:fructan 6-fructosyltransferase

PFT:

Transgenic line expressing PpFT1

WFT:

Transgenic line expressing wft1

WT:

Wild type

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Acknowledgments

We are grateful to Dr. John Vogel, US Department of Agriculture, Agricultural Research Service, Dr. Ichiro Mitsuhara, National Institute of Agrobiological Sciences, Japan and Dr. Masaru Iizuka, Kobe Shoin Women’s University, Japan for providing seeds of ‘Bd21-3’ of Brachypodium distachyon, the pMLH7133 plasmid vector and 6-kestotriose, respectively. We thank Ms. Yukari Ikenobe and Satomi Shimada for their technical assistance.

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Authors and Affiliations

  1. NARO Hokkaido Agricultural Research Center, 1 Hitsujigaoka, Toyohira, Sapporo, 062-8555, Japan

    Ken-ichi Tamura, Yasuharu Sanada, Kazuhiro Tase & Midori Yoshida

  2. NARO Kyushu Okinawa Agricultural Research Center, 2421 Suya, Koshi, Kumamoto, 861-1192, Japan

    Akira Kawakami

  3. Field Science Center for Northern Biosphere, Hokkaido University, Kita 11, Nishi 10, Kita, Sapporo, 060-0811, Japan

    Toshihiko Yamada

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  1. Ken-ichi Tamura
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  2. Yasuharu Sanada
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  3. Kazuhiro Tase
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  4. Akira Kawakami
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  5. Midori Yoshida
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  6. Toshihiko Yamada
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Correspondence to Ken-ichi Tamura.

Electronic supplementary material

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425_2013_2016_MOESM1_ESM.pptx

Suppl. Fig. S1 Anion exchange HPLC (HPAEC-PAD) analysis of water-soluble carbohydrates from the leaf blades of cold acclimated plants, treated with the recombinant Pp6-FEH1 enzyme. a Authentic standards. b, c Untreated and treated extracted solutions from fully expanded leaves of cold acclimated wild type plants. d, e Untreated and treated extracted solutions from a transgenic line expressing wft1, WFT1-20. f, g Untreated and treated extracted solutions from a transgenic line expressing PpFT1, PFT1-31. Chromatograms at the latter retention time are redrawn using an extended y axis scale (f and g). Abbreviations for each sugar peak are: G, glucose; F, fructose; S, sucrose; 1 K, 1-kestotriose; N, nystose

425_2013_2016_MOESM2_ESM.pptx

Suppl. Fig. S2 Low molecular parts of the anion exchange HPLC (HPAEC-PAD) analysis of water-soluble carbohydrates from the leaf blades of cold acclimated plants. a Authentic standards. b Wild type. c A transgenic line expressing wft1, WFT1-20. d A transgenic line expressing PpFT1, PFT1-31. Abbreviations for each sugar peak are: G, glucose; F, fructose; S, sucrose; 1 K, 1-kestotriose

425_2013_2016_MOESM3_ESM.pptx

Suppl. Fig. S3 HPLC chromatograms of water-soluble carbohydrates from the leaf blades of cold acclimated transgenic and wild type B. distachyon analyzed by the SE and LE separation mode with the RI detection. Extracted solutions from fully expanded leaves of cold acclimated wild type plants (a), plants from a transgenic line expressing wft1, WFT1-20 (b) and plants from a transgenic line expressing PpFT1, PFT1-31 (c) were analyzed. Abbreviations for each sugar peak are: G, glucose; F, fructose; S, sucrose; O, oligosaccharide; P, polysaccharide. The leftmost peak in each chromatogram does not show any sugars

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Tamura, Ki., Sanada, Y., Tase, K. et al. Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties. Planta 239, 783–792 (2014). https://doi.org/10.1007/s00425-013-2016-8

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