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Sugar beet M14 glyoxalase I gene can enhance plant tolerance to abiotic stresses

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Abstract

Glyoxalase I is the first enzyme of the glyoxalase system that can detoxify methylglyoxal, a cytotoxic compound increased rapidly under stress conditions. Here we report cloning and characterization of a glyoxalase I from sugar beet M14 line (an interspecific hybrid between a wild species Beta corolliflora Zoss and a cultivated species B. vulgaris L). The full-length gene BvM14-glyoxalase I has 1,449 bp in length with an open reading frame of 1,065 bp encoding 354 amino acids. Sequence analysis shows the conserved glyoxalase I domains, metal and glutathione binding sites and secondary structure (α-helixes and β-sheets). The BvM14-glyoxalase I gene was ubiquitously expressed in different tissues of sugar beet M14 line and up-regulated in response to salt, mannitol and oxidative stresses. Heterologous expression of BvM14-glyoxalase I could increase E. coli tolerance to methylglyoxal. Transgenic tobacco plants constitutively expressing BvM14-glyoxalase I were generated. Both leaf discs and seedlings showed significant tolerance to methylglyoxal, salt, mannitol and H2O2. These results suggest an important role of BvM14-glyoxalase I in cellular detoxification and tolerance to abiotic stresses.

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Acknowledgments

This research was supported by the National Science Foundation of China (Project 31071473: Studies on the function of the BvM14-cystatin in sugar beet M14 lines, and Project 30871566: Studies on floral organ-specific expressed proteins in sugar beet M14 lines), and the Graduate Innovation Project of Heilongjiang Province. The paper represents serial 006 from our innovation team at the Heilongjiang University (Hdtd2010-05).

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Authors and Affiliations

  1. Key Laboratory of Molecular Biology, College of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China

    Chuan Wu, Chunquan Ma, Yu Pan, Shilong Gong, Chenxi Zhao, Sixue Chen & Haiying Li

  2. Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32611, USA

    Sixue Chen

  3. Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education Heilongjiang University, Harbin, 150500, China

    Haiying Li

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  1. Chuan Wu
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  4. Shilong Gong
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Correspondence to Haiying Li.

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Supplemental Fig. 1. Phylogenetic tree of BvM14-glyoxalase I with other plant glyoxalase I proteins. AtGlyI (Arabidopsis thaliana, gi|21537360|), VvGlyI (Vitis vinifera, gi|225445150|), TaGlyI (Triticum aestivum, gi|7619802|), OsGlyI (Oryza sativa Japonica Group, gi|46485858|), ZmGlyI (Zea mays, gi|226500882|), MtGlyI (Medicago truncatula, gi|144924819|), GhGlyI (Gossypium hirsutum, gi|211906514|), and BvM14-GlyI (M14 line).

Supplemental Fig. 2. Heterologous expression of BvM14-glyoxalase I in E. coli cells. E. coli cells carrying pET28a vector and pET28a-BvM14-Gly I were inoculated in LB medium containing 50 µg/ml kanamycin and induced with different concentrations of IPTG. M, protein marker. Lanes 1-3 were total protein, supernatant and precipitate from the E. coli cell carrying pET28a vector induced with 0.1 mM IPTG. Lanes 4-6, 7-9, 10-12 were total protein, supernatant and precipitate from the E. coli cell carrying pET28a-BvM14-Gly I induced with 0.1, 0.5 and 1.0 mM IPTG, respectively.

Supplemental Fig. 3. Activity of BvM14-glyoxalase I in transgenic tobacco plants and stress tolerance analysis. (a) RT-PCR analyses of BvM14-glyoxalase I in wild type and G1 to G7 transgenic tobacco lines expressing BvM14-glyoxalase I. WT, wild-type. Actin was used as an equal loading control. (b) Activity of BvM14-glyoxalase I in transgenic tobacco lines. The results are mean and standard deviation of three different experiments. (c) Survival rate, fresh weight and root length analyses of G2 line offsprings and WT plants growing on medium containing MG, NaCl, Mannitol and H2O2.

Supplemental Fig. 4. Absorbance of Evans blue stain retained in leaf disks of wild type relative to transgenic tobacco constitutively expressing BvM14-glyoxalase I. Evan’s blue was extracted and determined spectrophotometrically at 595 nm. (A) leaf disks treated with 5 and 10 mM MG for 24 and 48 h. (B) leaf disks treated with 400 mM and 800 mM NaCl for 24 and 48 h. (C) leaf disks treated with 200 mM and 400 mM mannitol for 24 and 48 h. (D) leaf disks treated with 10 mM and 20 mM H2O2 for 24 and 48 h. Data represent mean and standard deviation calculated from three replicate experiments (n = 3).

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Wu, C., Ma, C., Pan, Y. et al. Sugar beet M14 glyoxalase I gene can enhance plant tolerance to abiotic stresses. J Plant Res 126, 415–425 (2013). https://doi.org/10.1007/s10265-012-0532-4

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