Heterologous expression of a Glyoxalase I gene from sugarcane confers tolerance to several environmental stresses in bacteria

Abstract

Glyoxalase I belongs to the glyoxalase system that detoxifies methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates. The concentration of MG increases rapidly under stress conditions. In this study, a novel glyoxalase I gene, designated as SoGloI was identified from sugarcane. SoGloI had a size of 1,091 bp with one open reading frame (ORF) of 885 bp encoding a protein of 294 amino acids. SoGloI was predicted as a Ni²⁺-dependent GLOI protein with two typical glyoxalase domains at positions 28-149 and 159-283, respectively. SoGloI was cloned into an expression plasmid vector, and the Trx-His-S-tag SoGloI protein produced in Escherichia coli was about 51 kDa. The recombinant E. coli cells expressing SoGloI compared to the control grew faster and tolerated higher concentrations of NaCl, CuCl₂, CdCl₂, or ZnSO₄. SoGloI ubiquitously expressed in various sugarcane tissues. The expression was up-regulated under the treatments of NaCl, CuCl₂, CdCl₂, ZnSO₄ and abscisic acid (ABA), or under simulated biotic stress conditions upon exposure to salicylic acid (SA) and methyl jasmonate (MeJA). SoGloI activity steadily increased when sugarcane was subjected to NaCl, CuCl₂, CdCl₂, or ZnSO₄ treatments. Sub-cellular observations indicated that the SoGloI protein was located in both cytosol and nucleus. These results suggest that the SoGloI gene may play an important role in sugarcane's response to various biotic and abiotic stresses.

Keywords: Expression analysis; Functional validation; SoGloI; Stress tolerance; Sugarcane.

Figure 1. Multiple protein sequence alignment of SoGloI protein with glyoxalase I proteins from other species.

The species name and accession numbers are as follows: Oryza sativa (OsglyI-11.2 and O. Zn-GloI with Acc. No. Os08g09250 and EEC78918.1), Arabidopsis thaliana (AtGLYI2, AtGLYI3 and AtGLYI6 with Acc. No. NP_172291.1, NP_172648.1, and NP_176896.1 respectively), Pseudomonas aeruginosa (GloA1, GloA2 and GloA3 with Acc. No. AAG06912, AAG04099 and AAG08496 respectively), Glycine max (G. Zn-GloI and G. Ni-GloI with Acc. No. XP_003539194.1 and XP_003528689.1) and Escherichia coli (GlxI with Acc. No. AAC27133). The letters A, B and C represent extended sequences found in Zn²⁺-dependent GLOI, but absent in Ni²⁺-dependent GLOI.

Figure 2. Heterologous expression of recombinant SoGloI in Escherichia coli Rosetta cells (red arrow).

Lane designation: M, protein size makers with 30, 40, 55, and 70 kDa indicated; 1, blank; 2, control without IPTG induction; 3, control with induction for 8 h; 4, pET 32a-SoGloI without induction; 5 to 8, pET32a-SoGloI with induction for 2, 4, 6 and 8 h, respectively.

Figure 3. Growth of pET32a- SoGloI-expressing (row a) and pET 32a (row b) Escherichia coli Rosetta cells on LB agar plates with various concentrations of salt or heavy metal ions indicated above the figures.

(A) Control; (B) NaCl; (C) CuCl₂; (D) CdCl₂, and (E) ZnSO₄.

Figure 4. Growth assessments of SoGloI overexpressing Escherichia coli Rosetta cells under salt and heavy metal treatments.

(A) under 250 mM NaCl treatment from 0 h to 24 h; (B) under 750 µM CuCl₂ treatment from 0 h to 28 h; (C) under 750 µM CdCl₂ treatment from 0 h to 26 h; (D) under 750 µM ZnSO₄ treatment from 0 h to 28 h. All data were measured every two hours. All data points are mean ± SE (n = 3).

Figure 5. Expression patterns of SoGloI in sugarcane.

(A) SoGloI expression in different sugarcane tissues collected in the field; (B) SoGloI expression in greenhouse-grown sugarcane plantlets under NaCl, CuCl₂, CdCl₂, and ZnSO₄ treatments; (C) SoGloI expression in greenhouse-grown sugarcane plantlets under SA, MeJA, and ABA treatments; (D) glyoxalase I activity under NaCl, CuCl₂, CdCl₂, and ZnSO₄ treatments. Different letters indicate a significant difference at 5% level (p ≤ 0.05). Each value represents the average of three biological repeats ± SE (n = 3).

Figure 6. Determination of subcellular localization of ScGloI in tobacco (Nicotiana benthamiana) protoplasts under a fluorescence microscopy.

(A) bright field vision of pCXSN-GFP; (B) fluorescence vision of pCXSN-GFP; (C) merged vision of A and B; (D) bright field vision of pCXSN-SoGloI-GFP; (E) fluorescence vision of pCXSN-SoGloI-GFP; (F) merged vision of D and E. The emission wavelength is 515 nm and the excitation wavelength is between 470 nm and 490 nm.