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

Plant material

Sugarcane genotype YCE 05-179 was used in this study. Plants were maintained in a genetic nursery at the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, China. In addition, tissue culture-derived young, healthy plantlets of YCE 05-179 were also involved in the study.

Cloning of SoGloI gene

A BLAST search of the sugarcane database (taxid: 286192; taxid: 4547; taxid: 128810; taxid: 62335) of GenBank (http://www.ncbi.nlm.nih.gov/) was conducted with the sugarcane EST, CA140600.1 (Wu et al., 2013b). Several highly homologous sugarcane ESTs were obtained and aligned. A 1,446 bp cDNA of sugarcane Glo I gene, designated as SoGloI, was assembled using the CAP3 Sequence Assembly Program (http://doua.prabi.fr/software/cap3). SoGloI cDNA sequence was amplified in a 25 µL reaction mixture on ABI Veriti96 PCR with primers G-F and G-R (Table 1) under a thermal cycling program: 94 °C, 4 min; 35 cycles of (94 °C, 1 min; 54 °C, 1 min; 72 °C, 1.5 min); and 72 °C, 10 min. Reaction mixture contained 2.5 µL 10 × PCR buffer (plus Mg²⁺), 2.5 µL dNTPs (2.5 mM), 1.0 µL first-strand cDNA, 1.0 µL each of forward and reverse primers (10 µM) and 0.125 µL Ex-Taq enzyme (5 U µL⁻¹) (Takara, Shanghai, China). Amplified SoGloI cDNA product was separated through 1% agarose gel electrophoresis, gel purified using Omega Gel Extraction Kit (Omega, Shanghai, China), and cloned into the PMD 18-T vector (Takara, Shanghai, China). Putative recombinants were confirmed by PCR, of which six were sequenced (Shenggong Co., Ltd., Shanghai, China).

Bioinformatics analysis

The ORF of the full-length SoGloI was predicted using the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Molecular weight and isoelectric point was calculated using Compute pI/Mw tool (http://www.expasy.ch/tools/pi_tool.html). Protein domain was predicted using the SMART Program (http://smart.embl-heidelberg.de/). Protein hydrophilicity was analyzed by Protscale Program (http://www.expasy.ch/tools/protscale.html). Prediction of signal peptides and analysis of trans-membrane helix domain were conducted using the SignalP and TMHMM-2.0 Programs (http://www.cbs.dtu.dk/services/). A multiple protein-sequence alignment was carried out using the DNAMAN^® Program (Guo et al., 2012; Liu et al., 2010; Su et al., 2013).

Expression of SoGloI in field-grown sugarcane plants

Five tissue samples, white young roots, leaf (+1), leaf sheath (+1), buds (6th-8th from the base), and internodes (6th and 7th from the base) were collected from 7- to 8-month-old plants in the field nursery. All samples except buds were cut into small pieces, wrapped up within tinfoil, and immediately flash-frozen in liquid nitrogen. The frozen samples were stored in a −80 °C freezer until RNA extraction. Total RNA samples were isolated from the frozen buds using the TRIzol^® kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA samples were dissolved in diethylpyrocarbonate-treated H₂O. RNA concentration was quantified by measuring absorbance at OD₂₆₀ and OD₂₈₀ using Synergy H1 Microplate Reader Multi-Mode (Bio-Tek, Winooski, VT, USA). RNA quality was also assessed by 1.0% denaturing agarose gel electrophoreses.

First-strand cDNA was synthesized from 1 µg RNA in 20 µL reaction mixture by reverse transcription PCR (RT-PCR) using the Prime-Script^® 1st Strand cDNA Synthesis Kit (Takara, Shanghai, China) on ABI Veriti96 PCR (ABI, Foster City, CA, USA). For real-time quantitative PCR (RT-qPCR), a total of 1 µg RNA in 20 µL reaction mixtures was used for the first-strand cDNA synthesis using the Prime-Script^® RT Reagent Perfect Real Time Kit (Takara, China) on ABI Veriti96 PCR. Phosphoglyceraldehyde dehydrogenase (GAPDH) (CA254672) gene was used as the internal control. The primers of GAPDH-QF/GAPDH-QR were listed in Table 1 (Iskandar et al., 2004; Que et al., 2009a; Que et al., 2009b).

SoGloI expression in greenhouse-grown sugarcane plantlets under different stress treatments

In the greenhouse, tissue culture-derived 4-month old healthy plantlets were rinsed with agar medium and transplanted into every other column of 46 mL-wells in two 96-well (8 × 12) plastic trays, containing only distilled water. The water was changed every morning. After 10 days, the plantlets were transplanted to 100-mL flat-bottomed glass tubes and subjected to seven different stress treatments in three replicates. Three plantlets of each treatment was treated with the following solutions: 100 µM methyl jasmonate (MeJA, dissolved with 0.1% (v/v) ethanol and 0.05% (v/v) Tween-20); 5.0 mM salicylic acid (SA, dissolved with 0.05% (v/v) Tween-20); 100 µM abscisic acid (ABA) for 6, 12, and 24 h (Li et al., 2009; Su et al., 2013); 250 mM NaCl; 500 µM CdCl₂; 100 µM CuCl₂; or 100 µM ZnSO₄ for 12, 24 and 48 h (Damaj et al., 2010; Guo et al., 2012; Que et al., 2009a; Que et al., 2009b), respectively. Plantlets grown in distilled water were used as the control.

SoGloI expression in response to MeJA, SA, ABA (0, 6, 12, 24 hour-post-treatment or hpt) and NaCl, CdCl₂, CuCl₂, ZnSO₄ (0, 12, 24, 48 hpt) was analyzed by RT-qPCR on a 7500 RT-qPCR system (ABI, Foster City, CA, USA). 25S rRNA (BQ536525) gene was chosen as internal control and. The primers of 25S-QF/25S-QR were listed in Table 1. The SoGloI-specific primer pair of G-QF/G-QR was designed by Primer Premier 5.0 software (Premier Biosoft International, CA) (Table 1). RT-qPCR was carried out with FastStart Universal SYBR Green Master (ROX) (Roche, Shanghai, China) in a 25 µL volume containing 12.5 µL FastStart Universal SYBR Green PCR Master (ROX), 0.5 µM of each primer and 1.0 µL template (100 × diluted cDNA). RT-qPCR with sterile dH₂O as template was the control. The RT-qPCR thermal cycle program included 2 min at 50 °C; 10 min at 95 °C; and 40 cycles of (15 s at 94 °C and 1 min at 60 °C). The reactions were repeated three times for each sample. The 2^−ddCt method was used to calculate relative gene expression levels (Livak & Schmittgen, 2001).

Expression of pET32a-SoGloI in E. coli Rosetta Cells (DE3)

A SoGloI gene fragment was amplified from the cDNA clone with primers G-32aF/G-32aR (Table 1). The SoGloI PCR fragment was digested with EcoR I and Xho I (NEB, USA) and subsequently sub-cloned into the EcoR I–Xho I sites of pET32a (+) (Guo et al., 2012) to produce pET32a-SoGloI. After sequence verification, the pET32a-SoGloI was transformed into Rosetta Cells (DE3) (Tiangen BioTech Co. Ltd., Sichuan, China). Expression of pET32a-SoGloI was induced in 1 mM isopropyl β-D-thiogalactoside (IPTG) for 8 h at 37 °C and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 0, 2, 4, and 8 h. Non-transformed (blank) and the vector pET32a-transformed Rosetta cells (control) were used as the controls.

Spot assays were performed to assess the response of pET32a-SoGloI transformed E. coli cells to NaCl, CdCl₂, CuCl₂ or ZnSO₄ treatments. When the E. coli culture mixture reached OD₆₀₀ = 0.6, 1 mM IPTG was added into the LB medium and the culture mixture was incubated for 12 h at 28 °C. Then the cultures were first diluted to 0.6 (OD₆₀₀), and further diluted to two levels (10⁻³ and 10⁻⁴) (Guo et al., 2012). Thereafter, 10 µL each of the diluted cultures was spotted on LB plates containing 170 µg mL⁻¹ chloramphenicol and 80 µg mL⁻¹ ampicillin, along with each test chemical. The concentrations of the chemicals used were NaCl at 250, 500 and 750 mM, CdCl₂ at 250, 500 and 750 µM, CuCl₂ at 250, 500 and 750 µM, and ZnSO₄ at 250, 500 and 750 µM, respectively (Guo et al., 2012; Su et al., 2013). All plates were incubated overnight at 37 °C.

Assay of sugarcane glyoxalase I enzyme activity

Entire flash-frozen 4-month old plantlets (100 mg wet weight) were pulverized in liquid N₂ in a mortar. Protein was extracted with an extraction buffer containing 0.1 M potassium phosphate buffer (PPB, pH 7.5), 50% (v/v) glycerol, 16 mM MgSO₄, 0.2 mM Phenylmethanesulfonyl fluoride (PMSF) and 0.2% (v/v) polyvinylpyrrolidone (PVP40). The extract was centrifuged twice at 13,000 rpm at 4 °C for 30 min to obtain the crude protein extract from the supernatant (Zeng et al., 2016). The supernatant was used as the cytosolic extract for the assessment of glyoxalase activity, and protein concentration was determined by the Bradford method (Bradford, 1976) using bovine serum albumin (BSA) as the standard. SoGloI activity assay was carried out following Hossain et al. (2009) and Hasanuzzaman, Hossain & Fujita (2011). Briefly, the assay mixture contained 100 mM K-phosphate buffer (PPB, pH 7.0), 15 mM magnesium sulfate, 1.7 mM GSH, and 3.5 mM MG in a final volume of 0.8 mL. Thioester formation was measured by the increase in absorbance at 240 nm for 1 min. The enzyme activity was calculated using an extinction coefficient (ε) of 3.37 mM⁻¹ cm⁻¹.

Sub-cellular localization

The SoGloI gene was sub-cloned into the Xcm I/BamH I restriction sites of pCXSN to construct a fusion protein expression vector of 35S::SoGloI::GFP. The GFP-containing pCXSN vector was a gift of Songbiao Chen, Institute of Biotechnology, Fujian Academy of Agricultural Sciences. The pCXSN-SoGloI recombinant plasmids were transformed into Agrobacterium tumefaciens cells, strain GV 3101 (Chen et al., 2006). The transgenic GV 3101 cells were inoculated into LB medium containing kanamycin (50 µg mL⁻¹) and rifampicin (34 µg mL⁻¹). The culture was incubated overnight at 28 °C with shaking at 200 rpm. The culture was then centrifuged at 5,000× g to harvest the Agrobacterium cells followed by, re-suspension in 10 mM MgCl₂ and 10 mM fatty acid methyl ester sulfonate (MES). The concentration of the bacterial suspension was measured and adjusted to OD₆₀₀ = 0.6 with Murashige and Skoog (MS) liquid medium supplemented with 200 mM acetosyringone. The resulting culture was incubated at 28 °C for 3 h (Yang et al., 2014). Then, 1 mL of the bacterial culture was infiltrated into 4-week old tobacco leaves with disposable syringes. The injection sites were marked. Injected plants were incubated under a 12 h-light/12 h-dark cycle at 28 °C for three days (Su et al., 2013). Then, the protoplasts were isolated from well-expanded leaves following the rice protoplast isolation protocol of Chen et al. (2006). Briefly, the leaves were cut into 1-mm strips and placed in a dish containing 12 mL of K3 medium (3 mM MES, 7 mM CaCl₂, 0.35 M mannitol, 0.7 mM NaH₂PO₄, 0.35 M sorbitol, 20 mM KCl, pH 5.6) supplemented with 0.4 M sucrose, 1.5% cellulase R-10 (Yakult Honsha, Japan) and 0.3% macerozyme R-10 (Yakult). The leaf tissue was vacuum-infiltrated for 30 min at 20 mm Hg and digested at room temperature with gentle shaking for 4 h to produce protoplasts. Then, the K3 medium was replaced with 12 mL of W5 solution (2 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, pH 5.8). The protoplasts were collected by centrifugation at 300× g for 4 min at 4 °C and re-suspended in 1 mL WI solution (4 mM MES, 0.5 M mannitol, 20 mM KCl, pH 5.7). The sub-cellular location of the SoGloI gene was observed using fluorescence microscopy (Ci-L; Nikon, Tokyo, Japan).

Sequence analysis of SoGloI

The full-length of SoGloI (GenBank Accession Number: KC857628) was 1,091 bp with one ORF of 885 bp (Fig. S1). The deduced SoGloI protein had 294 amino acids with a predicted molecular mass of 32.9 kDa and a pI value of 5.45. Two glyoxalase domains were found, located at 28–149 and 159–283, respectively (Fig. S2). The ProtScale predicted that SoGloI was a hydrophilic protein (Fig. S3). TMHMM Server v.2.0 and SignalP 4.1 Server program predicted that SoGloI was not a trans-membrane protein (Figs. S4, S5).

In order to predict the metal dependency of SoGloI protein, a multiple sequence alignment of SoGloI with other known GloI proteins from Oryza sativa (OsglyI-11.2 and O. Zn-GloI), Arabidopsis thaliana (AtGLYI2, AtGLYI3 and AtGLYI6), Pseudomonas aeruginosa (GloA1, GloA2 and GloA3), Glycine max (G. Zn-GloI and G. Ni-GloI) and Escherichia coli (GlxI) was done using DNAMAN^® (Fig. 1). SoGloI shared 87.8%, 69.7%, 57.3%, and 72.0% amino acid sequence identities with OsglyI-11.2, AtGLYI3, AtGLYI6, and G. Ni-GloI, respectively (Fig. 1). Extended amino acid sequences (the letters A, B and C in Fig. 1) of OsglyI-11.2, AtGLYI3, AtGLYI6, GloA1, GloA2, G. Ni-GloI, and GlxI were missing in SoGloI, a characteristic of Ni²⁺-dependent GloI. Contrarily, these extended amino acid sequences were present in AtGLYI2, GloA3, O. Zn-GloI and G. Zn-GloI, which were Zn²⁺-dependent GloI.

Expression of SoGloI in E. coli

Upon IPTG induction, the recombinant SoGloI gene expressed well in Rosetta cells (Fig. 2, Lanes 5–8) to yield SoGloI protein that was 51 kDa in size and carried a Trx-His-S-tag of 18.3 kDa. Moreover, gradually increased amounts of SoGloI protein were also observed when the IPTG induction was extended from 2 h to 8 h.

In the spot assays, SoGloI-expressing Rosetta cells (Figs. 3A–3E, Row a) grew more rapidly on LB agar plates containing NaCl, CuCl₂, CdCl₂ or ZnSO₄ than control Rosetta cells (pET 32a) (Figs. 3A–3E, Row b). Compared to control cells, SoGloI-expressing cells tolerated salt up to 250 mM (Fig. 3B) as well as heavy metal ions up to 750 µM (Figs. 3C–3E). Consistently, SoGloI-expressing cells also grew faster than the control cells in LB liquid media containing 250 mM NaCl, 750 µM CuCl₂, 750 µM CdCl₂ or 750 µM ZnSO₄(Fig. 4). These results may demonstrate that the recombinant SoGloI protein enhanced the growth of Rosetta cells under stress conditions.

Expression patterns of SoGloI in sugarcane tissues

RT-qPCR was conducted to detect both tissue-specific and stress-related expression of SoGloI. The SoGloI gene was ubiquitously expressed in five tissues of 7- to 8-month old plants collected from the field. The highest level was detected in buds, followed by leaves, roots, leaf sheaths, and internodes (Fig. 5A).

SoGloI expression patterns in healthy 4-month old plantlets under NaCl, CuCl₂, CdCl₂, ZnSO₄, SA, MeJA, and ABA treatments were shown in Figs. 5B and 5C. Under NaCl, CuCl₂, CdCl₂, and ZnSO₄ treatments, SoGloI expression was up-regulated steadily from 0 to 48 hpt. The peak level of SoGloI expression was about 3.1-, 2.9- 2.8- and 1.9-fold of the level in control, respectively (Fig. 5B). In contrast, under SA and MeJA treatments, SoGloI expression decreased after peaking at 6 hpt. The maximum level of SoGloI expression was detected at 6 hpt, which was about 2.6- and 2.1-fold of the level in control, respectively (Fig. 5C). Similarly, the peak level of SoGloI expression was detected at 12 hpt under ABA treatments, which was 2.4-fold of the level in control (Fig. 5C). Thus, SoGloI gene has been found to provide tolerance to multiple abiotic stresses.

Glyoxalase I activity in sugarcane under NaCl, CuCl₂, CdCl₂ or ZnSO₄ treatment

As shown in Fig. 5D, under NaCl, CuCl₂, CdCl₂, and ZnSO₄ treatments, the glyoxalase I activity was increased steadily from 0 to 48 hpt. Under a 250 mM NaCl treatment, the glyoxalase I activity was about 1.8-, 2.2-, and 2.3-fold at 12, 24, and 48 hpt comparing to control, respectively. At 48 hpt, the level of glyoxalase I activity reached 0.3230 µmol min⁻¹ mg⁻¹. Under a 750 µM CuCl₂ treatment, the level of glyoxalase I activity was about 2.0-, 2.7-, and 3.0-fold of the level in control, with 0.4128 µmol min⁻¹ mg⁻¹ protein produced at 48 hpt. Similarly, under a 750 µM CdCl₂ treatment, the glyoxalase I activity was 2.1-, 3.1- and 4.2-fold comparing to control, with a highest activity of 0.5730 µmol min⁻¹ mg⁻¹ protein at 48 hpt. Under 750 µM ZnSO₄ treatment, the glyoxalase I activity at 12, 24, and 48 hpt was about 1.3-, 2.6-, and 3.1-fold of the level at 0 hpt, and 0.2883 µmol min⁻¹ mg⁻¹ protein produced at 48 hpt. Thus, glyoxalase I activity was increased in varying degrees under salt and heavy metal ions stress conditions.

Determination of subcellular localization of ScGloI

To further understand the function of SoGloI gene, its subcellular localization was determined. The SoGloI gene was inserted into a plant expression vector pCXSN between the 35S promoter and GFP. The recombinant pCXSN-SoGloI-GFP construct was then introduced into tobacco leaves through Agrobacterium-mediated transformation. As shown in Fig. 6, green fluorescence signals were observable in the cytosol and nucleus of both pCXSN-SoGloI-GFP and the pCXSN-GFP transformed tobacco protoplasts.