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

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.


INTRODUCTION
Ubiquitously occurring in nature, the glyoxalase pathway involves a two-step catalytic reaction. In the first step, glyoxalase I (GLOI, lactoylglutathione lyase; EC 4.4.1.5) catalyzes the isomerization of hemithioacetal formed spontaneously between methylglyoxal (MG) and reduces glutathione (GSH) to S-D-lactoylglutathione (S-LG) (Thornalley, 1993). In the second step, S-LG is hydrolyzed by glyoxalase II (GLOII, hydroxyacylglutathione hydrolase; EC 3.1.2.6) to produce GSH and D-lactate (Yadav et al., 2005a;Yadav et al., 2005b). Under normal physiological conditions, MG is produced primarily through glycolysis at the triose-phosphate step (Phillips & Thornalley, 1993), and to a much lesser extent, through catabolism of amino acids (threonine and glycine) and acetone (Yadav et al., 2005a;Yadav et al., 2005b;Yadav et al., 2007). Under abiotic stresses, however, the concentration of MG in plants can significantly increase by 2-6 folds (Yadav et al., 2005a). The high-level accumulation of MG is toxic to cells, as it can react with DNA to form modified guanylate residues (Papoulis, Al-Abed & Bucala, 1995). MG also can react with proteins to form glycosylamine derivatives of arginine, lysine and hemithioacetal with cysteine residues (Lo et al., 1994). Apart from the direct effect of MG, its intermediate compound S-LG, a substrate for glyoxalase II, is also cytotoxic at higher concentrations by inhibiting DNA synthesis (Thornalley, 1996). Therefore, glutathione-based detoxification of harmful metabolites is one of the main roles of both glyoxalase enzymes (Thornalley, 1990).
The glyoxalase I enzyme is broadly categorized into Zn 2+ -or Ni 2+ -dependent class of metal activation. Previous studies have been showed that the Zn 2+ -dependent GLOI enzymes are thought to be of eukaryotic origin (Frickel et al., 2001;Ridderström & Mannervik, 1996), while Ni 2+ -dependent GLOI enzymes are thought to be of prokaryotic origin (Sukdeo et al., 2004). The coexistence of both Zn 2+ -dependent and Ni 2+ -dependent GLOI enzymes in Pseudomonas aeruginosa (Sukdeo & Honek, 2007) and the characterization of a Ni 2+ -dependent GLOI enzyme from rice (Mustafiz et al., 2014) have led to the discouragement of the view that Zn 2+ -dependent GLOI belongs to eukaryotes and Ni 2+ -dependent GLOI exists only in prokaryotes (Jain et al., 2016). In plants, the metal specificity of each member of the GLOI family is an important determinant of its catalytic efficiency (Kaur et al., 2017;Mustafiz et al., 2014).
Sugarcane (Saccharum spp. hybrids) is a cash crop mainly used for sugar, biofuel and other food industries such as industrial alcohol in tropical and subtropical regions. It is one of the world's largest crops. According to the FAO, sugarcane was cultivated in 101 countries on about 26.1 million hectares of land in 2012 (Que et al., 2014). However, the yields of sugarcane are often influenced by many diseases and various environmental stresses, such as smut, rust, ratoon stunting disease (RSD), salt, heavy metal and drought. Sugarcane is reportedly susceptible to salt and shows toxicity symptoms, low sprout emergence, nutritional imbalance, and overall biomass reduction (Akhtar, Wahid & Rasul, 2003;Plaut, Meinzer & Federman, 2000;Wahid, Rao & Rasul, 1997). Though sugarcane plants can overcome a short period of water deficit during the late, sucrose accumulating growth stage, an extended period of drought can cause a significant loss in cane and sugar yields (Begcy et al., 2012). RSD causes significant yield losses, 12%-37% under normal conditions and up to 60% under drought conditions. Moreover, it may also lead to variety deterioration (Bailey & Bechet, 1997;James, 1996;Que et al., 2008). Sugarcane smut also causes serious losses in cane and sugar yields (Hoy et al., 1986;Padmanaban, Alexander & Shanmugan, 1998;Que et al., 2012).
In our previous study, we constructed a sugarcane cDNA library from Sporisorium scitamineum-infected buds (Wu et al., 2013b). An expressed sequence tag of 613 bp (GenBank Accession Number: CA140600.1) had a high similarity to the GLOI gene of Zea mays (GenBank Accession Number: EU966885.1) (Wu et al., 2013b). To study stress response of GLOI in sugarcane, we cloned the entire sugarcane Glyoxalase I gene, designated as SoGloI. We determined the sub-cellular location of the SoGloI 's protein using tobacco protoplasts and investigated growth patterns of Escherichia coli Rosetta cells producing the SoGloI recombinant protein in response to salt and heavy metal ion stresses. We also assessed SoGloI expression and glyoxalase I enzyme in sugarcane in response to simulated biotic and abiotic stresses. The results provided valuable information for the improvement of stress resistance in sugarcane.

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.

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.  et al., 2004;Que et al., 2009a;Que et al., 2009b).
Spot assays were performed to assess the response of pET32a-SoGloI transformed E. coli cells to NaCl, CdCl 2 , CuCl 2 or ZnSO 4 treatments. When the E. coli culture mixture reached OD 600 = 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 (OD 600 ), 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, CdCl 2 at 250, 500 and 750 µM, CuCl 2 at 250, 500 and 750 µM, and ZnSO 4 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 N 2 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 MgSO 4 , 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) andHasanuzzaman, 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 MgCl 2 and 10 mM fatty acid methyl ester sulfonate (MES). The concentration of the bacterial suspension was measured and adjusted to OD 600 = 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 CaCl 2 , 0.35 M mannitol, 0.7 mM NaH 2 PO 4 , 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).

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.

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, CuCl 2 , CdCl 2 , ZnSO 4 , SA, MeJA, and ABA treatments were shown in Figs. 5B and 5C. Under NaCl, CuCl 2 , CdCl 2 , and ZnSO 4 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, CuCl 2 , CdCl 2 or ZnSO 4 treatment
As shown in Fig. 5D, under NaCl, CuCl 2 , CdCl 2 , and ZnSO 4 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 CuCl 2 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 CdCl 2 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 ZnSO 4 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.

DISCUSSION
Glyoxalase I functions to detoxify the potent cytotoxic compound MG (Thornalley, 1993).
In response to stress conditions, cells undergo active metabolism to produce more MG through leakages in the glycolysis and TCA cycle (Umea et al., 1994). GlyI, the first enzyme of the glyoxalase system, plays a critical role in controlling MG levels and cytotoxicity (Wu et al., 2013a). The GloI gene has been cloned and characterized from several plant species. However, the glyoxalase I gene was never cloned and characterized in sugarcane. In the present study, a length GloI gene, designated as SoGloI, was isolated from a smut-resistant sugarcane cultivar YCE 05-179. The GloI enzyme requires Ni 2+ or/and Zn 2+ for its catalytic activity (Sukdeo et al., 2004). Sukdeo & Honek (2007) reported that Pseudomonas aeruginosa, a gamma proteobacteria, encodes both Ni 2+ and Zn 2+ forms of the enzyme; GloA1, GloA2 (both Ni binding), and GloA3 (Zn binding). Jain et al. (2016) also found three active GLYI enzymes (AtGLYI2, AtGLYI3 and AtGLYI6) belonging to different metal activation classes coexisting in Arabidopsis thaliana. AtGLYI2 was found to be Zn 2+ -dependent, whereas AtGLYI3 and AtGLYI6 were Ni 2+ -dependent. Ni 2+ -dependent GloI is present as a two-domain protein in all eukaryotes. Among the early branching eukaryotes, the group of algae appears to be the first to encode this gene (Kaur et al., 2013). In this study, a sugarcane SoGloI gene was found to encode two glyoxalase domains as well (Fig. S2). Besides, the multiple protein sequence alignment of SoGloI with those from other species indicated that SoGloI was a Ni 2+ -dependent enzyme (Fig. 1). The result was similar to OsGLYI -11.2 (Mustafiz et al., 2014), who's expression was substrate inducible. However, unlike other eukaryotic Zn 2+ -dependent glyoxalases, OsGLYI -11.2 is a Ni 2+ -dependent monomeric enzyme. Plant glyoxalase system in different tissues plays an important role at various vegetative and reproductive stages (Mustafiz et al., 2011). GLOI gene is required for cell division and proliferation; a higher enzyme activity has been found in rapidly dividing cells of cell suspensions, seedlings, and root tips (Lin et al., 2010;Wu et al., 2013a). In this study, SoGloI was constitutively expressed in various tissues of sugarcane genotype YCE 05-179, with the highest level in buds, followed by leaves, roots, leaf sheaths, and internodes (Fig. 5A).
To date, only a few reports have shown that GLOI gene is associated with disease resistance in plants. For instance, a maize Glx-I gene enhances the host defense against Aspergillus flavus through the detoxification of MG, a major product of A. flavus (Chen et al., 2004). The expression of wheat TaGly I is up-regulated 2.3-fold upon infection by Fusarium graminearum (Lin et al., 2010). In our previous study, SoGloI expression was up-regulated during infection with S. scitamineum, the pathogen of sugarcane smut (Wu et al., 2013b). In the present study, we used SA and MeJA to simulate biotic stress. Consistently, under SA and MeJA treatments, the SoGloI expression peaked at 6 hpt, when its activity reached 2.6-and 2.1-fold higher than that of the control, respectively (Fig. 5C). These results suggest that SoGloI expression can increase significantly under pathogenic stresses; however, the exact role of SoGloI in pathogenic resistance process needs to be further investigated.
Glyoxalase I genes also have been implicated to enhance plant tolerance to salt stress. The expression of Gly, a glyoxalase I gene of B. juncea, is up-regulated after exposure to a high concentration of salt (Veena, Reddy & Sopory, 1999). The mRNA and polypeptide levels of GLX1, a glyoxalase I gene of tomato, increased by two to three folds in roots, internodes and leaves when the plants were treated with 10 g/L NaCl (Espartero, Sanchez-Aguayo & Pardo, 1995). The expression of two other glyoxalase I genes, Bv M14-glyoxalase I of sugar beet (Wu et al., 2013a) and TaGly I of wheat (Lin et al., 2010), also significantly enhanced hosts' tolerance to salt stress. In this study, SoGloI -expressing Rosetta cells grown on agar plates tolerated high concentrations of NaCl up to 250 mM (Fig. 3B) and grew faster in LB liquid medium containing 250 mM NaCl (Fig. 4A). SoGloI expression was increased steadily from 0 to 48 hpt in sugarcane under salt stress (Fig. 5B). Under salt stress, glyoxalase I activity also elevated (Fig. 5D). Taken together, the results indicate the expression level of SoGloI can be significantly up-regulated under salt stress; however, more research is needed to reveal the underlying mechanism.
Zinc (Zn 2+ ), a micronutrient, is necessary for plant growth, but an excessive amount of Zn 2+ can inhibit plant growth (Sun et al., 2006;Zarcinas et al., 2004). A few studies have demonstrated that plant GloI genes enhance host tolerance to Zn 2+ . Singla-Pareek et al. (2006) showed that GlyI from B. juncea enhanced host Zn 2+ tolerance to toxic levels in the transgenic tobacco. The expression of TaGly I, a glyoxalase I gene of T. aestivum, is induced continuously under 20 mM ZnCl 2 treatment. Compared to control, the increase in TaGly I expression is nearly 1.5-fold at 24 h (Lin et al., 2010). In the present study, SoGloIexpressing E. coli Rosetta cells were able to tolerate high concentrations of ZnSO 4 up to 750 µM (Fig. 3E) and also grew faster in LB liquid medium containing 750 µM ZnSO 4 (Fig. 4D).
Consistently, under ZnSO4 stress, the SoGloI expression in sugarcane was up-regulated steadily from 0 to 48 hpt, when its level and enzyme activity were 1.9-fold and 3.1-fold higher than that of the control (Figs. 5B, 5D). These results showed that SoGloI gene can enhance tolerance to excessive zinc stress even in a heterologous host system. Over-expression of glyoxalase I has been shown to confer tolerance to other heavy metals, such as cadmium or lead (Singla-Pareek et al., 2006). The level of expression and activity of SoGloI in E. coli (Figs. 3D, 4C) under CdCl 2 treatment (Figs. 5B, 5D) also supported this notion about tolerance to cadmium. Our work further showed that SoGloI expression and its enzyme activity were increased significantly under CuCl 2 treatment (Figs. 5B, 5D). All these findings suggest that SoGloI may be a good candidate gene for engineering to develop heavy metal resistant sugarcane cultivars.
As is known, sugarcane is a polyploidy and aneuploidy crop (Scortecci et al., 2012), in which low transformation efficiency remains one of the major limiting factors on transgenic sugarcane production (Dal-Bianco et al., 2012;Gómez-Merino, Trejo-Téllez & Sentíes-Herrera, 2014;Scortecci et al., 2012). This has also limited the functional analysis of isolated sugarcane genes; nonetheless, a model plant species (Arabidopsis thaliana, Nicotiana benthamiana or Brachypodium distachyon) with a shorter life cycle and simpler genome can be explored as an alternative host for transforming and assessing the functional properties of isolated sugarcane genes, such as SoGloI.

CONCLUSIONS
This is the first report on the cloning and characterization of glyoxalase I (SoGloI ) gene in sugarcane. We isolated and characterized SoGloI gene and demonstrated the enzyme activity of glyoxalase I protein. We found that SoGloI expression and SoGloI enzymatic activity were elevated significantly when sugarcane tissues were subject to simulated biotic and abiotic stress conditions, such as high concentrations of salt or heavy metal ions. The findings have opened up a new research avenue for sugarcane to grow in polluted or salty environments via genetic engineering and breeding of SoGloI to enhance host resistance.