Plants generate pathogenesis-related (PR) proteins in response to pathogen infection. Beta-1,3-glucanase (EC 3.2.1.39), a well-known example of PR proteins, is widely distributed in higher plants (Leubner-Metzger, 2012). As reported, the disease-resistant effect of beta-1,3-glucanase can catalyze the hydrolytic cleavage of beta-1,3-glucans, a major structural component present in the fungal cell wall, and inhibit the growth of pathogens (Chen et al., 2006; Shi et al., 2006; Singh et al., 2014). Additionally, its hydrolysate oligosaccharide can be used as an elicitor to induce a chain of systemic resistance in plants, such as promoting the generation of many PR proteins and defense-related products (Chen et al., 2006; Shi et al., 2006; Singh et al., 2014). So far, various beta-1,3-glucanase genes from Arabidopsis thaliana (Escobar et al., 2003), Oryza sativa (Romero et al., 1998), Triticum aestivum (Kemp et al., 1999; Liu et al., 2010), and Zea mays (Jondle et al., 1989) have been induced by pathogen attack.

In addition to the proposed role in defense response, beta-1,3-glucanase may also be involved in diverse plant physiological and developmental processes, such as seed and pollen germination (Morohashi and Matsushima, 2000; Wan et al., 2011), bud dormancy (Rinne et al., 2001), flower growth and fruit ripening (Akiyama et al., 2004; Tao et al., 2013). The expression of beta-1,3-glucanase genes has been shown to be regulated by certain environmental stresses, such as pathogen infection (Gu et al., 2008; Liu et al., 2010), wounding (Wu and Bradford, 2003), salt (Su et al., 2013), and plant hormone stimuli (Akiyama and Pillai, 2001; Wu and Bradford, 2003; Liu et al., 2010). Liu et al. (2010) detected the beta-1,3-glucanase gene, TaGlu, in wheat that was induced by the stripe rust pathogen Puccinia striiformis f. sp. tritici, salicylic acid (SA), methyl jasmonate (MeJA), and ethylene (ET). Gu et al. (2008) demonstrated that the overexpression of tobacco chitinase I gene and the beta-1,3-glucanase gene in sugarcane (Saccharum spp.) had different inhibitory efficiencies on the growth of the sugarcane smut pathogen (Sporisorium scitamineum). Wu and Bradford (2003) found that the GluB gene showed a tissue-specific regulation in tomato seeds and leaves, and its gene expression level was slightly up-regulated by MeJA and wounding during tomato seed germination. Akiyama and Pillai (2001) have reported that the expression of an endo-1,3-beta-glucanase (OsGLN1) from rice was up-regulated by abscisic acid (ABA) and drought stress.

Beta-1,3-glucanases with multiple structural isoforms and are divided into four subfamilies (A, B, C, and D) according to molecular size, isoelectric point (pI), primary structure, cellular localization, and expression patterns (Romero et al., 1998). Thirteen beta-1,3-glucanase genes, among which nine were classified into subfamily A (Gns2, Gns3, Gns4, Gns5, and Gns6), subfamily B (Gns1), subfamily C (Gns7 and Gns8) and subfamily D (Gns9) according to their structure and function, have been identified from the rice genome (Romero et al., 1998). Six highly similar endo-beta-1,3-glucanase genes (TaGlb2a, TaGlb2b, TaGlb2c, TaGlb2d, TaGlb2e, and TaGlb2f) from wheat clustered within subfamily A were cloned and characterized. These six TaGlb2 genes were phylogenetically related to each other and were differentially regulated during development processes and in response to powdery mildew (Erysiphe graminis) and head blight (Fusarium graminearum) pathogens (Higa-Nishiyama et al., 2006). Shi et al. (2006) isolated two beta-1,3-glucanase genes (FaBG2-2 and FaBG2-3) from strawberry and observed different expression levels of these two genes under Colletotrichum fragariaeor and C. acutatum infection.

In our previous study, two sugarcane beta-1,3-glucanase genes ScGluA1 (GenBank Acc No. KC848050, subfamily A) and ScGluD1 (GenBank Acc No. KC848051, subfamily D) were detected from sugarcane post-inoculation with S. scitamineum (Su et al., 2013). We have also validated that both genes were located in the apoplast. Analysis of prokaryotic expression and gene expression patterns analysis indicated that ScGluA1 showed positive response to biotic and abiotic stimuli; however, ScGluD1 did not (Su et al., 2013). In this report, a new sugarcane beta-1,3-glucanase subfamily D gene, ScGluD2 was cloned and identified from the smut-resistant cultivar Yacheng05-179 infected by S. scitamineum for 2 days. Its phylogenetic features, subcellular localization, and expression profiles in sugarcane against S. scitamineum and different chemicals stimuli were analyzed. In addition, the transient expression of ScGluD2 in Nicotiana benthamiana was further investigated by conductivity measurement, DAB (3,3′-diaminobenzidine) staining, the detection of immunity associated marker genes expression, and the pathogen infection test according to a previous study (Choi et al., 2012), which reflect the function of ScGluD2 protein. This study aims to obtain a better understanding of the role of ScGluD2 in response to various adversity stresses.

Sugarcane accounts for 92% of the total sugar production in China. Sugarcane smut, which is caused by S. scitamineum, occurs widely in sugarcane planting areas and has become one of the most difficult fungal diseases to control (Sundar et al., 2012; Que et al., 2014b). To date, a limited number of disease resistant genes have been characterized in sugarcane. Beta-1,3-glucanase is known to be a typical PR2 protein which could be induced by pathogenic infection and plays a role in plant defense response (Cheong et al., 2000; Leubner-Metzger, 2012). In plant genomes, beta-1,3-glucanases are encoded as relatively large gene families which can be divided into four classes (Dobnik et al., 2013). The major achievement of this study was the isolation and identification of a novel beta-1,3-glucanase gene ScGluD2 from sugarcane. The cDNA of ScGluD2 has a complete ORF that encoded beta-1,3-glucanase protein of 494 amino acids. As reported, the X8 module is found at the C terminus of family 17 glycosyl hydrolases of beta-1,3-glucanase (Henrissat and Davies, 2000). This domain is characterized by a conserved distribution of six cys residues and a phe residue and is possibly involved in carbohydrate binding (Simpson et al., 2009). Similarly, the X8 domain with a 6Cys-box was present at the C terminal region of ScGluD2, suggesting beta-1,3-glucan-binding function.

Previous reports have noted an increase in the expression of various beta-1,3-glucanases in plants during pathogenic infection (Kemp et al., 1999; Liu et al., 2010; Su et al., 2013). Kemp et al. (1999) observed that the activity of beta-1,3-glucanase was induced in three near-isogenic wheat lines after being infected with P. recondita f. sp. tritici. A higher level of beta-1,3-glucanase activity was also detected in infected than in the non-infected wheat plant by Western blot analysis (Kemp et al., 1999). Sugarcane buds serve as the invasion route of smut pathogen. Spore germination occurs on the sugarcane internodal surface and then forms the appressoria on the inner scales of young buds (Sundar et al., 2012). After the teliospore deposition 6–36 h, S. scitamineum enters into the bud meristem (Alexander and Ramakrishnan, 1980; Sundar et al., 2012). It is worth mentioning in the present study that the elevated expression levels of ScGluD2 in sugarcane smut-resistant cultivars were presented in the early stage (1 or 3 days) more than in the susceptible ones (Figure 3), suggesting that ScGluD2 may participate in the resistance of sugarcane against S. scitamineum infection. This was similar to the findings reported by Liu et al. (2010), in which the transcripts of TaGlu gene were much higher in the incompatible interaction than in the compatible interaction between wheat and P. striiformis f. sp. tritici. Our previous study also demonstrated that beta-1,3-glucanase activity in the resistant sugarcane cultivar (Yacheng05-179) increased faster and lasted longer than that of the susceptible one (Liucheng03-182), and the transcripts of ScGluA1 and ScGluD1 were up-regulated and slightly down-regulated post-inoculation with S. scitamineum, respectively (Su et al., 2013). Although from the same subfamily D (Figure 2), the opposite responses to S. scitamineum stress between ScGluD2 and ScGluD1 implied there might be a functional diversity in the sugarcane beta-1,3-glucanase multigene family. Similarly, two highly homologous beta-1,3-glucanase genes, FaBG2-2 and FaBG2-3, were identified from strawberry, while the gene expression level of FaBG2-3 was remarkably higher than that of FaBG2-1 under C. fragariaeor and C. acutatum stresses (Shi et al., 2006).

In this study, ScGluD2 was induced by smut pathogen infection in sugarcane, which in turn warrants an investigation on the role of its encoding gene in further transient expression. The overexpression of ScGluD2 in N. benthamiana (Figures 6 and 7) indicated that it may be involved in plant defense. Levine et al. (1994) reported a close relationship between HR and the accumulation of H₂O₂. Bolwell et al. (2002) indicated that H₂O₂ accumulation could be considered as an early signal molecule in the interaction between plant and pathogen and has direct antimicrobial effect by inducing gene expression and hypersensitive cell death (HCD). Plant defense responses mediated by signal transduction pathways (such as reactive oxygen species, SA, JA, and ET) lead to the reinforcement of cell walls, the production of PR proteins and antimicrobial metabolites, and even the HR that limits the development of the pathogen (Dangl and Jones, 2001; Hwang and Hwang, 2011). These findings were also similar to those observed in this study whereby the increased ion conductivity (Figure 6A) and H₂O₂ accumulation (Figure 6B), the up-regulation of the HR marker genes NtHSR201 and NtHSR203, the JA associated genes NtPR-1a/c and NtPR2, and the ET synthesis-dependent genes NtEFE26 and NtAccdeaminase (Figure 6D), as well as the antimicrobial action on tobacco pathogens P. solanacearum and B. cinerea (Figure 7) in ScGluD2-expressing leaves. Similarly, Mercado et al. (2015) demonstrated that transgenic strawberry plants expressing the beta-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in increased tolerance to crown rot disease. Liu et al. (2013) reported that the crude protein extract of transgenic tobacco lines that carrying the PpGlu gene from the fruit of Pyrus pyrifolia Nakai cv. Huobali inhibited the hyphal growth of the Phomopsis sp., Alternaria sp., and Fusarium sp.

In addition to the pathogen, various abiotic stresses can induce different expression patterns of plant beta-1,3-glucanases at the transcription or protein level (Akiyama and Pillai, 2001; Wu and Bradford, 2003). Choudhury et al. (2010) found that the transcript of β-1,3-gluc during ripening in banana fruit was strongly increased under ET treatment and partially induced by ABA stress. On the other hand, β-1,3-gluc was not induced under wound treatment, and was even negatively regulated under auxin, cold and white light treatments. The expression of OsGLN1 from rice was up-regulated under ABA and drought stresses (Akiyama and Pillai, 2001). The levels of GluB transcripts were increased by MeJA and wounding in tomato seeds (Wu and Bradford, 2003). These findings suggest different functional properties among the members of beta-1,3-glucanases gene family in plants. Our group has also previously reported that the transcripts of ScGluA1 were up-regulated under SA, MeJA, ABA, NaCl, CdCl₂, and drought stresses, whereas ScGluD1 was nearly down-regulated (Su et al., 2013). In this study, the expression level of ScGluD2 was partially increased under NaCl treatment, and steadily up-regulated upon ABA, H₂O₂, and CdCl₂ stimuli (Figure 4). It is known that the broad-spectrum plant hormone ABA regulates plant development and physiology and responds to various environmental stresses such as salinity, drought, hypoxic and heavy metals contamination, and pathogen infection (Finkelstein et al., 2002; Pompeu et al., 2016). Scores of stress-responsive genes are up-regulated by ABA in osmotic imbalance (Ingram and Bartels, 1996). Oxidative stress is reported to be highly controlled by phytohormones (Pompeu et al., 2016). Cadmium (Cd) is one of the most toxic heavy metals which negatively affecting plant metabolism mainly by inducing oxidative stress (Cuypers et al., 2010; Pompeu et al., 2016). Han et al. (2012) indicated that ABA induced the tolerance of T. aestivum seedlings response to Cd stress. Our data suggests that ABA may be a signal molecule regulating oxidative stress and may play a role in the salt and heavy metal stress-induced stimulation of ScGluD2 transcripts. This is similar to the observations of Cheong et al. (2000) in which the expression of SGN1 from soybean was strongly induced by a variety of defense-related signals including H₂O₂, wounding, the fungal elicitor from Phytophthora parasitica (Pmg), and the pathogen P. syringae pv. glycinea (Psg). However, the SGN1 transcripts were barely induced upon SA, JA, and ET stresses (Cheong et al., 2000).

As is known, sugarcane is a highly polyploidy and aneuploidy crop (Scortecci et al., 2012). Selection of new resistant sugarcane clones with high yield and sucrose content needs a large segregation population of progeny and takes more than 10 years in conventional breeding (Dal-Bianco et al., 2012; Scortecci et al., 2012). Most modern sugarcane cultivars originate from crosses between a small number of original ancestor clones, resulting in a narrow genetic base (Jackson, 2005). These constraints create challenges for sugarcane genetic improvement (Lakshmanan et al., 2005; Scortecci et al., 2012). There are two major advantages for transgenic sugarcane, including the following: (i) sugarcane is an industrial crop and its sucrose production, which is chemically refined at 107°C, is the only consumer good that results in low-risk transgenic sugarcane; (ii) the short breeding process for its asexual reproduction to routinely multiply and maintain the genetically modified clones makes the transgenic technology using candidate genes an effective approach in transmitting commercially desirable traits to an elite variety (Falco et al., 2000; Henry and Kole, 2010; Dal-Bianco et al., 2012; Gómez-Merino et al., 2014). To date, a number of transgenic sugarcane transformed with genes expressing sugar improvement (Chong et al., 2007; Wu and Birch, 2007), herbicide resistance (Gallo-Meagher and Irvine, 1996; Manickavasagam et al., 2004), insect resistance to sugarcane stem borer (Diatraea saccharalis F.; Arencibia et al., 1997; Weng et al., 2006; Gao et al., 2016) and wooly aphid (Ceratovacuna lanigera Z.; Zhangsun et al., 2008), disease resistance to Sugarcane mosaic virus (SCMV; Gilbert et al., 2005) and Xanthomonas albilineans (Zhang et al., 1999), abiotic stress tolerance to drought (Zhang et al., 2006; Molinaria et al., 2007), and recombination protein of ER-targeted human cytokine protein GM-CSF (Wang et al., 2005) and aromatic hydroxybenzoic acid (pHBA; Petrasovits et al., 2007) have been successfully developed. Although traits of interest are being tested in various sugarcane cultivated countries, no commercial transgenic sugarcane line has been reported (Cheavegatti-Gianotto et al., 2011; Dal-Bianco et al., 2012; Ye et al., 2016), except for three drought-tolerant sugarcane transgenic events, NXI-1T, NXI-4T, and NXI-6T, which have been approved in Indonesia⁸ but without commercial cultivation.

The process of transformation has limitation because of its complex engineering system, involving the selection of receptor cells, exogenous gene, transformation methods, and screening system (Chen et al., 2011). As reported, there are several problems in the field of transgenic sugarcane research such as low transformation efficiency and transgene expression, single target gene and promoter, and the rarely investigated biosafety issue (Chen et al., 2011; Dal-Bianco et al., 2012; Scortecci et al., 2012). Therefore, the focus of future sugarcane transformation research should take into account the following aspects: (i) screening suitable recipient materials and optimizing the transformation efficiency, (ii) isolating new promoter regions (both constitutive and inducible) and vectors to improve the expression efficiency of the target gene, (iii) co-transforming multi-gene to achieve an elite transgenic sugarcane cultivar with a variety of traits at the same time, (iv) employing the security screening or no screening marking transformation method to make the transgenic sugarcane be more secure (Chen et al., 2011; Dal-Bianco et al., 2012; Scortecci et al., 2012; Gómez-Merino et al., 2014). Future developments would be expected to widen the application prospect of transgenic sugarcane. At present, although low transformation efficiency remains one of the major limiting factors in sugarcane production (Dal-Bianco et al., 2012; Scortecci et al., 2012; Gómez-Merino et al., 2014), the process of commercialization for transgenic sugarcane largely depends on mining for functional genes, particularly key functional genes that can significantly improve important agronomic or industrial traits. The findings of the present study suggest that elevated levels of ScGluD2 transcripts reflect a stress response. Whether this gene plays a direct causal role in defense against smut pathogen and/or salt and heavy metal stimuli needs further investigation. Besides, using model plant species systems that possess shorter life cycles and simpler genomes such as Brachypodium distachyon and Setaria italica, which belonging to Gramineae together with sugarcane, may be utilized as an alternative in future transgenic sugarcane experiments for the functional identification of new genes (Dal-Bianco et al., 2012).

The present study showed that a ScGluD2 cDNA encoded a novel sugarcane acidic subfamily D beta-1,3-glucanase. It contained a X8 domain at the C terminus and was localized to the plasma membrane, indicating beta-1,3-glucan-binding function. The ScGluD2 expression was rapidly induced by smut pathogen infection at the early stage in sugarcane smut-resistant cultivar-S. scitamineum interaction, suggesting its involvement in the defense against the invasion of S. scitamineum. The transient overexpression and the antimicrobial action test in N. benthamiana aim to further but partly determine the function of ScGluD2 protein, which indicated that it is related to plant resistance to pathogen inoculations. In addition, we speculated its potential role in protecting sugarcane from salt and heavy metal stresses based on the observation that ScGluD2 was up-regulated by ABA, H₂O₂, NaCl, and CdCl₂. Notably, ScGluD2 is differently regulated in comparison with sugarcane beta-1,3-glucanase genes ScGluA1 and ScGluD1 due to their different responses to adverse stimuli. These results revealed that ScGluD2 is a novel sugarcane stress-related gene involved in the defense response against smut pathogen infection and salt and heavy metal stresses.

YS, LX, and YQ conceived and designed the experiments. YS, ZW, FL, ZL, QP, and JG performed the experiments. YS analyzed the data and wrote the paper. LX and YQ revised the paper. All authors read and approved the final version of the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.