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The glyoxalase system, involving Glyoxalase I (GlyI) and Glyoxalase II (Gly II), plays a vital role in abiotic stress tolerance in plants. A novel enzyme Glyoxalase III (Gly III) was found recently from bacteria, yeast, and plant species. This enzyme provides a new way to detoxify Methylglyoxal (MG), a cytotoxic α-oxoaldehyde, which, in excess, can cause complete cell destruction by forming Reactive Oxygen Species (ROS) and Advanced Glycation End products (AGEs) or DNA/RNA mutation. In this background, the current study examined sugarcane transgenic events that exhibit an increase in expression of EaGly III, to assess their performance in terms of germination and biomass production during formative stage under stress conditions. Southern blot analysis outcomes confirmed the integration of transgene in the transgenic plants. The results from quantitative RT-PCR analyses confirmed high expression levels of EaGly IIIin transgenic events compared to wild type (WT) under salinity (100 and 200 mM NaCl) and drought (withholding watering) conditions. Transgenic events exhibited enhanced biomass productivity ranged between 0.141 Kg/pot and 0.395 Kg/pot under 200 mM salinity and 0.262 Kg/pot and 0.666 Kg/pot under drought stress. Further, transgenic events observed significantly higher germination rates under salinity and drought conditions compared to that of WT. Subcellular localization prediction by EaGlyIII-GFPfusion expression in sugarcane callus showed that it is distributed across the cytoplasm, thus indicating its widespread activity within the cell. These results strongly suggest that enhancing EaGly IIIactivity is a useful strategy to improve the salinity and drought-tolerance in sugarcane as well as other crops.

Plant nuclear factor (NF-Y) is a transcription activating factor, consisting of three subunits, and plays a key regulatory role in many stress-responsive mechanisms including drought and salinity stresses. NF-Ys function both as complex and individual subunits. Considering the importance of sugarcane as a commercial crop with high socio-economic importance and the crop being affected mostly by water deficit stress and salinity stress causing significant yield loss, nuclear transcriptional factorNF-YB2was focused in this study. Plant nuclear factor subunit B2 from Erianthus arundinaceus(EaNF-YB2), a wild relative of sugarcane which is known for its drought and salinity stress tolerance, and commercial Saccharumhybrid Co 86032 (ShNF-YB2) was isolated and characterized.Both EaNF-YB2and ShNF-YB2genes are 543 bp long that encodes for a polypeptide of 180 amino acid residues. Comparison of EaNF-YB2and ShNF-YB2gene sequences revealed nucleotide substitutions at nine positions corresponding to three synonymous and six nonsynonymous amino acid substitutions that resulted in variations in physiochemical properties. However, multiple sequence alignment (MSA) of NF-YB2 proteins showed conservation of functionally important amino acid residues. In silico analysis revealed NF-YB2 to be a hydrophilic and intracellular protein, and EaNF-YB2 is thermally more stable than that of ShNF-YB2. Phylogenetic analysis suggested the lower rate of evolution of NF-YB2. Subcellular localization in sugarcane callus revealed NF-YB2 localization at nucleus that further evidenced it to be a transcription activation factor. Comparative RT-qPCR experiments showed a significantly higher level of NF-YB2expression in E. arundinaceuswhen compared to that in the commercial Saccharumhybrid Co 86032 under drought and salinity stresses. Hence, EaNF-YB2could be an ideal candidate gene, and its overexpression in sugarcane through genetic engineering approach might enhance tolerance to drought and salinity stresses.