Transgenic sugarcane overexpressing Glyoxalase III improved germination and biomass production at formative stage under salinity and water-deficit stress conditions.

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 III in 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-GFP fusion 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 III activity is a useful strategy to improve the salinity and drought-tolerance in sugarcane as well as other crops.

Isolation of 5' regulatory region of COLD1 gene and its functional characterization through transient expression analysis in tobacco and sugarcane.

Chilling Tolerant Divergence 1 (COLD1) gene consists of Golgi pH Receptor (GPHR) as well as Abscisic Acid-linked G Protein-Coupled Receptor (ABA_GPCR), which are the major transmembrane proteins in plants. This gene expression has been found to be differentially regulated, under various stress conditions, in wild Saccharum-related genera, Erianthus arundinaceus, compared to commercial sugarcane variety. In this study, Rapid Amplification of Genomic Ends (RAGE) technique was employed to isolate the 5' upstream region of COLD1 gene to gain knowledge about the underlying stress regulatory mechanism. The current study established the cis-acting elements, main promoter regions, and Transcriptional Start Site (TSS) present within the isolated 5' upstream region (Cold1P) of COLD1, with the help of specific bioinformatics techniques. Phylogenetic analysis results revealed that the isolated Cold1P promoter is closely related to the species, Sorghum bicolor. Cold1P promoter-GUS gene construct was generated in pCAMBIA 1305.1 vector that displayed a constitutive expression of the GUS reporter gene in both monocot as well as dicot plants. The histochemical GUS assay outcomes confirmed that Cold1P can drive expression in both monocot as well as dicot plants. Cold1P's activities under several abiotic stresses such as cold, heat, salt, and drought, revealed its differential expression profile in commercial sugarcane variety. The highest activity of the GUS gene was found after 24 h of cold stress, driven by the isolated Cold1P promoter. The outcomes from GUS fluorimetric assay correlated with that of the GUS expression findings. This is the first report on Cold1P isolated from the species, E. arundinaceus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03650-8.

Overexpression of Glyoxalase III gene in transgenic sugarcane confers enhanced performance under salinity stress.

The glyoxalase pathway is a check point to monitor the elevation of methylglyoxal (MG) level in plants and is mediated by glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes in the presence of glutathione. Recent studies established the presence of unique DJ-1/PfpI domain containing protein named glyoxalase III (Gly III) in prokaryotes, involved in the detoxification of MG into D-lactic acid through a single step process. In the present study, eleven transgenic sugarcane events overexpressing EaGly III were assessed for salinity stress (100 mM and 200 mM NaCl) tolerance. Lipid peroxidation as well as cell membrane injury remained very minimal in all the transgenic events indicating reduced oxidative damage. Transgenic events exhibited significantly higher plant water status, gas exchange parameters, chlorophyll, carotenoid, and proline content, total soluble sugars, SOD and POD activity compared to wild type (WT) under salinity stress. Histological studies by taking the cross section showed a highly stable root system in transgenic events upon exposure to salinity stress. Results of the present study indicate that transgenic sugarcane events overexpressing EaGly III performed well and exhibited improved salinity stress tolerance.

Ectopic expression of DJ-1/PfpI domain containing Erianthus arundinaceus Glyoxalase III (EaGly III) enhances drought tolerance in sugarcane.

KEY MESSAGE: Sugarcane transgenic overexpressing EaGly III from Erianthus arundinaceus showed enhanced water deficit stress tolerance. Methylglyoxal (MG), an α-ketoaldehyde formed from either glycolysis or TCA cycle, is capable of causing total cellular damage via the generation of reactive oxygen species (ROS), advanced glycation end products (AGEs) and nucleic acid degradation. Glyoxalase pathway is a ubiquitous pathway known for detoxification of MG, involving key enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Recently, a novel and an additional enzyme in glyoxalase pathway, viz., glyoxalase III (Gly III), has been discovered which possesses DJ-1/PfpI domain recognized for detoxifying MG in a single step process without requirement of any coenzyme. In the present study, a Gly III gene isolated from Erianthus arundinaceus, a wild relative of sugarcane, overexpressed in commercially cultivated sugarcane hybrid Co 86032 was assessed for drought tolerance. Morphometric observations revealed that transgenic sugarcane overexpressing EaGly III acquired drought tolerance trait. Oxidative damage caused by triggering generation of ROS has been determined to be low in transgenic plants as compared to wild type (WT). Transgenics resulted in higher relative water content, chlorophyll content, gas exchange parameters, photosynthetic efficiency, proline content and soluble sugars upon water deficit stress. In addition, higher and stable level of superoxide dismutase and peroxidase activities were observed along with minimal lipid peroxidation during drought stress signifying the tolerance mechanism exhibited by transgenic events. There was no significant structural change observed in the root anatomy of transgenic plants. Altogether, EaGly III gene could be considered as a potential candidate for conferring water deficit stress tolerance for sugarcane and other agricultural crops.

Isolation, characterization and expression analysis of stress responsive plant nuclear transcriptional factor subunit (NF-YB2) from commercial Saccharum hybrid and wild relative Erianthus arundinaceus.

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 factor NF-YB2 was 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 Saccharum hybrid Co 86032 (ShNF-YB2) was isolated and characterized. Both EaNF-YB2 and ShNF-YB2 genes are 543 bp long that encodes for a polypeptide of 180 amino acid residues. Comparison of EaNF-YB2 and ShNF-YB2 gene 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-YB2 expression in E. arundinaceus when compared to that in the commercial Saccharum hybrid Co 86032 under drought and salinity stresses. Hence, EaNF-YB2 could be an ideal candidate gene, and its overexpression in sugarcane through genetic engineering approach might enhance tolerance to drought and salinity stresses.

Differential gene expression profiling through transcriptome approach of Saccharum spontaneum L. under low temperature stress reveals genes potentially involved in cold acclimation.

Sugarcane (Saccharum sp.) is predominantly grown in both tropics and subtropics in India, and the subtropics alone contribute more than half of sugarcane production. Sugarcane active growth period in subtropics is restricted to 8-9 months mainly due to winter's low temperature stress prevailing during November to February every year. Being a commercial crop, tolerance to low temperature is important in sugarcane improvement programs. Development of cold tolerant sugarcane varieties require a deep knowledge on molecular mechanism naturally adapted by cold tolerant genotypes during low temperature stress. To understand gene regulation under low temperature stress, control and stressed (10 °C, 24 h) leaf samples of cold tolerant S. spontaneum IND 00-1037 collected from high altitude region in Arunachal Pradesh were used for transcriptome analysis using the Illumina NextSeq 500 platform with paired-end sequencing method. Raw reads of 5.1 GB (control) and 5.3 GB (stressed) obtained were assembled using trinity and annotated with UNIPROT, KEGG, GO, COG and SUCEST databases, and transcriptome was validated using qRT-PCR. The differential gene expression (DGE) analysis showed that 2583 genes were upregulated and 3302 genes were down-regulated upon low temperature stress. A total of 170 cold responsive transcriptional factors belonging to 30 families were differentially regulated. CBF6 (C-binding factor), a DNA binding transcriptional activation protein associated with cold acclimation and freezing tolerance was differentially upregulated. Many low temperature responsive genes involved in various metabolic pathways, viz. cold sensing through membrane fluidity, calcium and lipid signaling genes, MAP kinases, phytohormone signaling and biosynthetic genes, antioxidative enzymes, membrane and cellular stabilizing genes, genes involved in biosynthesis of polyunsaturated fatty acids, chaperones, LEA proteins, soluble sugars, osmoprotectants, lignin and pectin biosynthetic genes were also differentially upregulated. Potential cold responsive genes and transcriptional factors involved in cold tolerance mechanism in cold tolerant S. spontaneum IND 00-1037 were identified. Together, this study provides insights into the cold tolerance to low temperature stress in S. spontaneum, thus opening applications in the genetic improvement of cold stress tolerance in sugarcane.

Construction of a novel synthetic root-specific promoter and its characterization in transgenic tobacco plants.

Synthetic promoter technology offers a framework for designing expression cassettes that could provide precise control of transgene expression. Such artificially designed promoters enable defined transgene regulation, reduce unwanted background expression, and can overcome homology-dependent gene silencing in transgenic plants. In the present study, a synthetic root-specific module was designed using characterized cis-acting elements, fused with minimal promoter (86 bp) from PortUbi882 promoter, and cloned in pCAMBIA1305.1 by replacing CaMV 35S promoter so as to drive GUS expression. Two constructs were made; one had the synthetic module at the 5' end of the minimal promoter (SynR1), whereas in the other construct, the module was present in both 5' and 3' ends (SynR2). Furthermore, the synthetic promoter constructs were transformed in tobacco wherein SynR1 promoter drove constitutive expression, whereas SynR2 conferred root-specific expression though slight leaky expression was present in stem. GUS assay in the roots of transgenic tobacco plants (T1) indicated that SynR2 promoter expressed significantly higher GUS activity than the CaMV 35S promoter. The real-time quantitative PCR (RT-qPCR) analysis of GUS gene further confirmed that SynR2 promoter conferred 2.1-fold higher root-specific expression when compared to CaMV 35S promoter.