Identification and expression analysis of nuclear factor Y transcription factor genes under drought, cold and Eldana infestation in sugarcane (Saccharum spp. hybrid).

BACKGROUND: The Nuclear Factor Y (NF-Y) transcription factor (TF) gene family plays a crucial role in plant development and response to stress. Limited information is available on this gene family in sugarcane. OBJECTIVES: To identify sugarcane NF-Y genes through bioinformatic analysis and phylogenetic association and investigate the expression of these genes in response to abiotic and biotic stress. METHODS: Sugarcane NF-Y genes were identified using comparative genomics from functionally annotated Poaceae and Arabidopsis species. Quantitative PCR and transcriptome analysis assigned preliminary functional roles to these genes in response to water deficit, cold and African sugarcane borer (Eldana saccharina) infestation. RESULTS: We identify 21 NF-Y genes in sugarcane. Phylogenetic analysis revealed three main branches representing the subunits with potential discrepancies present in the assignment of numerical names of some NF-Y putative orthologs across the different species. Gene expression analysis indicated that three genes, ShNF-YA1, A3 and B3 were upregulated and two genes, NF-YA4 and A7 were downregulated, while three genes were upregulated, ShNF-YB2, B3 and C4, in the plants exposed to water deficit and cold stress, respectively. Functional involvement of NF-Y genes in the biotic stress response were also detected where three genes, ShNF-YA6, A3 and A7 were downregulated in the early resistant (cv. N33) response to Eldana infestation whilst only ShNF-YA6 was downregulated in the susceptible (cv. N11) early response. CONCLUSIONS: Our research findings establish a foundation for investigating the function of ShNF-Ys and offer candidate genes for stress-resistant breeding and improvement in sugarcane.

Genetic manipulation of trehalose-6-phosphate synthase results in changes in the soluble sugar profile in transgenic sugarcane stems.

Trehalose is a non-reducing disaccharide widely distributed in nature. The trehalose biosynthetic intermediate, trehalose 6-phosphate (Tre6P) is an essential regulatory and signaling molecule involved in both regulation of carbon metabolism and photosynthesis. To investigate the effect of altered trehalose synthesis on sucrose accumulation in sugarcane (Saccharum spp. hybrid), we independently overexpressed the Escherichia coli otsA (trehalose-6-phosphate synthase; TPS) and otsB (trehalose-6-phosphate phosphatase; TPP) genes and additionally partially silenced native TPS expression. In mature cane, sucrose levels in the otsA transgenic plants were lowered, whereas sucrose levels in the otsB transgenic plants were increased. Partial silencing of TPS expression in sugarcane transformed with a TPS-targeted microRNA recombinant construct was confirmed in leaf and mature internode tissue of transgenic plants. Most of the silencing transgenic lines accumulated trehalose at lower levels than the wild-type (WT) plants. The immature stalk tissue of these transgenic lines had lower levels of glucose and fructose, whereas the mature internode tissue had lower sucrose and glucose levels, when compared with the WT. Furthermore, various minor metabolites and sugars were detected in the sugarcane plants, which mostly decreased as the stalk tissue of the cane matured. The results demonstrate that manipulation of Tre6P/trehalose metabolism has the potential to modify the profile of soluble sugars accumulated in sugarcane stems.

The SlNAC2 transcription factor from tomato confers tolerance to drought stress in transgenic tobacco plants.

Drought is a key environmental factor that restricts crop growth and productivity. Plant responses to water-deficit stress at the whole plant level are mediated by stress-response gene expression through the action of transcription factors (TF). The NAC (NAM/ATAF/CUC) transcription factor family has been well documented in its role in improving plant abiotic stress tolerance. In the present study we evaluated the effects of overexpression of SlNAC2 TF on the photosynthetic machinery, relative water content (RWC), reactive oxygen species, antioxidants and proline levels in tobacco plants exposed to a water-deficit treatment. Shoot growth and seed formation were also evaluated before, during and following water-deficit to determine any morphological consequences of transgene expression. The transgenic plants maintained higher RWC and chlorophyll levels over 21 days after withholding water and stomatal conductance until the 16th day of water-deficit. Overexpression of SlNAC2 in tobacco increased proline levels, improved seed setting and delayed leaf senescence of the transgenic plants. Reactive oxygen species accumulated at lower levels in the dehydrated transgenic plants but no significant difference in superoxide dismutase and catalase content were seen between the genotypes. The conversion of glutathione to oxidized glutathione was significantly higher in the transgenic plants, supported by increased glutathione reductase transcript levels. Our results indicate that overexpression of SlNAC2 in tobacco improved survival during and recovery from water-deficit stress, without an associated biomass penalty under irrigation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00996-2.

SlNAC2 overexpression in Arabidopsis results in enhanced abiotic stress tolerance with alteration in glutathione metabolism.

Plant NAC (NAM, ATAF, and CUC) transcription factors (TF) have important roles to play in abiotic stress responses through activation of a battery of functional genes/transcriptional regulators responsible for stress tolerance. Here we report the cloning of a novel Solanum lycopersicum L., NAC2 TF having 960 nucleotides long CDS (GenBank: KT740994.1). Phylogenetic analysis depicted the similarity of SlNAC2 to other orthologs. SlNAC2 was overexpressed in Arabidopsis thaliana to assess and characterize its role in plant abiotic stress responses. The transgenic events were first confirmed by genomic DNA PCR and qRT PCR; then the T3 generation plants were used for stress assays. Soil stress assay depicted better survivability of the transgenic plants under both salt (NaCl) and drought (PEG) stress. The transgenic plants showed enhanced endurance; with better antioxidative response, reduced accumulation of reactive oxygen species (ROS) molecules and better retention of water in tissue. This study for the very first time analyzed the different stakeholders of the glutathione metabolism in SlNAC2 overexpressing transgenic lines on exposure to both salinity and PEG stress. The expression of the two genes (ɤ-ECS, GS) responsible for glutathione biosynthesis increased with SlNAC2 overexpression. Further glutathione reductase responsible for reduction of glutathione disulfide (GSSG) to glutathione (GSH) also increased significantly which suggested the regulation of glutathione metabolism as a mechanism for the osmotic stress tolerance conferred to plants upon NAC overexpression.

Expression of a Small Ubiquitin-Like Modifier Protease Increases Drought Tolerance in Wheat (Triticum aestivum L.).

Post-translation modification of proteins plays a critical role in cellular signaling processes. In recent years, the SUMO (Small Ubiquitin-Like Modifier) class of molecules has emerged as an influential mechanism for target protein management. SUMO proteases play a vital role in regulating pathway flux and are therefore ideal targets for manipulating stress-responses. In the present study, the expression of an Arabidopsis thaliana cysteine protease (OVERLY TOLERANT TO SALT-1, OTS1) in wheat (Triticum aestivum L.) has led to improved plant growth under water stress conditions. Transformed wheat (pUBI-OTS1) displayed enhanced growth and delayed senescence under water deficit when compared with untransformed Gamtoos-R genotype or plants carrying an empty vector. Transformed pUBI-OTS1 plants also maintained a high relative moisture content (RMC), had a higher photosynthesis rate, and also had a higher total chlorophyll content when compared to untransformed plants or plants carrying an empty vector. SUMOylation of total protein also increased in untransformed plants but not in the AtOTS1 transformed plants. Our results suggest that SUMO-proteases may influence an array of mechanisms in wheat to the advantage of the crop to be more tolerant to water stress caused by drought. This is the first report to elucidate SUMOylation effects in the hexaploid crop wheat (T. aestivum L.).

Repression of Sex4 and Like Sex Four2 Orthologs in Potato Increases Tuber Starch Bound Phosphate With Concomitant Alterations in Starch Physical Properties.

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.

In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase.

Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 µg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 µg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus.

The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg-MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin-infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites.

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.

Oryzacystatin I expression in transformed tobacco produces a conditional growth phenotype and enhances chilling tolerance.

A recent strategy for pest control in plants has involved transformation with genes encoding cysteine proteinase inhibitors (cystatins). Little is known, however, about the effects of constitutive cystatin expression on whole plant physiology. The present study using oryzacystatin I (OC-I) expression in transformed tobacco was designed to resolve this issue and also to test the effects on abiotic stress tolerance. All transformed plants expressing OC-I showed a conditional phenotype. A marked effect on stem elongation was observed in plants grown under low light intensities. After 7 weeks of growth at low light, the plants expressing OC-I were smaller with fewer expanded leaves and a slightly lower total biomass than empty vector controls or wild type plants. Maximal rates of photosynthesis (A(max)) were also decreased, the inhibitory effect being greatest in the plants with highest OC-I expression. After 12 weeks of growth at low light, however, the plants expressing OC-I performed better in terms of shoot biomass production, which was nearly double that of the empty vector or wild type controls. All plants showed similar responses to drought, however photosynthesis was better protected against chilling injury in plants constitutively expressing OC-I. Photosynthetic CO(2) assimilation was decreased in all plants following exposure to 5 degrees C, but the inhibition was significantly less in the OC-I expressing plants than in controls. The transformed tobacco plants expressing OC-I therefore show a phenotype-environment interaction with important implications for biotechnological applications.