Molecular cloning and in silico analysis of heat stress responsive gene ClpB1 from Ziziphus nummularia genotypes

Heat stress is one of the most destructive abiotic stresses which adversely affect crop plants, resulting in reduced potential yield. Plants that are able to tolerate heat stress possess an intrinsic mechanism which needs to be unravelled at molecular level so as to decipher the role of gene and metabolic pathways involved in heat stress tolerance. To understand the molecular mechanism of heat stress tolerance, studies on isolation and characterization of gene for abiotic stress tolerance, ClpB1 were performed in Ziziphus nummularia (Burm. f.) Wight & Arn, an inherently abiotic stress tolerant plant. Differential expression studies of gene ClpB1 by qRT-PCR in contrasting genotypes of Z. nummularia (genotype Jaisalmer: heat tolerant and genotype Godhra: heat sensitive) was carried out. CDS (Coding DNA sequence) of gene ClpB1 from the genotypes Z. nummularia J and Z. nummularia G were cloned and characterized. These genes ZnJClpB1 (ACNO: MN398267) and ZnGClpB1 (ACNO: MN398268) showed 1.09 and 2.3% dissimilarity at nucleotide and amino acid level, respectively. Computational based analysis revealed the presence of larger functional AAA lid 9 domains in ZnJClpB1 as compared to ZnGClpB1. Phylogenetic relationship and structure modeling was performed to understand isoform type and basic molecular functioning and of gene ZnClpB1 from Z. nummularia genotypes. Possibly, it is the first report on cloning, characterization and comparative in silico based analysis of gene ZnClpB1 in Z. nummularia. Gene ZnClpB1 would be a prospective resource for developing abiotic stress tolerant crops by transgenic or breeding approach

Approaches in Enhancing Antioxidant Defense in Plants

This Special Issue, “Approaches in Enhancing Antioxidant Defense in Plants” published 13 original research works and a couple of review articles that discuss the various aspects of plant oxidative stress biology and ROS metabolism, as well as the physiological mechanisms and approaches to enhancing antioxidant defense and mitigating oxidative stress. These papers will serve as a foundation for plant oxidative stress tolerance and, in the long term, provide further research directions in the development of crop plants’ tolerance to abiotic stress in the era of climate change

Oxidative Stress in Plants

Plants are continuously exposed to different environmental stress conditions that have a huge impact on agriculture worldwide, consequently leading to massive economic losses. These adverse conditions alter the metabolism of reactive oxygen and nitrogen species (ROS and RNS). High concentrations of these reactive species—that exceed the capacity of antioxidant defense enzymes—disturb redox homeostasis, which can trigger damage to such macromolecules as membrane lipids, proteins, and nucleic acids, ultimately resulting in nitro-oxidative stress and plant cell death. Significant progress has been made to understand how plants persist in these stressful environments which could be vital to improving plant crop yield. This Special Issue “Oxidative Stress in Plants” includes both original research articles and detailed reviews that aim to better understand the nitro-oxidative stress networks in higher plants, and the addressed topics provide updated and new knowledge about ROS and RNS metabolism in plant responses to abiotic stress as well as the modulation of antioxidant systems in the control of ROS production and accumulation

Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions

Overall, the 19 contributions in this Special Issue “Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions” discuss the various aspects of salt stress responses in plants. It also discusses various mechanisms and approaches to conferring salt tolerance on plants. These types of research studies provide further directions in the development of crop plants for the saline environment in the era of climate change

Towards a Better Understanding of the Molecular Mechanisms Underlying Plant Development and Stress Response

The spectacular array of diverse plant forms as well as the predominantly sessile life style of plants raises two questions that have been fascinating to scientists in the field of plant biology for many years: 1) how do plants develop to a specific size and shape? 2) how do plants respond to environmental stresses given its immobility? Plant organ development to a specific size and shape is controlled by cell proliferation and cell expansion. While the cell proliferation process is extensively studied, the cell expansion process remains largely unknown, and can be affected by several factors, such as cell wall remodeling and the incorporation of new wall materials. To better understand the genetic basis of plant development, we identified an Arabidopsis T-DNA insertion mutant named development related Myb-like 1 (drmy1), which showed altered size and shape in both vegetative and reproductive organs due to defective cell expansion. We further demonstrated that the defective cell expansion in the drmy1 mutant is linked to the change in cell wall composition. Complementation testing by introduction of DRMY1 into the mutant background rescued the phenotype, indicating that DRMY1 is a functional regulator of plant organ development. The DRMY1 protein contains a single Myb-like DNA binding domain and is localized in the nucleus, and may cooperate with other transcription factors to regulate downstream gene expression as DRMY1 itself does not possess transactivation ability. DRMY1 expression analysis revealed that its expression is reduced by the plant hormone ethylene (a negative regulator of cell expansion) while induced by ABA (a positive regulator of cell expansion). Furthermore, whole transcriptome profiling suggested that DRMY1 might control cell expansion directly by regulating genes related to cell wall biosynthesis/remodeling and ribosome biogenesis or indirectly through regulating genes involved in ethylene and ABA signaling pathways. Plants cannot “escape” from salinity stress but have evolved different mechanisms for salt tolerance over the time of adaptation to salinity. About 1% of plant species named halophytes can survive and thrive in environments containing high salt concentrations, which makes it important to understand their salt tolerance mechanisms and the responsible genes. Here, we investigated salt tolerance mechanisms in Supreme, the most salt-tolerant cultivar of a halophytic warm-seasoned perennial grass, Seashore paspalum (Paspalum vaginatum) at the physiological and transcriptomic levels by comparative study with another cultivar Parish, which possesses moderate salinity tolerance. Our results suggest that Na+ accumulation under normal conditions and further increased accumulation under high salinity conditions (400 mM NaCl), possibly by vacuolar sequestration is a crucial mechanism for salinity tolerance in Supreme. Our data suggests that Na+ accumulation in Supreme under normal conditions might trigger the secondary messenger Ca2+ for signal transduction and the resulting upregulation of salt stress related transcription factors in addition to serving as cheap osmolytes to facilitate water uptake. Moreover, the retention of K+ under salt treatment, which can counteract the toxicity of Na+, is a protective mechanism for both cultivars. A strong oxidation-reduction process and nucleic acid binding activity under high salinity conditions are two other contributors to the salinity tolerance in both cultivars. We also identified ion transporters including NHXs and H+-PPases for Na+ sequestration and K+ uptake transporters, which can be used as candidate genes for functional studies and potential targets to engineer plants for enhanced salinity tolerance, opening new avenues for future research

Genomic Resource Development for European Hazelnut (Corylus avellana L.)

European hazelnut (Corylus avellana L.) is an important crop Oregon's Willamette Valley, producing 99% of the hazelnuts grown in North America and brings over US $60 million dollars to the region annually. Hazelnuts are rich in fiber and vitamins, as well in demand by consumers due to their popularity as the predominant flavor in a multitude of confectionary pastes and chocolate spreads. Breeding efforts are focused on developing hazelnut cultivars with enhanced agronomic traits of interest including size, blanching ability, and resistance to disease. Molecular markers have been developed for hazelnut and placed on a genetic linkage map, linking DNA marker sequences that segregate with phenotypic traits of interest. The objective of this study was to utilize highthroughput sequencing technologies to sequence the hazelnut genome, allowing for trait and marker discovery on a genome-wide scale. We have established genomic resources for hazelnut, including genomic and transcriptomic sequences to allow breeders the opportunity to exploit the wealth of genetic diversity when choosing germplasm for crosses. We chose the Eastern Filbert Blight (EFB) resistant diploid hazelnut cultivar 'Jefferson' (OSU 703.007) as the reference. The 'Jefferson' transcriptome assembly is represented by 28,255 transcript contigs, having an average length of 532bp, and an N50 of 961bp. These transcripts were characterized using both BLASTX protein homology and gene ontology (GO) classifications, with the majority of the predicted proteins to have high conservation with the most closely related plant sequences of Vitis, Populus, and Ricinus. A survey of gene classes enriched among tissue types further validates the assembly and transcript models. The draft genome assembly for 'Jefferson' was assembled de novo using Illumina short read technology into 36,641 contigs and scaffolds, with half of the assembly contained in scaffolds and contigs greater than 21.5 Kb. We captured approximately 91% (345 Mb) of the flow-cytometrydetermined genome size and identified 34,910 putative gene loci which were functionally annotated to identify candidates for future molecular validation. The majority of the annotated genes share homology with the best annotated and related genera Vitis, Prunus, Populus, and Ricinus. We also resequenced seven additional European hazelnut cultivars, detecting millions of variants between one of more of these genomes and that of 'Jefferson'. These variants were annotated based on the functional consequence each polymorphism on the affected loci. In addition, we utilized Genotyping-by-Sequence (GBS) technologies to produce a high-density genetic map within 138 individuals of an F1 hazelnut mapping population, representing a five-fold increase in marker density over the previous maps. Hazelnut genome sequencing has provided new resources to the scientific community and promises to accelerate trait discovery and enhance future breeding efforts, and serve as a tool for gene discovery and functional studies

In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability

The interest on alkaline-stable lipases by the scientific community is increasing due to its great potential use. As most industrial processes are performed under highly basic conditions, alkaline-stable lipases become hugely valued biocatalysts. In this study, three aspartic acid residues at positions 51, 122 and 247 in the outer loop of LipKV1 from Acinetobacter haemolyticus was computationally mutated into lysine using the SWISS-MODEL program, followed by energy minimization of the protein models. PROCHECK, ERRAT and Verify3D refined models of LipKV1 and Mut-LipKV1 indicated that the Mut-LipKV1 protein conformation is in a good condition. The study found that the overall electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0 −10.0). Molecular dynamics (MD) simulation of Lip-KV1 and Mut-LipKV1 protein models under different alkaline pHs using GROMACS version 2018.6 revealed that Mut-LipKV1 was more stable at the high pH 9.0 (RMSD ~0.3 nm, RMSF ~0.05 – 0.2 nm), compared the optimal pH 8.0 of LipKV1 (RMSD 0.3 nm, RMSF 0.05 – 0.20 nm). Molecular docking using AutoDock Vina with tributyrin as the substrate identified detailed changes that occurred post mutation. The highest binding affinity (−4.1 kcal/mol) with Mut-LipKV1 which occurred at pH 9.0 was from a single hydrogen bond with His289. MD simulations showed that configurations which formed between Mut-LipKV1-tributyrin (RMSD 0.3 nm; RMSF 0.05 − 0.3 nm) and the LipKV1-tributyrin complexes (RMSD 0.35 nm; RMSF 0.05 − 0.4 nm) were comparably stable at pH 8.0. Furthermore, MM-PBSA calculation validated that the Mut-LipKV1-tributyrin complex at pH 8.0 (-44.01 kcal/mol) showed comparable binding free energy to LipKV1-tributyrin complex (−43.83 kcal/mol). Whereas the lowest binding free energy for Mut-LipKV1-tributyrin complex was simulated at pH 12.0 (−44.04 kcal/mol). Thus, adaptive strategy of replacing the outer loop surface aspartic acid to lysine in LipKV1 successfully broadened pH stability of Mut-LipKV1 towards higher pH, raising it from pH 8.0 − 11.0 to pH 8.0 − 12.0 in the mutant lipase. In a nutshell, this research offered a considerable insight for further improving the alkaline tolerance of lipases