Mitochondria-Targeted SmsHSP24.1 Overexpression Stimulates Early Seedling Vigor and Stress Tolerance by Multi-Pathway Transcriptome-Reprogramming.

Among the diverse array of heat shock proteins across the three domains of life, mitochondria-targeted small heat shock proteins (sHSPs) are evolved in the plant lineage. However, they remained mysterious and understudied. In this study, we reported a systematic study of a novel mitochondria-targeted nuclear sHSP from eggplant (Solanum melongena L.; SmsHSP24.1). Differential expression of SmsHSP24.1 indicated its positive role exerted during stress conditions. Escherichia coli-BL21 cell line overexpressing the SmsHSP24.1 showed excellent thermo-tolerance ability, tolerating up to 52°C. Spectrometry and electron microscopy revealed a multimeric structure of the protein which acted as a molecular chaperone at high temperatures. Overexpression of SmsHSP24.1 significantly enhanced resistance against heat, drought, and salt stresses and showed rapid germination in constitutively overexpressed eggplant lines. RNA-seq analysis reveals an apparent upregulation of a set of reactive oxygen species (ROS) scavenging enzymes of the glutathione (GHS) pathway and mitochondrial electron transport chain (ETC). Significant upregulation was also observed in auxin biosynthesis and cell-wall remodeling transcripts in overexpressed lines. qPCR, biochemical and physiological analysis further aligned with the finding of transcriptome analysis and suggested an essential role of SmsHSP24.1 under various stress responses and positive physiological influence on the growth of eggplants. Therefore, this gene has immense potential in engineering stress-resilient crop plants.

Overexpression of improved EPSPS gene results in field level glyphosate tolerance and higher grain yield in rice.

Glyphosate is a popular, systemic, broad-spectrum herbicide used in modern agriculture. Being a structural analog of phosphoenolpyruvate (PEP), it inhibits 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) which is responsible for the biosynthesis of aromatic amino acids and various aromatic secondary metabolites. Taking a lead from glyphosate-resistant weeds, two mutant variants of the rice EPSPS gene were developed by amino acid substitution (T173I + P177S; TIPS-OsEPSPS and G172A + T173I + P177S; GATIPS-OsEPSPS). These mutated EPSPS genes were overexpressed in rice under the control of either native EPSPS or constitutive promoters (maize ubiquitin [ZmUbi] promoter). The overexpression of TIPS-OsEPSPS under the control of the ZmUbi promoter resulted in higher tolerance to glyphosate (up to threefold of the recommended dose) without affecting the fitness and related agronomic traits of plants in both controlled and field conditions. Furthermore, such rice lines produced 17%-19% more grains compared to the wild type (WT) in the absence of glyphosate application and the phenylalanine and tryptophan contents in the transgenic seeds were found to be significantly higher in comparison with WT seeds. Our results also revealed that the native promoter guided expression of modified EPSPS genes did not significantly improve the glyphosate tolerance. The present study describing the introduction of a crop-specific TIPS mutation in class I aroA gene of rice and its overexpression have potential to substantially improve the yield and field level glyphosate tolerance in rice. This is the first report to observe that the EPSPS has role to play in improving grain yield of rice.

Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses.

Glutamine synthetase (GS) is a key enzyme involved in the nitrogen metabolism of higher plants. Abiotic stresses have adverse effects on crop production and pose a serious threat to global food security. GS activity and expression is known to be significantly modulated by various abiotic stresses. However, very few transgenic overexpression studies of GS have studied its impact on abiotic stress tolerance. GS is also the target enzyme of the broad spectrum herbicide Glufosinate (active ingredient: phosphinothricin). In this study, we investigated the effect of concurrent overexpression of the rice cytosolic GS1 (OsGS1;1) and chloroplastic GS2 (OsGS2) genes in transgenic rice on its tolerance to abiotic stresses and the herbicide Glufosinate. Our results demonstrate that the co-overexpression of OsGS1;1 and OsGS2 isoforms in transgenic rice plants enhanced its tolerance to osmotic and salinity stress at the seedling stage. The transgenic lines maintained significantly higher fresh weight, chlorophyll content, and relative water content than wild type (wt) and null segregant (ns) controls, under both osmotic and salinity stress. The OsGS1;1/OsGS2 co-overexpressing transgenic plants accumulated higher levels of proline but showed lower electrolyte leakage and had lower malondialdehyde (MDA) content under the stress treatments. The transgenic lines showed considerably enhanced photosynthetic and agronomic performance under drought and salinity stress imposed during the reproductive stage, as compared to wt and ns control plants. The grain filling rates of the transgenic rice plants under reproductive stage drought stress (64.6 ± 4.7%) and salinity stress (58.2 ± 4.5%) were significantly higher than control plants, thereby leading to higher yields under these abiotic stress conditions. Preliminary analysis also revealed that the transgenic lines had improved tolerance to methyl viologen induced photo-oxidative stress. Taken together, our results demonstrate that the concurrent overexpression of OsGS1;1 and OsGS2 isoforms in rice enhanced physiological tolerance and agronomic performance under adverse abiotic stress conditions, apparently acting through multiple mechanistic routes. The transgenic rice plants also showed limited tolerance to the herbicide Glufosinate. The advantages and limitations of glutamine synthetase overexpression in crop plants, along with future strategies to overcome these limitations for utilization in crop improvement have also been discussed briefly.

Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation.

KEY MESSAGE: The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.