Methylglyoxal-glyoxalase system as a possible selection module for raising marker-safe plants in rice.

Methylglyoxal (MG) is ubiquitously produced in all living organisms as a byproduct of glycolysis, higher levels of which are cytotoxic, leading to oxidative stress and apoptosis in the living systems. Though its generation is spontaneous but its detoxification involves glyoxalase pathway genes. Based on this understanding, the present study describes the possible role of MG as a novel non-antibiotic-based selection agent in rice. Further, by metabolizing MG, the glyoxalase pathway genes viz. glyoxalase I (GLYI) and glyoxalase II (GLYII), may serve as selection markers. Therefore, herein, transgenic rice harboring GLYI-GLYII genes (as selection markers) were developed and the effect of MG as a selection agent was assessed. The 3 mM MG concentration was observed as optimum for the selection of transformed calli, allowing efficient callus induction and proliferation along with high regeneration frequency (55 ± 2%) of the transgenic calli. Since the transformed calli exhibited constitutively higher activity of GLYI and GLYII enzymes compared to the wild type calli, the rise in MG levels was restricted even upon exogenous addition of MG during the selection process, resulting in efficient selection of the transformed calli. Therefore, MG-based selection method is a useful and efficient system for selection of transformed plants without significantly compromising the transformation efficiency. Further, this MG-based selection system is bio-safe and can pave way towards better public acceptance of transgenic plants.

Manipulation of glyoxalase pathway confers tolerance to multiple stresses in rice.

Crop plants face a multitude of diverse abiotic and biotic stresses in the farmers' fields. Although there now exists a considerable knowledge of the underlying mechanisms of response to individual stresses, the crosstalk between response pathways to various abiotic and biotic stresses remains enigmatic. Here, we investigated if the cytotoxic metabolite methylglyoxal (MG), excess of which is generated as a common consequence of many abiotic and biotic stresses, may serve as a key molecule linking responses to diverse stresses. For this, we generated transgenic rice plants overexpressing the entire two-step glyoxalase pathway for MG detoxification. Through assessment of various morphological, physiological and agronomic parameters, we found that glyoxalase-overexpression imparts tolerance towards abiotic stresses like salinity, drought and heat and also provides resistance towards damage caused by the sheath blight fungus (Rhizoctonia solani) toxin phenylacetic acid. We show that the mechanism of observed tolerance of the glyoxalase-overexpressing plants towards these diverse abiotic and biotic stresses involves improved MG detoxification and reduced oxidative damage leading to better protection of chloroplast and mitochondrial ultrastructure and maintained photosynthetic efficiency under stress conditions. Together, our findings indicate that MG may serve as a key link between abiotic and biotic stress response in plants.

Silicon nutrition stimulates Salt-Overly Sensitive (SOS) pathway to enhance salinity stress tolerance and yield in rice.

In rice (Oryza sativa), Si nutrition is known to improve salinity tolerance; however, limited efforts have been made to elucidate the underlying mechanism. Salt-Overly Sensitive (SOS) pathway contributes to salinity tolerance in plants in a major way which works primarily through Na+ exclusion from the cytosol. SOS1, a vital component of SOS pathway is a Na+/H+ antiporter that maintains ion homeostasis. In this study, we evaluated the effect of overexpression of Oryza sativa SOS1 (OsSOS1) in tobacco (cv. Petit Havana) and rice (cv. IR64) for modulating its response towards salinity further exploring its correlation with Si nutrition. OsSOS1 transgenic tobacco plants showed enhanced tolerance to salinity as evident by its high chlorophyll content and maintaining favorable ion homeostasis under salinity stress. Similarly, transgenic rice overexpressing OsSOS1 also showed improved salinity stress tolerance as shown by higher seed germination percentage, seedling survival and low Na+ accumulation under salinity stress. At their mature stage, compared with the non-transgenic plants, the transgenic rice plants showed better growth and maintained better photosynthetic efficiency with reduced chlorophyll loss under stress. Also, roots of transgenic rice plants showed reduced accumulation of Na+ leading to reduced oxidative damage and cell death under salinity stress which ultimately resulted in improved agronomic traits such as higher number of panicles and fertile spikelets per panicle. Si nutrition was found to improve the growth of salinity stressed OsSOS1 rice by upregulating the expression of Si transporters (Lsi1 and Lsi2) that leads to more uptake and accumulation of Si in the rice shoots. Metabolite profiling showed better stress regulatory machinery in the transgenic rice, since they maintained higher abundance of most of the osmolytes and free amino acids.

An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars.

BACKGROUND: Rice genome sequencing projects have generated remarkable amount of information about genes and genome architecture having tremendous potential to be utilized in both basic and applied research. Success in transgenics is paving the way for preparing a road map of functional genomics which is expected to correlate action of a gene to a trait in cellular and organismal context. However, the lack of a simple and efficient method for transformation and regeneration is a major constraint for such studies in this important cereal crop. RESULTS: In the present study, we have developed an easy, rapid and highly efficient transformation and regeneration protocol using mature seeds as explants and found its successful applicability to a choice of elite indica rice genotypes. We have optimized various steps of transformation and standardized different components of the regeneration medium including growth hormones and the gelling agent. The modified regeneration medium triggers production of large number of shoots from smaller number of calli and promotes their faster growth, hence significantly advantageous over the existing protocols where the regeneration step requires maximum time. Using this protocol, significantly higher transformation efficiency (up to 46%) and regeneration frequency (up to 92% for the untransformed calli and 59% for the transformed calli) were achieved for the four tested cultivars. We have used this protocol to produce hundreds of independent transgenic lines of different indica rice genotypes. Upon maturity, these transgenic lines were fertile thereby indicating that faster regeneration during tissue culture did not affect their reproductive potential. CONCLUSIONS: This speedy, yet less labor-intensive, protocol overcomes major limitations associated with genetic manipulation in rice. Moreover, our protocol uses mature seeds as the explant, which can easily be obtained in quantity throughout the year and kept viable for a long time. Such an easy, efficient and generalized protocol has the potential to be a major tool for crop improvement and gene-function studies on the model monocot plant rice.

Metabolic engineering of glyoxalase pathway for enhancing stress tolerance in plants.

Glyoxalase system consists of two enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I detoxifies methylglyoxal (MG), a cytotoxic byproduct of glycolysis, to S-lactoylglutathione (SLG) where it uses one molecule of reduced glutathione. Subsequently, SLG is converted to lactate by Gly II and one molecule of reduced glutathione is recycled back into the system. The level of MG, which is produced ubiquitously in all living organisms, is enhanced upon exposure to different abiotic stresses in plants. Overexpression of glyoxalase pathway genes in transgenic plants has been found to keep a check on the MG level under stress conditions, regulate glutathione homeostasis, and the transgenic plants are able to survive and grow under various abiotic stresses.