Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples.

The impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing offers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modification of plant's genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the final product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01423-y.

A rapid and robust colorimetric method for measuring relative abundance of auxins in plant tissues.

INTRODUCTION: Auxin estimation in plant tissues is a crucial component of auxin signaling studies. Despite the availability of various high-throughput auxin quantification methods like LC-MS, GC-MS, HPLC, biosensors, and DR5-gus/gfp-based assays, auxin quantification remains troublesome because these techniques are very expensive and technology intensive and they mostly involve elaborate sample preparation or require the development of transgenic plants. OBJECTIVES: To find a solution to these problems, we made use of an old auxin detection system to quantify microbe derived auxins and modified it to effectively measure auxin levels in rice plants. MATERIALS AND METHODS: Auxins from different tissues of rice plants, including root samples of seedlings exposed to IAA/TIBA or subjected to different abiotic stresses, were extracted in ethanol. The total auxin level was measured by the presently described colorimetric assay and counterchecked by other auxin estimation methods like LC-MS or gus staining of DR5-gus overexpressing lines. RESULTS: The presented colorimetric method could measure (1) the auxin levels in different tissues of rice plants, thus identifying the regions of higher auxin abundance, (2) the differential accumulation of auxins in rice roots when auxin or its transport inhibitor was supplied exogenously, and (3) the levels of auxin in roots of rice seedlings subjected to various abiotic stresses. The thus obtained auxin levels correlated well with the auxin levels determined by other methods like LC-MS or gus staining and the expression pattern of auxin biosynthesis pathway genes. CONCLUSIONS: The auxin estimation method described here is simple, rapid, cost-effective, and sensitive and allows for the efficient detection of relative auxin abundances in plant tissues.

A simplified and improved protocol of rice transformation to cater wide range of rice cultivars.

The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76-78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69-83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.

Revisiting the role of MAPK signalling pathway in plants and its manipulation for crop improvement.

The mitogen-activated protein kinase (MAPK) pathway is an important signalling event associated with every aspect of plant growth, development, yield, abiotic and biotic stress adaptation. Being a central metabolic pathway, it is a vital target for manipulation for crop improvement. In this review, we have summarised recent advancements in understanding involvement of MAPK signalling in modulating abiotic and biotic stress tolerance, architecture and yield of plants. MAPK signalling cross talks with reactive oxygen species (ROS) and abscisic acid (ABA) signalling events in bringing about abiotic stress adaptation in plants. The intricate involvement of MAPK pathway with plant's pathogen defence ability has also been identified. Further, recent research findings point towards participation of MAPK signalling in shaping plant architecture and yield. These make MAPK pathway an important target for crop improvement and we discuss here various strategies to tweak MAPK signalling components for designing future crops with improved physiology and phenotypes.

Characterization and expression profiling of PIN auxin efflux transporters reveal their role in developmental and abiotic stress conditions in rice.

The auxin efflux transporter proteins called PINs ferry auxin from its source to sinks in particular directions depending on their polar localizations in the plasma membrane, thus facilitating the development of the entire plant architecture. The rice genome has 12 PIN genes distributed over eight chromosomes. To study their roles in plant development, abiotic stress responsiveness, and shaping an auxin-dependent root architecture, a genome-wide analysis was carried out. Based on phylogeny, cellular localization, and hydrophilic loop domain size, the PINs were categorized into canonical and noncanonical PINs. PINs were found expressed in all of the organs of plants that emphasized their indispensable role throughout the plant's life cycle. We discovered that PIN5C and PIN9 were upregulated during salt and drought stress. We also found that regardless of its cellular level, auxin functioned as a molecular switch to turn on auxin biosynthesis genes. On the contrary, although PIN expression was upregulated upon initial treatment with auxin, prolonged auxin treatment not only led to their downregulation but also led to the development of auxin-dependent altered root formation in rice. Our study paves the way for developing stress-tolerant rice and plants with a desirable root architecture by genetic engineering.

Sugar transporters and their molecular tradeoffs during abiotic stress responses in plants.

Sugars as photosynthates are well known as energy providers and as building blocks of various structural components of plant cells, tissues and organs. Additionally, as a part of various sugar signaling pathways, they interact with other cellular machinery and influence many important cellular decisions in plants. Sugar signaling is further reliant on the differential distribution of sugars throughout the plant system. The distribution of sugars from source to sink tissues or within organelles of plant cells is a highly regulated process facilitated by various sugar transporters located in plasma membranes and organelle membranes, respectively. Sugar distribution, as well as signaling, is impacted during unfavorable environments such as extreme temperatures, salt, nutrient scarcity, or drought. Here, we have discussed the mechanism of sugar transport via various types of sugar transporters as well as their differential response during environmental stress exposure. The functional involvement of sugar transporters in plant's abiotic stress tolerance is also discussed. Besides, we have also highlighted the challenges in engineering sugar transporter proteins as well as the undeciphered modules associated with sugar transporters in plants. Thus, this review provides a comprehensive discussion on the role and regulation of sugar transporters during abiotic stresses and enables us to target the candidate sugar transporter(s) for crop improvement to develop climate-resilient crops.

Phytohormone signaling and crosstalk in regulating drought stress response in plants.

Phytohormones are ubiquitously involved in plant biological processes and regulate cellular signaling pertaining to unheralded environmental cues, such as salinity, drought, extreme temperature and nutrient deprivation. The association of phytohormones to nearly all the fundamental biological processes epitomizes the phytohormone syndicate as a candidate target for consideration during engineering stress endurance in agronomically important crops. The drought stress response is essentially driven by phytohormones and their intricate network of crosstalk, which leads to transcriptional reprogramming. This review is focused on the pivotal role of phytohormones in water deficit responses, including their manipulation for mitigating the effect of the stressor. We have also discussed the inherent complexity of existing crosstalk accrued among them during the progression of drought stress, which instigates the tolerance response. Therefore, in this review, we have highlighted the role and regulatory aspects of various phytohormones, namely abscisic acid, auxin, gibberellic acid, cytokinin, brassinosteroid, jasmonic acid, salicylic acid, ethylene and strigolactone, with emphasis on drought stress tolerance.

Imperative role of sugar signaling and transport during drought stress responses in plants.

Cellular sugar status is essentially maintained during normal growth conditions but is impacted negatively during various environmental perturbations. Drought presents one such unfavorable environmental cue that hampers the photosynthetic fixation of carbon into sugars and affects their transport by lowering the cellular osmotic potential. The transport of cellular sugar is facilitated by a specific set of proteins known as sugar transporters. These transporter proteins are the key determinant of influx/ efflux of various sugars and their metabolite intermediates that support the plant growth and developmental process. Abiotic stress and especially drought stress-mediated injury results in reprogramming of sugar distribution across the cellular and subcellular compartments. Here, we have reviewed the imperative role of sugar accumulation, signaling, and transport under typical and atypical stressful environments. We have discussed the physiological effects of drought on sugar accumulation and transport through different transporter proteins involved in monosaccharide and disaccharide sugar transport. Further, we have illustrated sugar-mediated signaling and regulation of sugar transporter proteins along with the overall crosstalk of this signaling with the phytohormone module of abiotic stress response under osmotic stress. Overall, the present review highlights the critical role of sugar transport, distribution and signaling in plants under drought stress conditions.

Transcription factors as key molecular target to strengthen the drought stress tolerance in plants.

Amid apprehension of global climate change, crop plants are inevitably confronted with a myriad of abiotic stress factors during their growth that inflicts a serious threat to their development and overall productivity. These abiotic stresses comprise extreme temperature, pH, high saline soil, and drought stress. Among different abiotic stresses, drought is considered the most calamitous stressor with its serious impact on the crops' yield stability. The development of climate-resilient crops that withstands reduced water availability is a major focus of the scientific fraternity to ensure the food security of the sharply increasing population. Numerous studies aim to recognize the key regulators of molecular and biochemical processes associated with drought stress tolerance response. A few potential candidates are now considered as promising targets for crop improvement. Transcription factors act as a key regulatory switch controlling the gene expression of diverse biological processes and, eventually, the metabolic processes. Understanding the role and regulation of the transcription factors will facilitate the crop improvement strategies intending to develop and deliver agronomically-superior crops. Therefore, in this review, we have emphasized the molecular avenues of the transcription factors that can be exploited to engineer drought tolerance potential in crop plants. We have discussed the molecular role of several transcription factors, such as basic leucine zipper (bZIP), dehydration responsive element binding (DREB), DNA binding with one finger (DOF), heat shock factor (HSF), MYB, NAC, TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP), and WRKY. We have also highlighted candidate transcription factors that can be used for the development of drought-tolerant crops.

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.

Co-expression of P173S Mutant Rice EPSPS and igrA Genes Results in Higher Glyphosate Tolerance in Transgenic Rice.

Weeds and their devastating effects have been a great threat since the start of agriculture. They compete with crop plants in the field and negatively influence the crop yield quality and quantity along with survival of the plants. Glyphosate is an important broad-spectrum systemic herbicide which has been widely used to combat various weed problems since last two decades. It is very effective even at low concentrations, and possesses low environmental toxicity and soil residual activity. However, the residual concentration of glyphosate inside the plant has been of major concern as it severely affects the important metabolic pathways, and results in poor plant growth and grain yield. In this study, we compared the glyphosate tolerance efficiency of two different transgenic groups over expressing proline/173/serine (P173S) rice EPSPS glyphosate tolerant mutant gene (OsmEPSPS) alone and in combination with the glyphosate detoxifying encoding igrA gene, recently characterized from Pseudomonas. The molecular analysis of all transgenic plant lines showed a stable integration of transgenes and their active expression in foliar tissues. The physiological analysis of glyphosate treated transgenic lines at seed germination and vegetative stages showed a significant difference in glyphosate tolerance between the two transgenic groups. The transgenic plants with OsmEPSPS and igrA genes, representing dual glyphosate tolerance mechanisms, showed an improved root-shoot growth, physiology, overall phenotype and higher level of glyphosate tolerance compared to the OsmEPSPS transgenic plants. This study highlights the advantage of igrA led detoxification mechanism as a crucial component of glyphosate tolerance strategy in combination with glyphosate tolerant OsmEPSPS gene, which offered a better option to tackle in vivo glyphosate accumulation and imparted more robust glyphosate tolerance in rice transgenic plants.

Phosphite: a novel P fertilizer for weed management and pathogen control.

The availability of orthophosphate (Pi) is a key determinant of crop productivity because its accessibility to plants is poor due to its conversion to unavailable forms. Weed's competition for this essential macronutrient further reduces its bio-availability. To compensate for the low Pi use efficiency and address the weed hazard, excess Pi fertilizers and herbicides are routinely applied, resulting in increased production costs, soil degradation and eutrophication. These outcomes necessitate the identification of a suitable alternate technology that can address the problems associated with the overuse of Pi-based fertilizers and herbicides in agriculture. The present review focuses on phosphite (Phi) as a novel molecule for its utility as a fertilizer, herbicide, biostimulant and biocide in modern agriculture. The use of Phi-based fertilization will help to reduce the consumption of Pi fertilizers and facilitate weed and pathogen control using the same molecule, thereby providing significant advantages over current orthophosphate-based fertilization.

The development of a phosphite-mediated fertilization and weed control system for rice.

Fertilizers and herbicides are two vital components of modern agriculture. The imminent danger of phosphate reserve depletion and multiple herbicide tolerance casts doubt on agricultural sustainability in the future. Phosphite, a reduced form of phosphorus, has been proposed as an alternative fertilizer and herbicide that would address the above problems to a considerable extent. To assess the suitability of a phosphite-based fertilization and weed control system for rice, we engineered rice plants with a codon-optimized ptxD gene from Pseudomonas stutzeri. Ectopic expression of this gene led to improved root growth, physiology and overall phenotype in addition to normal yield in transgenic plants in the presence of phosphite. Phosphite functioned as a translocative, non-selective, pre- and post-emergent herbicide. Phosphite use as a dual fertilizer and herbicide may mitigate the overuse of phosphorus fertilizers and reduce eutrophication and the development of herbicide resistance, which in turn will improve the sustainability of agriculture.

Glutathione Peroxidase of Pennisetum glaucum (PgGPx) Is a Functional Cd2+ Dependent Peroxiredoxin that Enhances Tolerance against Salinity and Drought Stress.

Reactive oxygen species (ROS) arise in the plant system due to inevitable influence of various environmental stimuli. Glutathione peroxidases are one of the important ROS scavengers inside the cell. A glutathione peroxidase (PgGPx) gene was previously found from Pennisetum glauccum abiotic stressed cDNA library. Enzyme kinetics data revealed that PgGPx possessed preference towards thioredoxin rather than glutathione as electron donor and thus belongs to the functional peroxiredoxin group. Moreover, its activity was found to be dependent on divalent cations, especially Cd2+ and homology model showed the presence of Cd2+ binding site in the protein. Site directed mutagenesis study of PgGPx protein revealed the vital role of two conserved Cysteine residues for its enzymatic activity and structural folding. Expression analysis suggested that PgGPx transcript is highly up-regulated in response to salinity and drought stresses. When expressed ectopically, PgGPx showed enhanced tolerance against multiple abiotic stresses in prokaryotic E. coli and model plant, rice. Transgenic rice plants showed lesser accumulation of MDA and H2O2; and higher accumulation of proline as compared to wild type (WT) plants in response to both salinity and drought stresses that clearly indicates suppression of lipid peroxidation and ROS generation in transgenic lines. Moreover, transgenic plants maintained better photosynthesis efficiency and higher level of antioxidant enzyme activity as compared to WT plants under stress conditions. These results clearly indicate the imperative role of PgGPx in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance towards oxidative stress.

Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its ß-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.