Lysine is the first limiting essential amino acid in rice because it is present in the lowest quantity compared to all the other amino acids. Amino acids are the building block of proteins and play an essential role in maintaining the human body’s healthy functioning. Rice is a staple food for more than half of the global population; thus, increasing the lysine content in rice will help improve global health. In this paper, we studied the lysine biosynthesis pathway in rice (Oryza sativa) to identify the regulators of the lysine reporter gene LYSA (LOC_Os02g24354). Genetically intervening at the regulators has the potential to increase the overall lysine content in rice. We modeled the lysine biosynthesis pathway in rice seedlings under normal and saline (NaCl) stress conditions using Bayesian networks. We estimated the model parameters using experimental data and identified the gene DAPF(LOC_Os12g37960) as a positive regulator of the lysine reporter gene LYSA under both normal and saline stress conditions. Based on this analysis, we conclude that the gene DAPF is a potent candidate for genetic intervention. Upregulating DAPF using methods such as CRISPR-Cas9 gene editing strategy has the potential to upregulate the lysine reporter gene LYSA and increase the overall lysine content in rice.

Rice ( Oryza sativa L.), due to its tropical and subtropical origin, is adversely affected by low temperatures below 15°C, restricting growth and yield. During the seedling and vegetative stage, cold stress causes curled leaves, fewer tillers, retarded growth, chlorosis, and necrosis. Due to the significance of this trait, exploring the wealth of genetic resources from a wider pool of germplasm to identify new sources of tolerance will be a crucial step. Toward this goal, we performed a genome‐wide association study (GWAS) on a novel diversity panel of 238 rice accessions using the 7K SNP Cornell‐IR LD Rice (C7AIR) single nucleotide polymorphism (SNP) array. The experiment was conducted in controlled growth conditions, and rice accessions were evaluated for seven traits and four indices, which were recorded at three different time points, that is, before cold treatment, after cold treatment, and post‐recovery phase. A total of 77 significant GWAS‐quantitative trait loci (QTLs) were identified for 21 cold tolerance and related traits of interest. Out of these, 34 were detected after cold stress, and 43 were detected during the post‐recovery phase. A total of 42 QTL regions were significant at false discovery rate (FDR) < 0.001 and −log ₁₀ ( p ‐values) > 5. In addition, 31 novel GWAS‐QTLs, three colocalized GWAS‐QTL hotspots, and eight colocalized GWAS‐QTLs for two traits sharing the same genomic location were also identified. The results of this study may help in further elucidating the molecular mechanisms underlying cold tolerance as well as aid in developing climate‐smart rice varieties for chilling stress conditions.

Plants have evolved a complex, multilayered immune system that integrates molecular recognition, signaling pathways, epigenetic regulation, and small RNA-mediated control. Recent studies have shown that DNA-level regulatory mechanisms, such as RNA-directed DNA methylation (RdDM), histone modifications, and chromatin remodeling, are critical for modulating immune gene expression, allowing for rapid and accurate pathogen-defense responses. The epigenetic landscape not only maintains immunological homeostasis but also promotes stress-responsive transcription via stable chromatin modifications. These changes contribute to immunological priming, a process in which earlier exposure to pathogens or abiotic stress causes a heightened state of preparedness for future encounters. Small RNAs, including siRNAs, miRNAs, and phasiRNAs, are essential for gene silencing before and after transcription, fine-tuning immune responses, and inhibiting negative regulators. These RNA molecules interact closely with chromatin features, influencing histone acetylation/methylation (e.g., H3K4me3, H3K27me3) and guiding DNA methylation patterns. Epigenetically encoded immune memory can be stable across multiple generations, resulting in the transgenerational inheritance of stress resilience. Such memory effects have been observed in rice, tomato, maize, and Arabidopsis. This review summarizes new findings on short RNA biology, chromatin-level immunological control, and epigenetic memory in plant defense. Emerging technologies, such as ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing), ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing), bisulfite sequencing, and CRISPR/dCas9-based epigenome editing, are helping researchers comprehend these pathways. These developments hold an opportunity for establishing epigenetic breeding strategies that target the production of non-GMO, stress-resistant crops for sustainable agriculture.

Rice, being one of the largest grown staple sources of energy, feeds nearly half of the world’s population. Although rice grain provides nearly 90% of the total calories to the developing world, its nutritional value is reduced by its low lysine content. Of the total 20 essential amino acids, lysine is one of the most limiting factors as low levels of lysine can lead to malnutrition. Improving the nutritional composition of rice grains can be performed either by increasing lysine biosynthesis or suppressing its catabolism which would eventually increase the lysine levels in rice. Toward this goal, the CRISPR-Cas9 system which is a powerful, efficient, and versatile tool for genome editing in plants is used. In the present study, we aim to use this genome editing strategy to knockout key genes in the lysine catabolic pathway using the endogenous tRNA-processing system for increasing lysine levels in the rice grain. A vector construct containing gRNAs for the target genes was transformed into the U.S. elite rice variety Presidio using Agrobacterium-mediated transformation. We have successfully transformed 20 rice plants and 19 of them are confirmed through sequencing to be CRISPR-mutated plants. The Cas9 integration in these CRISPR edited plants will be segregated away in the subsequent generations. The screening and genotyping of six T1 lines are currently underway. We believe that this approach will help us to lay a foundation in addressing the nutritional value of rice by improving the lysine content. Keywords: staple food crops, lysine, limiting essential amino acid, malnutrition, multiplex CRISPR/Cas9 genome editing Rice, being one of the largest grown staple sources of energy, feeds nearly half of the world’s population. Although rice grain provides nearly 90% of the total calories to the developing world, its nutritional value is reduced by its low lysine content. Of the total 20 essential amino acids, lysine is one of the most limiting factors as low levels of lysine can lead to malnutrition. Improving the nutritional composition of rice grains can be performed either by increasing lysine biosynthesis or suppressing its catabolism which would eventually increase the lysine levels in rice. Toward this goal, the CRISPR-Cas9 system which is a powerful, efficient, and versatile tool for genome editing in plants is used. In the present study, we aim to use this genome editing strategy to knockout key genes in the lysine catabolic pathway using the endogenous tRNA-processing system for increasing lysine levels in the rice grain. A vector construct containing gRNAs for the target genes was transformed into the U.S. elite rice variety Presidio using Agrobacterium-mediated transformation. We have successfully transformed 20 rice plants and 19 of them are confirmed through sequencing to be CRISPR-mutated plants. The Cas9 integration in these CRISPR edited plants will be segregated away in the subsequent generations. The screening and genotyping of six T1 lines are currently underway. We believe that this approach will help us to lay a foundation in addressing the nutritional value of rice by improving the lysine content. Keywords: staple food crops, lysine, limiting essential amino acid, malnutrition, multiplex CRISPR/Cas9 genome editing