Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T₂ OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein–protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.

Proteins encoded by U-box type ubiquitin ligase (PUB) genes in rice are known to play an important role in plant responses to abiotic and biotic stresses. Functional analysis has revealed a detailed molecular mechanism involving PUB proteins in relation to abiotic and biotic stresses. In this study, characteristics of 77 OsPUB genes in rice were identified. Systematic and comprehensive analyses of the OsPUB gene family were then performed, including analysis of conserved domains, phylogenetic relationships, gene structure, chromosome location, cis-acting elements, and expression patterns. Through transcriptome analysis, we confirmed that 16 OsPUB genes show similar expression patterns in drought stress and blast infection response pathways. Numerous cis-acting elements were found in promoter sequences of 16 OsPUB genes, indicating that the OsPUB genes might be involved in complex regulatory networks to control hormones, stress responses, and cellular development. We performed qRT-PCR on 16 OsPUB genes under drought stress and blast infection to further identify the reliability of transcriptome and cis-element analysis data. It was confirmed that the expression pattern was similar to RNA-sequencing analysis results. The transcription of OsPUB under various stress conditions indicates that the PUB gene might have various functions in the responses of rice to abiotic and biotic stresses. Taken together, these results indicate that the genome-wide analysis of OsPUB genes can provide a solid basis for the functional analysis of U-box E3 ubiquitin ligase genes. The molecular information of the U-box E3 ubiquitin ligase gene family in rice, including gene expression patterns and cis-acting regulatory elements, could be useful for future crop breeding programs by genome editing.

The primary goals of rice breeding programs are grain quality and yield potential improvement. With the high demand for rice varieties of premium cooking and eating quality, we developed low-amylose content breeding lines crossed with Samgwang and Milkyqueen through the marker-assisted backcross (MABc) breeding program. Trait markers of the SSIIIa gene referring to low-amylose content were identified through an SNP mapping activity, and the markers were applied to select favorable lines for a foreground selection. To rapidly recover the genetic background of Samgwang (recurrent parent genome, RPG), 386 genome-wide markers were used to select BC₁F₁ and BC₂F₁ individuals. Seven BC₂F₁ lines with targeted traits were selected, and the genetic background recovery range varied within 97.4–99.1% of RPG. The amylose content of the selected BC₂F₂ grains ranged from 12.4–16.8%. We demonstrated the MABc using a trait and genome-wide markers, allowing us to efficiently select lines of a target trait and reduce the breeding cycle effectively. In addition, the BC₂F₂ lines confirmed by molecular markers in this study can be utilized as parental lines for subsequent breeding programs of high-quality rice for cooking and eating.

In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8–9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Nowadays, rice production faces significant challenges due to population pressure, global climate change, and outbreak of various pests and diseases. Breeding techniques used to improve rice traits include mutant breeding, cross breeding, heterogeneity, transformation, molecular markers, genome-wide association study (GWAS), and so on. Since the recently developed CRISPR/Cas9 technology can directly target a specific part of a desired gene to induce mutation, it can be used as a powerful means to expand genetic diversity of crops and develop new varieties. So far, CRISPR/Cas9 technology has been used for improving rice characteristics such as high yield, good quality, abundant nutrition, pest and disease resistance, herbicide resistance, and biotic and abiotic stress resistance. This review highlights the mechanisms and optimization of the CRISPR system and its application to rice crop, including resistance to biotic and abiotic stresses, and improved rice quality and yield.