Physicochemical Properties and Antioxidant Activity of CRISPR/Cas9-Edited Tomato SGR1 Knockout (KO) Line.

Tomatoes contain many secondary metabolites such as ß-carotene, lycopene, phenols, flavonoids, and vitamin C, which are responsible for antioxidant activity. SlSGR1 encodes a STAY-GREEN protein that plays a critical role in the regulation of chlorophyll degradation in tomato leaves and fruits. Therefore, the present study was conducted to evaluate the sgr1 null lines based on their physicochemical characteristics, the content of secondary metabolites, and the γ-Aminobutyric acid (GABA) content. The total soluble solids (TSS), titrated acidity (TA), and brix acid ratio (BAR) of the sgr1 null lines were higher than those of the wild type(WT). Additionally, the sgr1 null lines accumulated higher levels of flavor-inducing ascorbic acid and total carotenoids compared to WT. Also, the total phenolic content, total flavonoids, GABA content, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical content of the sgr1 null lines were higher than those of the WT. Therefore, these studies suggest that the knockout of the SGR1 gene by the CRISPR/Cas9 system can improve various functional compounds in tomato fruit, thereby satisfying the antioxidant properties required by consumers.

Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice.

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 T2OsPUB7 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.

Transcriptional Comparison of Genes Associated with Photosynthesis, Photorespiration, and Photo-Assimilate Allocation and Metabolic Profiling of Rice Species.

The ever-increasing human population alongside environmental deterioration has presented a pressing demand for increased food production per unit area. As a consequence, considerable research effort is currently being expended in assessing approaches to enhance crop yields. One such approach is to harness the allelic variation lost in domestication. This is of particular importance since crop wild relatives often exhibit better tolerance to abiotic stresses. Here, we wanted to address the question as to why wild rice species have decreased grain production despite being characterized by enhanced rates of photosynthesis. In order to do so, we selected ten rice species on the basis of the presence of genome information, life span, the prominence of distribution, and habitat type and evaluated the expression of genes in photosynthesis, photorespiration, sucrose and starch synthesis, sucrose transport, and primary and secondary cell walls. We additionally measured the levels of a range of primary metabolites via gas chromatography-mass spectrometry. The results revealed that the wild rice species exhibited not only higher photosynthesis but also superior CO2 recovery by photorespiration; showed greater production of photosynthates such as soluble sugars and starch and quick transportation to the sink organs with a possibility of transporting forms such as RFOs, revealing the preferential consumption of soluble sugars to develop both primary and secondary cell walls; and, finally, displayed high glutamine/glutamic acid ratios, indicating that they likely exhibited high N-use efficiency. The findings from the current study thus identify directions for future rice improvement through breeding.

Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography−tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.

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.

Molecular and Functional Analysis of U-box E3 Ubiquitin Ligase Gene Family in Rice (Oryzasativa).

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.

Trends and Prospects of Genetic and Molecular Research in Plants.

With the exponential advancements in biotechnology research, various studies are being conducted to overcome productivity limitations in crop breeding [...].

UVB Irradiation-Induced Transcriptional Changes in Lignin- and Flavonoid Biosynthesis and Indole/Tryptophan-Auxin-Responsive Genes in Rice Seedlings.

Global warming accelerates the destruction of the ozone layer, increasing the amount of UVB reaching the Earth's surface, which in turn alters plant growth and development. The effects of UVB-induced alterations of plant secondary and cell wall metabolism were previously documented; however, there is little knowledge of its effects on rice seedlings during the developmental phase of leaves. In this study, we examined secondary metabolic responses to UVB stress using a transcriptomic approach, focusing on the biosynthetic pathways for lignin, flavonoid, and indole/tryptophan-auxin responses. As new leaves emerged, they were irradiated with UVB for 5 days (for 3 h/day-1). The genes encoding the enzymes related to lignin (4CL, CAD, and POD) and flavonoid biosynthesis (CHS, CHI, and FLS) were highly expressed on day 1 (younger leaves) and day 5 (older leaves) after UVB irradiation. The expression of the genes encoding the enzymes related to tryptophan biosynthesis (AS, PRT, PRAI, IGPS, and TS) increased on day 3 of UVB irradiation, and the level of tryptophan increased and showed the same temporal pattern of occurrence as the expression of the cognate gene. Interestingly, the genes encoding BBX4 and BBX11, negative regulators of UVB signaling, and SAUR27 and SAUR55, auxin response enzymes, were downregulated on day 3 of UVB irradiation. When these results are taken together, they suggest that secondary metabolic pathways in rice seedlings are influenced by the interaction between UVB irradiation and the leaf developmental stage. Thus, the strategies of protection against, adaptation to, and mitigation of UVB might be delicately regulated, and, in this context, our data provide valuable information to understand UVB-induced secondary metabolism in rice seedlings.

Marker-Assisted Backcrossing (MABc) to Improve Eating Quality with Thin Seed Coat and Aleurone Layer of Non-Glutinous Japonica Variety in Rice.

Brown rice is composed of rice bran, pericarp, seed coat, and aleurone layers, and the rice bran layer contains a large number of substances useful for the human body, such as dietary fiber, α-tocopherol, α-tocotrienol, and vitamins. However, more than 90% of these substances are removed when polished, and white rice has the disadvantage of losing food-related ingredients, such as umami-related amino acids, when compared to the unpolished group. In this study, we tried to develop new breeding lines with a thinner seed coat and aleurone layer to provide high eating quality with softer chewing characteristics and processability in rice grain. We detected an SNP for foreground selection for the backcross population by comparing genome sequences between Samgwang and Seolgaeng and developed high eating quality brown rice breeding lines by applying marker-assisted backcrossing (MABC) breeding programs to backcross populations between Samgwang and Seolgaeng using KASP markers. SNP markers for foreground selection were identified to improve eating and processability through SNP mapping of Samgwang and Seolgaeng with SSIIa as a target gene in this study. Line selection according to genotype of KASP markers was successful in BC1F1 and BC2F1 generations, with the recurrent parent genome recovery ratio ranging from 91.22% to 98.65%. In BC2F1 seeds of the selected lines, thickness of the aleurone layer was found to range from 13.82 to 21.67 µm, which is much thinner than the 30.91 µm of the wild type, suggesting that selection by MABc could be used as an additional breeding material for the development of highly processed rice varieties. These lines will be useful to develop new brown rice varieties with softer chewing characteristics and processability in rice grain.

Development of SNP Markers from GWAS for Selecting Seed Coat and Aleurone Layers in Brown Rice (Oryza sativa L.).

Ninety-five percent of the general nutrients in rice are concentrated in the rice bran and germ, and many nutrients such as vitamins, minerals, dietary fiber, and essential fatty acids, as well as antioxidants such as tocopherol, are lost during milling. In this study, we investigated the thickness of seed coat and aleurone layers using a 294 rice core collection, and found candidate genes related to thickness of seed coat and aleurone layers, by performing a genome wide association study (GWAS) analysis using whole genome resequencing data. Two primer pairs that can be used as high-resolution melting (HRM) markers were developed. As a result of genotyping BC2F2 individuals derived from a cross between "Samgwang" and "Seolgaeng", and using corresponding HRM markers, it was possible to finally develop HRM markers for selecting seed coat and aleurone layer thickness. This is expected to be used as basic data for the application of gene editing using CRISPR/Cas9 technology and for establishing a breeding strategy for high eating quality rice using molecular genetic technology.

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene.

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, ß-carotene, and two ß-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and ß-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in ß-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the ß-carotene content of plants.

Transcriptional Changes of Cell Wall Organization Genes and Soluble Carbohydrate Alteration during Leaf Blade Development of Rice Seedlings.

Plant cell walls have two constituent parts with different components and developmental stages. Much of the mystery concerning the mechanisms of synthesis, decomposition, modification, and so forth, has been resolved using omics and microscopic techniques. However, it still remains to be determined how cell wall development progresses over time after leaf emergence. Our focus in the present study was to expand our knowledge of the molecular mechanisms associated with cell wall synthesis in rice leaf blade during three distinct stages (sink, sink-to-source transition, and source). The RNA-seq, quantitative reverse transcription PCR (qRT-PCR) and carbohydrate concentrations were evaluated using developing fifth leaf blades harvested at different time points. The results revealed that some of the essential genes for the primary cell wall (PCW) were highly upregulated in the sink-to-source transition compared to the sink stage, whereas those essential to the secondary cell wall (SCW) displayed relatively higher levels (p < 0.05) during the source stage. The concentrations of soluble carbohydrates differed via type rather than stage; we observed higher monosaccharides during the sink stage and higher di- and oligo-saccharides during the sink-to-source transition and source stages. In conclusion, our findings suggest that the transcriptional regulation of plant cell wall biosynthesis genes are both synchronistic with and independent of, and directly and indirectly governed by, the abundance of soluble carbohydrates in the developing leaf blade, and, finally, raffinose is likely to play a transport role comparable to sucrose.

Breeding of High Cooking and Eating Quality in Rice by Marker-Assisted Backcrossing (MABc) Using KASP Markers.

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.

Overexpression of Orange Gene (OsOr-R115H) Enhances Heat Tolerance and Defense-Related Gene Expression in Rice (Oryza sativa L.).

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.