The significant increase in grain yield and quality are often antagonistic but a constant demand for breeders and consumers. Some genes related to cytochrome P450 family are known for rice organ growth but their role in controlling grain yield is still unknown. Here, we generated new rice mutants with high yield and improved aroma by simultaneously editing three cytochrome P450 homoeologs (Os03g0603100, Os03g0568400, and GL3.2) and OsBADH2 with the CRISPR/Cas9 system, and RNA-sequencing and proteomic analysis were performed to unveil the subsequent changes. High mutation efficiency was achieved in both target sites of each gene and the mutations were predominantly only deletions, while insertions were rare, and no mutations were detected in the five most likely off-target sites against each sgRNA. Mutants exhibited increased grain size, 2-acetyl-1-pyrroline (2AP) content, and grain cell numbers while there was no change in other agronomic traits. Transgene-DNA-free mutant lines appeared with a frequency of 44.44% and homozygous mutations were stably transmitted, and bi-allelic and heterozygous mutations followed Mendelian inheritance, while the inheritance of chimeric mutations was unpredictable. Deep RNA sequencing and proteomic results revealed the regulation of genes and proteins related to cytochrome P450 family, grain size and development, and cell cycle. The KEGG and hub-gene and protein network analysis showed that the gene and proteins related to ribosomal and photosynthesis pathways were mainly enriched, respectively. Our findings provide a broad and detailed basis to understand the role of CRISPR/Cas9 in rice yield and quality improvement.

Rice leaf morphology is an essential agronomic trait to develop drought-tolerant genotypes for adequate and stable crop production in drought-prone areas. Here, rolled leaf mutant plants were acquired by CRISPR/Cas9-based mutagenesis of Semi-rolled leaf1,2 (SRL1 and SRL2) genes, and isobaric tags for relative and absolute quantification (iTRAQ) based proteomic analysis was performed to analyze the subsequent proteomic regulation events. Homozygous mutants exhibit decreased chlorophyll content, transpiration rate, stomatal conductance, vascular bundles (VB), stomatal number, and agronomic traits with increased panicle number and bulliform cells (BCs). Under drought stress, mutant plants displayed lower malondialdehyde (MDA) content while higher survival rate, abscisic acid (ABA) content, superoxide dismutase (SOD), catalase (CAT) activities, and grain filling percentage compare with their wild type (WT). Proteomic results revealed that 270 proteins were significantly downregulated, and 107 proteins were upregulated in the mutant line compared with WT. Proteins related to lateral organ boundaries’ (LOB) domain (LBD) were downregulated, whereas abiotic stress-responsive proteins were upregulated in the CRISPR mutant. LBD proteins (Q5KQR7, Q6K713, Q7XGL4, Q8LQH4), probable indole-3-acetic acid-amido synthetase (Q60EJ6), putative auxin transporter-like protein 4 (Q53JG7), Monoculm 1 (Q84MM9) and AP2 (Apetala2) domain-containing protein (Q10A97) were found to be hub-proteins. The hybrids developed from mutant restorers showed a semi-rolled leaf phenotype with increased panicle number, grain number per panicle, and yield per plant. Our findings reveal the intrinsic value of genome editing and expand the knowledge about the network of proteins for leaf rolling and drought avoidance in rice.

Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/ Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T 0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T 1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein–protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance.

the antioxidant mechanism is crucial for resisting oxidative damage induced by drought stress in plants. Different antioxidant mechanisms may contribute to the tolerance of cassava to drought stress, but for a specific genotype, the response is still unknown. The objective of this study was to investigate antioxidant response and physiological changes of four cassava genotypes under water stress conditions, by keeping the soil moisture content as 80% (control), 50% (medium), 20% (severe) of field capacity for a week. Genotypes RS01 and SC124 were keeping higher relative water content (RWC) and relative chlorophyll content (SPAD value) and less affected by oxidative stress than SC205 and GR4 under drought stress. RS01 just showed slight membrane damage and oxidative stress even under severe drought conditions. A principal component analysis showed that cassava plant water status was closely related to the antioxidant mechanism. Antioxidant response in genotypes RS01 and SC124 under drought stress might attribute to the increased accumulation of ascorbate (AsA) and glutathione (GSH) content and higher superoxide dismutase (SOD) and catalase (CAT) activities, which explained by the up-regulation of Mn-SOD and CAT genes. However, Genotypes SC205 and GR4 mainly depended on the accumulation of total phenolics (TP) and increased glutathione reductase (GR) activity, which attribute to the up-regulation of the GR gene. Our findings could provide vital knowledge for refining the tactics of cultivation and molecular breeding with drought avoidance in cassava. Drought stress is commonly induced by rainfall patterns, greenhouse effect and the variations of temperature. It is an important environmental stress factor that limits plant growth, regulation, and distribution 1-3. Compared with other abiotic stresses, drought stress exerts more restrictions on crop productivity 4 , especially on the marginal lands with poor soils and limited water resources. For resource-limited small farmers in these marginal areas, cassava (Manihot esculenta Crantz) is an important staple food crop due to its inherent tolerance to stressful environments 5. Because of its starchy roots, cassava is used for starch extraction and as feed resource and feedstock production in China and other Southeast Asian countries 6. Drought is one of the main constraints that limit cassava growth and production, particularly during the first three months after planting 7. Therefore, it is urgent to understand the mechanisms underlying drought tolerance of cassava at the seedling stage. Plants have developed defense mechanisms which enable them to adapt and survive under drought condition in their life cycle 8. The drought response of plants varies from species and the severity of the drought stress. The mechanisms of cassava resistant to water deficit include stomatal closure, decreased leaf area, the proper maintenance of net photosynthetic rate for prolonged drought, and the ability to explore water from deep soil layers 9. The defense strategies against drought ...

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Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx, major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T0 mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased (P < 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T1 plants were re-edited but T2 generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC2F1. The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD+-dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development.

Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.