Two genomic regions of a sodium azide induced rice mutant confer broad-spectrum and durable resistance to blast disease

Rice blast, one of the most destructive epidemic diseases, annually causes severe losses in grain yield worldwide. To manage blast disease, breeding resistant varieties is considered a more economic and environment-friendly strategy than chemical control. For breeding new resistant varieties, natural germplasms with broad-spectrum resistance are valuable resistant donors, but the number is limited. Therefore, artificially induced mutants are an important resource for identifying new broad-spectrum resistant (R) genes/loci. To pursue this approach, we focused on a broad-spectrum blast resistant rice mutant line SA0169, which was previously selected from a sodium azide induced mutation pool of TNG67, an elite japonica variety. We found that SA0169 was completely resistant against the 187 recently collected blast isolates and displayed durable resistance for almost 20 years. Linkage mapping and QTL-seq analysis indicated that a 1.16-Mb region on chromosome 6 (Pi169-6(t)) and a 2.37-Mb region on chromosome 11 (Pi169-11(t)) conferred the blast resistance in SA0169. Sequence analysis and genomic editing study revealed 2 and 7 candidate R genes in Pi169-6(t) and Pi169-11(t), respectively. With the assistance of mapping results, six blast and bacterial blight double resistant lines, which carried Pi169-6(t) and/or Pi169-11(t), were established. The complementation of Pi169-6(t) and Pi169-11(t), like SA0169, showed complete resistance to all tested isolates, suggesting that the combined effects of these two genomic regions largely confer the broad-spectrum resistance of SA0169. The sodium azide induced mutant SA0169 showed broad-spectrum and durable blast resistance. The broad resistance spectrum of SA0169 is contributed by the combined effects of two R regions, Pi169-6(t) and Pi169-11(t). Our study increases the understanding of the genetic basis of the broad-spectrum blast resistance induced by sodium azide mutagenesis, and lays a foundation for breeding new rice varieties with durable resistance against the blast pathogen.

Metabolic Disturbance Induced by the Embryo Contributes to the Formation of Chalky Endosperm of a Notched-Belly Rice Mutant

Grain chalkiness is a key quality trait of the rice grain, whereas its underlying mechanism is still not thoroughly understood because of the complex genetic and environmental interactions. We identified a notched-belly (NB) mutant that has a notched-line on the belly of grains. The line dissects the endosperm into two distinct parts, the upper translucent part, and the bottom chalky part in the vicinity of the embryo. Using this mutant, our previous studies clued the negative influence of embryo on the biochemical makeup of the endosperm, suggesting the need for the in-depth study of the embryo effect on the metabolome of developing endosperm. This study continued to use the NB mutant to evolve a novel comparison method to clarify the role of embryo in the formation of a chalky endosperm. Grain samples of the wild-type (WT) and NB were harvested at 10, 20, and 30 days after fertilization (DAF), and then divided into subsamples of the embryo, the upper endosperm, and the bottom endosperm. Using non-targeted metabolomics and whole-genome RNA sequencing (RNA-seq), a nearly complete catalog of expressed metabolites and genes was generated. Results showed that the embryo impaired the storage of sucrose, amino acid, starch, and storage proteins in the bottom endosperm of NB by enhancing the expression of sugar, amino acids, and peptide transporters, and declining the expression of starch, prolamin, and glutelin synthesis-related genes. Importantly, the competitive advantage of the developing embryo in extracting the nutrients from the endosperm, transformed the bottom endosperm into an “exhaustive source” by diverting the carbon (C) and nitrogen (N) metabolism from synthetic storage to secondary pathways, resulting in impaired filling of the bottom endosperm and subsequently the formation of chalky tissue. In summary, this study reveals that embryo-induced metabolic shift in the endosperm is associated with the occurrence of grain chalkiness, which is of relevance to the development of high-quality rice by balancing the embryo–endosperm interaction.

Down-regulation of OsMYB103L distinctively alters beta-1,4-glucan polymerization and cellulose microfibers assembly for enhanced biomass enzymatic saccharification in rice

Background As a major component of plant cell walls, cellulose provides the most abundant biomass resource convertible for biofuels. Since cellulose crystallinity and polymerization have been characterized as two major features accounting for lignocellulose recalcitrance against biomass enzymatic saccharification, genetic engineering of cellulose biosynthesis is increasingly considered as a promising solution in bioenergy crops. Although several transcription factors have been identified to regulate cellulose biosynthesis and plant cell wall formation, much remains unknown about its potential roles for genetic improvement of lignocellulose recalcitrance. Results In this study, we identified a novel rice mutant (Osfc9/myb103) encoded a R2R3-MYB transcription factor, and meanwhile generated OsMYB103L-RNAi-silenced transgenic lines. We determined significantly reduced cellulose levels with other major wall polymers (hemicellulose, lignin) slightly altered in mature rice straws of the myb103 mutant and RNAi line, compared to their wild type (NPB). Notably, the rice mutant and RNAi line were of significantly reduced cellulose features (crystalline index/CrI, degree of polymerization/DP) and distinct cellulose nanofibers assembly. These alterations consequently improved lignocellulose recalcitrance for significantly enhanced biomass enzymatic saccharification by 10–28% at p < 0.01 levels (n = 3) after liquid hot water and chemical (1% H2SO4, 1% NaOH) pretreatments with mature rice straws. In addition, integrated RNA sequencing with DNA affinity purification sequencing (DAP-seq) analyses revealed that the OsMYB103L might specifically mediate cellulose biosynthesis and deposition by regulating OsCesAs and other genes associated with microfibril assembly. Conclusions This study has demonstrated that down-regulation of OsMYB103L could specifically improve cellulose features and cellulose nanofibers assembly to significantly enhance biomass enzymatic saccharification under green-like and mild chemical pretreatments in rice. It has not only indicated a powerful strategy for genetic modification of plant cell walls in bioenergy crops, but also provided insights into transcriptional regulation of cellulose biosynthesis in plants.

SLL1-ZH Regulates Spikelets Architecture and Grain Yield in Rice

The spikelet developmental processes that control structure and floral organ identity play critical roles in rice grain yield formation. In this study, we characterized a novel rice mutant, SLL1-ZH, which exhibits a variety of defective agronomic characters, including semi-dwarf, rolling leaf, deformed panicles, and reduced grains production. Morphological analysis also revealed that the SLL1-ZH mutant shows numerous defects of floral organs, such as cracked glumes, hooked and thin lemmas, shrunken but thickened paleas, an indeterminate number of stamens and stigmas, and heterotopic ovaries. Map-based cloning identified a single nucleotide substitution (C to G) in the first exon of LOC_Os09g23200 that is responsible for the SLL1-ZH phenotype. In addition, qPCR analysis showed a significant change in the relative expression of SLL1-ZH in the mutant during inflorescence differentiation and in the different floral organs. Transcription of rice floral organ development-related factors also changed significantly in the mutant. Therefore, our results suggested that SLL1-ZH plays a great role in plant growth, spikelet development, and grain yield in rice.

Comparative Physiological, Biochemical, and Proteomic Responses of Photooxidation-Prone Rice Mutant 812HS under High Light Conditions

Photosynthetic efficiency decreases as light energy surpasses the photosynthesis capacity. This study was designed to investigate the potential effects of high-intensity light on the photooxidation-prone mutant 812HS of rice and its wild-type 812S during yellow and recovering stages. Results showed that in the yellowing stage, light oxidation occurs due to the exposure of mutant 812HS leaves to the high sunlight, which causes yellowing of the leaves, leading to a reduction in the photochemical activities, physiological mechanisms, and protein contents in mutant 812HS. In the recovery stage, mutant 812HS leaves were exposed to the maximum high brightness, the mutant’s leaves were draped with a dark cover to decrease the exposure of leaves of the plants from direct sunlight, which leads to the restoration of the green color again to the mutant 812HS leaves, leading to improving the performance of the photochemical activities, physiological mechanisms, and protein contents in mutant 812HS. Exposing leaves of mutant 812HS to high light at the yellow stage also resulted in a decrease in the net photosynthetic rate (Pn) in carotenoids content and chlorophyll a and b. Similarly, chlorophyll fluorescence of mutant 812HS decreased in (O-I-J-I-P) curves, and the ATP content, Mg2+-ATPase, and Ca2+-ATPase activities also decreased. An increase in energy dissipation was observed, while ABS/RC, DI0/RC, and TR0/RC values in mutant 812HS at the yellow stage increased. During photooxidation, an increase in O2•– and H2O2 contents was observed in mutant 812HS. While O2•– and H2O2 contents were decreased in mutant 812HS at the recovery stage. The rate of thylakoid membrane protein content was significantly decreased in mutant 812HS at the yellow stage, while at the recovery stage, there was no significant decrease. Our findings showed that photooxidation prompted oxidative damages and lipid peroxidation that caused severe damages to the membranes of the cell, photosynthetic pigments degradation, protein levels, and photosynthesis inhibition in mutant 812HS.

Radiosensitivity of rice varieties of Mira-1 and Bestari mutants using gamma rays irradiation

New rice varieties could be released by various plant breeding methods including mutation induction using gamma rays irradiation. Radiosensitivity and LD50 values (Lethal Dose, 50%) can be determined from the morphological response of plants to irradiation treatment in M1 generation. The research aims to determine the value of LD50 and the performance of rice mutant traits of Mira-1 and Bestari. The experiment was conducted using the Randomized Complete Block Design (RCBD) with two factors (rice varieties and gamma irradiation doses) and three replications. The plant traits observed were the percentage of germination, seedling height, and root length in the seedling phase. The results showed that LD50 values in the Mira-1 and Bestari varieties differed in all observed characters. The optimum dose to induce rice mutation of the varieties under investigation is within the range of 521.40 – 663.68 Gy.

Stability of Rice Mutant Lines By Linear Mixed Model And Suggestion A New Index Based For Yield Performance And Stability

Fourteen rice mutant lines with four cultivars were evaluated in a randomized complete block design with three replications in three locations in Iran (Rasht, ChaparSar and Fars province) during two growing seasons (2014-2016). In addition, a new index namely as equivalent index of stability and performance (EISP) is suggested for simultaneous evaluation of yield performance and stability. The heat map of yield performance and WAASB (weighted average of absolute scores based on BLUP (best linear unbiased prediction)) identified G3, G9, G6, G12 and G5 as highly productive and stable genotypes. Based on the analysis by multi-trait stability index (MTSI) G7, G5 and G1 were selected as superior genotypes. The top five superior genotypes based on harmonic mean and of the relative performance of genotypic values (HMRPGV) were G5, G12, G7, G2 and G1. For verification of EISP, its value was calculated for some of multi and univariate stability indices and identified genotypes G5 and G12 as the best ones. Principal component analysis indicated yield positively correlated with HMGV, RPGV, HMRPGV, EIS2P EIbP and EIPiP. In conclusion, G12, G5 and G9 had a significant advantage over all genotypes and could undergo selection or cultivar introduction processes.

A Ubiquitously Expressed UDP-Glucosyltransferase, UGT74J1, Controls Basal Salicylic Acid Levels in Rice

Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.