The zinc finger protein DHHC09 S-acylates the kinase STRK1 to regulate H2O2 homeostasis and promote salt tolerance in rice.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

Rapid improvement of grain appearance in three-line hybrid rice via CRISPR/Cas9 editing of grain size genes.

KEY MESSAGE: Genetic editing of grain size genes quickly improves three-line hybrid rice parents to increase the appearance quality and yield of hybrid rice. Grain size affects rice yield and quality. In this study, we used CRISPR/Cas9 to edit the grain size gene GW8 in the maintainer line WaitaiB (WTB) and restorer line Guanghui998 (GH998). The new slender sterile line WTEA (gw8) was obtained in the BC2F1 generation by transferring the grain mutation of the maintainer plant to the corresponding sterile line WantaiA (WTA, GW8) in the T1 generation. Two slender restorer lines, GH998E1 (gw8(II)) and GH998E2 (gw8(I)), were obtained in T1 generation. In the early stage, new sterile and restorer lines in grain mutations were created by targeted editing of GS3, TGW3, and GW8 genes. These parental lines were mated to detect the impact of grain-type mutations on hybrid rice yield and quality. Mutations in gs3, gw8, and tgw3 had a minimal impact on agronomic traits except the grain size and thousand-grain weight. The decrease in grain width in the combination mainly came from gw8/gw8, gs3/gs3 increased the grain length, gs3/gs3-gw8/gw8 had a more significant effect on the grain length, and gs3/gs3-gw8/gw8(I) contributed more to grain length than gs3/gs3-gw8/gw8(II). The heterozygous TGW3/tgw3 may not significantly increase grain length. Electron microscopy revealed that the low-chalky slender-grain variety had a cylindrical grain shape, a uniform distribution of endosperm cells, and tightly arranged starch grains. Quantitative fluorescence analysis of endospermdevelopment-related genes showed that the combination of slender grain hybrid rice caused by gs3 and gw8 mutations promoted endosperm development and improved appearance quality. An appropriate grain size mutation resulted in hybrid rice varieties with high yield and quality.

Molecular basis of genetic improvement for key rice quality traits in Southern China.

Grain qualities including milling quality, appearance quality, eating and cooking quality, and nutritional quality are important indicators in rice breeding. Significant achievements in genetic improvement of rice quality have been made. In this study, we analyzed the variation patterns of 16 traits in 1570 rice varieties and found significant improvements in appearance quality and eating and cooking quality, particularly in hybrid rice. Through genome-wide association study and allelic functional nucleotide polymorphisms analysis of quality trait genes, we found that ALK, FGR1, FLO7, GL7/GW7, GLW7, GS2, GS3, ONAC129, OsGRF8, POW1, WCR1, and Wx were associated with the genetic improvement of rice quality traits in Southern China. Allelic functional nucleotide polymorphisms analysis of 13 important rice quality genes, including fragrance gene fgr, were performed using the polymerase chain reaction amplification refractory mutation system technology. The results showed that Gui516, Gui569, Gui721, Ryousi, Rsimiao, Rbasi, and Yuehui9802 possessed multiple superior alleles. This study elucidates the phenotypic changes and molecular basis of key quality traits of varieties in Southern China. The findings will provide guidance for genetic improvement of rice quality and the development of new varieties.

Editing of rice (Oryza sativa L.) OsMKK3 gene using CRISPR/Cas9 decreases grain length by modulating the expression of photosystem components.

Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.

The genetic editing of GS3 via CRISPR/Cas9 accelerates the breeding of three-line hybrid rice with superior yield and grain quality.

Grain size is one of the major traits that determine rice grain yield and quality. The GS3 gene is the first major quantitative trait locus (QTL) that was identified in regulating rice grain length and weight. It was reported that the gs3 allele with a mutation in the organ size regulation (OSR) domain of the GS3 protein produced longer grains. In this study, we used the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele into our indica maintainer line, Mei1B, to enhance its grain yield and quality. Through molecular analysis and sequencing, a homologous edited-gs3 mutant line without any transgene was obtained in the T1 generation and was named Mei2B. A superior male sterile line Mei2A was generated by backcrossing the cytoplasmic male sterile (CMS) line Mei1A with Mei2B. Mei2B had a higher grain quality and yield compared to its wild-type Mei1B. Its grain length increased by 7.9%, its length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. In addition, genetic improvement of other quality traits including brown rice length (6.83 mm), brown rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both Mei1A and Mei2A to produce hybrid rice. Compared to two hybrids of Mei1A, the hybrids of Mei2A had longer grains, higher length/width ratio, TGW, and yield per plant. In addition, the hybrids of Mei2A showed a better grain appearance including better translucency, a lower chalky rice rate, and degree of chalkiness than the hybrids of Mei1A. These results demonstrated that the introduction of an elite gs3 allele into Mei1A via CRISPR/Cas9 gene editing technology led to significant genetic improvement of the rice grain. The resultant CMS line Mei2A(gs3) displayed much higher grain quality and yield than the original Mei1A. Therefore, our study demonstrated that the targeted genetic improvement via gene editing technology can enhance rice breeding, especially the breeding of three-line hybrid rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01290-z.

ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress.

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for a relative and absolute quantification (iTRAQ) labeling-based quantitative proteomics approach was used to detected changes in protein levels. Two-week-old seedlings of the cold tolerant rice variety Kongyu131 were treated at 8°C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to the control group. Functional analysis revealed that most of the regulated proteins are involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulated proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is that it provides new insights into cold stress responses in rice with respect to proteomics and provides candidate genes for cold-tolerance rice breeding.

Transcriptomic profiling of germinating seeds under cold stress and characterization of the cold-tolerant gene LTG5 in rice.

BACKGROUND: Low temperature is a limiting factor of rice productivity and geographical distribution. Wild rice (Oryza rufipogon Griff.) is an important germplasm resource for rice improvement. It has superior tolerance to many abiotic stresses, including cold stress, but little is known about the mechanism underlying its resistance to cold. RESULTS: This study elucidated the molecular genetic mechanisms of wild rice in tolerating low temperature. Comprehensive transcriptome profiles of two rice genotypes (cold-sensitive ce 253 and cold-tolerant Y12-4) at the germinating stage under cold stress were comparatively analyzed. A total of 42.44-68.71 million readings were obtained, resulting in the alignment of 29,128 and 30,131 genes in genotypes 253 and Y12-4, respectively. Many common and differentially expressed genes (DEGs) were analyzed in the cold-sensitive and cold-tolerant genotypes. Results showed more upregulated DEGs in the cold-tolerant genotype than in the cold-sensitive genotype at four stages under cold stress. Gene ontology enrichment analyses based on cellular process, metabolic process, response stimulus, membrane part, and catalytic activity indicated more upregulated genes than downregulated ones in the cold-tolerant genotype than in the cold-sensitive genotype. Quantitative real-time polymerase chain reaction was performed on seven randomly selected DEGs to confirm the RNA Sequencing (RNA-seq) data. These genes showed similar expression patterns corresponding with the RNA-Seq method. Weighted gene co-expression network analysis (WGCNA) revealed Y12-4 showed more positive genes than 253 under cold stress. We also explored the cold tolerance gene LTG5 (Low Temperature Growth 5) encoding a UDP-glucosyltransferase. The overexpression of the LTG5 gene conferred cold tolerance to indica rice. CONCLUSION: Gene resources related to cold stress from wild rice can be valuable for improving the cold tolerance of crops.

Development of molecular marker and introgression of Bph3 into elite rice cultivars by marker-assisted selection.

The brown planthopper (BPH) is a serious insect pest of rice and a substantial threat to rice production. Identification of new BPH resistance genes and their transfer into modern rice cultivars are effective breeding approaches to reduce the damage caused by BPH. In this study, we mapped a BPH resistance gene to a 50-kb genomic interval between two InDel markers 4M03980 and 4M04041 on the short arm of chromosome 4 in indica rice cultivar BP60, where the BPH resistance gene was mapped in Rathu Heenati by Liu et al. (2015) and named "Bph3". This region contains two annotated genes Os04g0201900 and Os04g0202300, which encode lectin receptor kinases responsible for BPH resistance. We also developed a molecular marker "MM28T" for Bph3, and introgression Bph3 into susceptible rice restorer lines Guihui582 and Gui7571 by the marker-assisted selection (MAS) approach. The BPH resistance level is significantly enhanced in the Bph3-introgression lines, the resistance scores decrease from 8.2 to 3.6 for Guihui582 and decrease from 8.7 to around 3.8 for Gui7571. Therefore, developing molecular markers for the BPH resistance gene Bph3 and using them for molecular breeding will facilitate the creation of BPH-resistance rice cultivars to reduce damage caused by BPH.

Mapping and Identifying a Candidate Gene Plr4, a Recessive Gene Regulating Purple Leaf in Rice, by Using Bulked Segregant and Transcriptome Analysis with Next-Generation Sequencing.

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1-pl2, pl1-pl3, and pl1-pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9-31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

Unscented information filtering phase unwrapping algorithm for interferometric fringe patterns.

This paper proposes a new phase unwrapping (PU) algorithm based on an unscented information filter for interferometric fringes. The proposed algorithm is the result of combining an unscented information filter with a Levenberg-Marquardt method, a robust phase gradient estimator called the amended matrix pencil model, and an efficient quality-guided strategy based on heapsort. The unscented information filter, a new type of filter that has recently been well applied to traditional nonlinear object tracking fields, is introduced to estimate the unambiguous unwrapped phase of wrapped phase images for the first time, to the best of our knowledge. First, a recursive PU procedure based on an unscented information filter is established to perform PU and noise filtering at the same time by combining the unscented information filter and the amended matrix pencil model, where the amended matrix pencil model is applied to acquire phase gradient information needed for the recursive PU procedure. Second, the above recursive PU procedure is further optimized to improve the accuracy of the phase estimate by inserting the Levenberg-Marquardt method. This is also the first time that the Levenberg-Marquardt method is combined with the unscented information filter for the unwrapping of interferometric fringes, to the best of our knowledge. Finally, the efficient quality-guided strategy based on heapsort is used to efficiently route the path of the unwrapping procedure and to guide the proposed method to efficiently unwrap wrapped pixels along the path from the high-reliance region to the low-reliance region of the wrapped fringes. Results obtained with synthetic data and real data show more acceptable solutions with the proposed method, compared to some of the most used algorithms.