[Genetic dissection of rice resistance to bacterial blight].

Bacterial blight, a major disease in rice, poses a serious impact on rice production. In this study, a doubled haploid (DH) population derived from a cross between the introduced japonica cultivar 'Maybelle' and the indica landrace 'Baiyeqiu' was used to investigate the pathogenicity of four pathogen races causing bacterial blight. The results showed that the pathogenicity of all the pathogen races exhibited continuous, transgressive distribution in the DH population. Moreover, strong correlations existed between every two pathogen races, with the correlation coefficients ranging from 0.3 to 0.6. A total of 12 quantitative trait loci (QTLs) distributed on chromosomes 1, 2, 3, 5, 6, 7, 9, and 12 were detected for rice bacterial blight, explaining 4.95% to 16.05% of the phenotype. Among these QTLs, a major QTL located in the interval RM6024-RM163 on chromosome 5 was detected in three pathogen races. In addition, the pyramiding of the positive alleles can apparently improve the rice resistance to bacterial blight. This study is of great significance for broadening the genetic resources with resistance to bacterial blight in China.

[Mapping of QTL associated with rice cooking quality and candidate gene analysis].

Excavating the quantitative trait locus (QTL) associated with rice cooking quality, analyzing candidate genes, and improving cooking quality-associated traits of rice varieties by genetic breeding can effectively improve the taste of rice. In this study, we used the indica rice HZ, the japonica rice Nekken2 and 120 recombinant inbred lines (RILs) populations constructed from them as experimental materials to measure the gelatinization temperature (GT), gel consistency (GC) and amylose content (AC) of rice at the maturity stage. We combined the high-density genetic map for QTL mapping. A total of 26 QTLs associated with rice cooking quality (1 QTL associated with GT, 13 QTLs associated with GC, and 12 QTLs associated with AC) were detected, among which the highest likelihood of odd (LOD) value reached 30.24. The expression levels of candidate genes in the localization interval were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and it was found that the expression levels of six genes were significantly different from that in parents. It was speculated that the high expression of LOC_Os04g20270 and LOC_Os11g40100 may greatly increase the GC of rice, while the high expression of LOC_Os01g04920 and LOC_Os02g17500 and the low expression of LOC_Os03g02650 and LOC_Os05g25840 may reduce the AC. The results lay a molecular foundation for the cultivation of new high-quality rice varieties, and provide important genetic resources for revealing the molecular regulation mechanism of rice cooking quality.

[Exploring the innovative talents training mode in new era].

Innovation is an important way to promote economic development and social progress. Recent years have seen rapid development of biological sciences. In response to social demands and the needs for developing an innovative country, fostering innovative talents in the field of biosciences has become a significant initiative supported by national policies and the needs from talent market. Taking the innovative talent training mode implemented by Zhejiang Normal University in the field of biological sciences as an example, this paper comprehensively introduces several key aspects of the mode. This includes establishing a mentorship system as the foundation, carrying out curriculum reform through project competitions and practical platforms, and promoting synergy among industry, academia, and research in talent training. This training mode has achieved positive results in practice, promoting the training of outstanding innovative talents in biological science majors, and may facilitate the reform of talent training in similar majors.

WLP3 Encodes the Ribosomal Protein L18 and Regulates Chloroplast Development in Rice.

Plastid ribosomal proteins play a crucial role in the growth and development of plants, mainly in the gene expression and translation of key genes in chloroplasts. While some information is known about the regulatory processes of plastid ribosomal proteins in various plant species, there is limited knowledge about the underlying mechanisms in rice. In this study, ethyl methanesulfonate (EMS) mutagenesis was used to generate a new mutant called wlp3 (white leaf and panicle3), characterized by white or albino leaves and panicles, which exhibited this phenotype from the second leaf stage until tillering. Furthermore, after a certain period, the newly emerging leaves developed the same phenotype as the rice variety ZH11, while the albino leaves of wlp3 showed an incomplete chloroplast structure and significantly low chlorophyll content. A transition mutation (T to C) at position 380 was identified in the coding region of the LOC_Os03g61260 gene, resulting in the substitution of isoleucine by threonine during translation. WLP3 encodes the ribosomal L18 subunit, which is localized in the chloroplast. Complementation experiments confirmed that LOC_Os03g61260 was responsible for the albino phenotype in rice. WLP3 has high expression in the coleoptile, leaves at the three-leaf stage, and panicles at the heading stage. Compared to the wild-type (WT), wlp3 exhibited reduced chlorophyll synthesis and significantly decreased expression levels of genes associated with plastid development. Yeast two-hybrid (Y2H) analysis revealed that WLP3 interacts with other ribosomal subunits, to influence chloroplast development. These results contribute to a better understanding of the underlying molecular mechanisms of chloroplast development and plastid gene translation.

The Mining of Genetic Loci and the Analysis of Candidate Genes to Identify the Physical and Chemical Markers of Anti-Senescence in Rice.

Premature senescence is a common occurrence in rice production, and seriously affects rice plants' nutrient utilization and growth. A total of 120 recombinant inbred lines (RILs) were obtained from successive self-crossing of F12 generations derived from Huazhan and Nekken2. The superoxide dismutase (SOD) activity, malondialdehyde (MDA), content and catalase (CAT) activity related to the anti-senescence traits and enzyme activity index of rice were measured for QTL mapping using 4858 SNPs. Thirteen QTLs related to anti-senescence were found, among which the highest LOD score was 5.70. Eighteen anti-senescence-related genes were found in these regions, and ten of them differed significantly between the parents. It was inferred that LOC_Os01g61500, LOC_Os01g61810, and LOC_Os04g40130 became involved in the regulation of the anti-senescence molecular network upon upregulation of their expression levels. The identified anti-senescence-related QTLs and candidate genes provide a genetic basis for further research on the mechanism of the molecular network that regulates premature senescence.

Identification of QTLs Conferring Resistance to Bacterial Diseases in Rice.

Bacterial panicle blight, bacterial leaf streak, and bacterial brown stripe are common bacterial diseases in rice that represent global threats to stable rice yields. In this study, we used the rice variety HZ, Nekken and their 120 RIL population as experimental materials. Phenotypes of the parents and RILs were quantitatively analyzed after inoculation with Burkholderia glumae, Xanthomonas oryzae pv. oryzicola, and Acidovorax avenae subsp. avenae. Genetic SNP maps were also constructed and used for QTL mapping of the quantitative traits. We located 40 QTL loci on 12 chromosomes. The analysis of disease resistance-related candidate genes in the QTL regions with high LOD value on chromosomes 1, 3, 4, and 12 revealed differential expression before and after treatment, suggesting that the identified genes mediated the variable disease resistance profiles of Huazhan and Nekken2. These results provide an important foundation for cloning bacterial-resistant QTLs of panicle blight, leaf streak, and brown stripe in rice.

Advances in cloning functional genes for rice yield traits and molecular design breeding in China.

Rice, a major food crop in China, contributes significantly to international food stability. Advances in rice genome sequencing, bioinformatics, and transgenic techniques have catalyzed Chinese researchers' discovery of novel genes that control rice yield. These breakthroughs in research also encompass the analysis of genetic regulatory networks and the establishment of a new framework for molecular design breeding, leading to numerous transformative findings in this field. In this review, some breakthroughs in rice yield traits and a series of achievements in molecular design breeding in China in recent years are presented; the identification and cloning of functional genes related to yield traits and the development of molecular markers of rice functional genes are summarized, with the intention of playing a reference role in the following molecular design breeding work and how to further improve rice yield.

Long-term spatiotemporal and highly specific imaging of the plasma membrane of diverse plant cells using a near-infrared AIE probe.

Fluorescent probes are valuable tools to visualize plasma membranes intuitively and clearly and their related physiological processes in a spatiotemporal manner. However, most existing probes have only realized the specific staining of the plasma membranes of animal/human cells within a very short time period, while almost no fluorescent probes have been developed for the long-term imaging of the plasma membranes of plant cells. Herein, we designed an AIE-active probe with NIR emission to achieve four-dimensional spatiotemporal imaging of the plasma membranes of plant cells based on a collaboration approach involving multiple strategies, demonstrated long-term real-time monitoring of morphological changes of plasma membranes for the first time, and further proved its wide applicability to plant cells of different types and diverse plant species. In the design concept, three effective strategies including the similarity and intermiscibility principle, antipermeability strategy and strong electrostatic interactions were combined to allow the probe to specifically target and anchor the plasma membrane for an ultralong amount of time on the premise of guaranteeing its sufficiently high aqueous solubility. The designed APMem-1 can quickly penetrate cell walls to specifically stain the plasma membranes of all plant cells in a very short time with advanced features (ultrafast staining, wash-free, and desirable biocompatibility) and the probe shows excellent plasma membrane specificity without staining other areas of the cell in comparison to commercial FM dyes. The longest imaging time of APMem-1 can be up to 10 h with comparable performance in both imaging contrast and imaging integrity. The validation experiments on different types of plant cells and diverse plants convincingly proved the universality of APMem-1. The development of plasma membrane probes with four-dimensional spatial and ultralong-term imaging ability provides a valuable tool to monitor the dynamic processes of plasma membrane-related events in an intuitive and real-time manner.

[Application of CRISPR-Cas9 gene editing technology in crop breeding].

The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.

A base substitution in OsphyC disturbs its Interaction with OsphyB and affects flowering time and chlorophyll synthesis in rice.

BACKGROUND: Phytochromes are important photoreceptors in plants, and play essential roles in photomorphogenesis. The functions of PhyA and PhyB in plants have been fully analyzed, while those of PhyC in plant are not well understood. RESULTS: A rice mutant, late heading date 3 (lhd3), was characterized, and the gene LHD3 was identified with a map-based cloning strategy. LHD3 encodes phytochrome C in rice. Animo acid substitution in OsphyC disrupted its interaction with OsphyB or itself, restraining functional forms of homodimer or heterodimer formation. Compared with wild-type plants, the lhd3 mutant exhibited delayed flowering under both LD (long-day) and SD (short-day) conditions, and delayed flowering time was positively associated with the day length via the Ehd1 pathway. In addition, lhd3 showed a pale-green-leaf phenotype and a slower chlorophyll synthesis rate during the greening process. The transcription patterns of many key genes involved in photoperiod-mediated flowering and chlorophyll synthesis were altered in lhd3. CONCLUSION: The dimerization of OsPhyC is important for its functions in the regulation of chlorophyll synthesis and heading. Our findings will facilitate efforts to further elucidate the function and mechanism of OsphyC and during light signal transduction in rice.

OsSPL88 Encodes a Cullin Protein that Regulates Rice Growth and Development.

Plant lesion mimics refer to necrotic spots spontaneously produced by the plant without mechanical damage, pathogen invasion, and adversity stress. Here, we isolated and characterized two rice (Oryza sativa L) mutants, namely, spl88-1 (spotted leaf88-1) and spl88-2 (spotted leaf88-2), which were identified from an ethyl methanesulfonate-mutagenized japonica cultivar Xiushui 11 population. Physiological and biochemical experiments indicated that more ROS accumulated in spl88-1 and spl88-2 than in wild type. spl88-1 and spl88-2 displayed spontaneous cell death and enhanced their resistance to bacterial blight by affecting the expression of defense-related genes. We isolated SPL88 by map-based cloning, which encoded a highly conserved Cullin protein. A single base deletion was detected in spl88-1 and spl88-2, in which the 132nd base C of SPL88-1 and the 381th base T of SPL88-2 were deleted, causing premature termination of protein translation. SPL88 was expressed in root, stem, leaf, leaf sheath, and panicle. The Cullin protein was localized in the cytoplasm and nucleus. The aforementioned results indicate that SPL88 regulates the growth and development of rice by affecting the expression of defense-related genes.

Advance of Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 System and Its Application in Crop Improvement.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is the third generation of novel targeted genome editing technology after zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). It is also one of the most promising techniques for mutating and modifying genes. The CRISPR-Cas9 system has the advantages of simplicity, high efficiency, high specificity, and low production cost, thus greatly promoting the study of gene function. Meanwhile, it has attracted the attention of biologists. After the development and improvement in recent years, CRISPR-Cas9 system has become increasingly mature and has been widely used in crop improvement. Firstly, this review systematically summarizes the generation and advantages of CRISPR-Cas9 system. Secondly, three derivative technologies of the CRISPR-Cas9 system are introduced. Thirdly, this review focuses on the application of CRISPR-Cas9 system in gene knockout, gene knock-in, and gene regulation, as well as the improvement of yield, quality, and biological resistance of important crops such as rice, wheat, soybean, corn, and potato. Finally, this review proposes the potential challenges of CRISPR-Cas9 system, and discusses the future development of CRISPR-Cas9 system.

UDP-N-acetylglucosamine pyrophosphorylase enhances rice survival at high temperature.

High-temperature stress inhibits normal cellular processes and results in abnormal growth and development in plants. However, the mechanisms by which rice (Oryza sativa) copes with high temperature are not yet fully understood. In this study, we identified a rice high temperature enhanced lesion spots 1 (hes1) mutant, which displayed larger and more dense necrotic spots under high temperature conditions. HES1 encoded a UDP-N-acetylglucosamine pyrophosphorylase, which had UGPase enzymatic activity. RNA sequencing analysis showed that photosystem-related genes were differentially expressed in the hes1 mutant at different temperatures, indicating that HES1 plays essential roles in maintaining chloroplast function. HES1 expression was induced under high temperature conditions. Furthermore, loss-of-function of HES1 affected heat shock factor expression and its mutation exhibited greater vulnerability to high temperature. Several experiments revealed that higher accumulation of reactive oxygen species occurred in the hes1 mutant at high temperature. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and comet experiments indicated that the hes1 underwent more severe DNA damage at high temperature. The determination of chlorophyll content and chloroplast ultrastructure showed that more severe photosystem defects occurred in the hes1 mutant under high temperature conditions. This study reveals that HES1 plays a key role in adaptation to high-temperature stress in rice.

Quantitative trait locus mapping and candidate gene analysis for salt tolerance at bud stage in rice.

Soil salinization has a serious influence on rice yield and quality. How to enhance salt tolerance in rice is a topical issue. In this study, 120 recombinant inbred line populations were generated through nonstop multi-generation selfing using a male indica rice variety Huazhan (Oryza sativa L. subsp. indica cv. 'HZ') and a female variety of Nekken2 (Oryza sativa L. subsp. japonica cv. 'Nekken2') as the parents. Germination under 80 mM NaCl conditions was measured and analyzed, and quantitative trait locus (QTL) mapping was completed using a genetic map. A total of 16 salt-tolerance QTL ranges were detected at bud stage in rice, which were situated on chromosomes 3, 4, 6, 8, 9, 10, 11, and 12. The maximum limit of detection was 4.69. Moreover, the qST12.3 was narrowed to a 192 kb region on chromosome 12 using map-based cloning strategy. Statistical analysis of the expression levels of these candidate genes under different NaCl concentrations by qRT-PCR revealed that qST12.3 (LOC_Os12g25200) was significantly down-regulated with increasing NaCl concentration, and the expression level of the chlorine-transporter-encoding gene LOC_Os12g25200 in HZ was significantly higher than that of Nekken2 under 0 mM NaCl. Sequencing analysis of LOC_Os12g25200 promoter region indicated that the gene expression difference between parents may be due to eight base differences in the promoter region. Through QTL mining and analysis, a plurality of candidate genes related to salt tolerance in rice was obtained, and the results showed that LOC_Os12g25200 might negatively regulate salt tolerance in rice. The results provide the basis for further screening and cultivation of salt-tolerant rice varieties and have laid the foundation for elucidating further molecular regulation mechanisms of salt tolerance in rice.

Identification and Characterization of Short Crown Root 8, a Temperature-Sensitive Mutant Associated with Crown Root Development in Rice.

Crown roots are essential for plants to obtain water and nutrients, perceive environmental changes, and synthesize plant hormones. In this study, we identified and characterized short crown root 8 (scr8), which exhibited a defective phenotype of crown root and vegetative development. Temperature treatment showed that scr8 was sensitive to temperature and that the mutant phenotypes were rescued when grown under low temperature condition (20 °C). Histological and EdU staining analysis showed that the crown root formation was hampered and that the root meristem activity was decreased in scr8. With map-based cloning strategy, the SCR8 gene was fine-mapped to an interval of 126.4 kb on chromosome 8. Sequencing analysis revealed that the sequence variations were only found in LOC_Os08g14850, which encodes a CC-NBS-LRR protein. Expression and inoculation test analysis showed that the expression level of LOC_Os08g14850 was significantly decreased under low temperature (20 °C) and that the resistance to Xanthomonas oryzae pv. Oryzae (Xoo) was enhanced in scr8. These results indicated that LOC_Os08g14850 may be the candidate of SCR8 and that its mutation activated the plant defense response, resulting in a crown root growth defect.

The ell1 mutation disrupts tryptophan metabolism and induces cell death.

Tryptophan metabolism pathways are important components of the plant immune system; for example, serotonin is derived from tryptophan, and plays a vital role in rice (Oryza sativa) innate immunity. Recently, we isolated a rice mutant, early lesion leaf 1 (ell1), which exhibits lesions. RNA-seq analysis revealed that KEGG pathways related to amino acid metabolism were significantly enriched in the transcripts differentially expressed in this mutant. Furthermore, measurements of free amino acid contents revealed the accumulated tryptophan of ell1 mutant. In addition, the transcript levels of genes related to tryptophan biosynthesis were significantly enhanced in the ell1 mutant. These results revealed that ELL1 plays a critical role in tryptophan metabolism. Based on these findings, it is revealed that loss of ELL1 function may disrupt tryptophan metabolism, thereby inducing cell death and forming lesions in rice.

PHOTO-SENSITIVE LEAF ROLLING 1 encodes a polygalacturonase that modifies cell wall structure and drought tolerance in rice.

The biosynthesis and modification of cell wall composition and structure are controlled by hundreds of enzymes and have a direct consequence on plant growth and development. However, the majority of these enzymes has not been functionally characterised. Rice mutants with leaf-rolling phenotypes were screened in a field. Phenotypic analysis under controlled conditions was performed for the selected mutant and the relevant gene was identified by map-based cloning. Cell wall composition was analysed by glycome profiling assay. We identified a photo-sensitive leaf rolling 1 (psl1) mutant with 'napping' (midday depression of photosynthesis) phenotype and reduced growth. The PSL1 gene encodes a cell wall-localised polygalacturonase (PG), a pectin-degrading enzyme. psl1 with a 260-bp deletion in its gene displayed leaf rolling in response to high light intensity and/or low humidity. Biochemical assays revealed PG activity of recombinant PSL1 protein. Significant modifications to cell wall composition in the psl1 mutant compared with the wild-type plants were identified. Such modifications enhanced drought tolerance of the mutant plants by reducing water loss under osmotic stress and drought conditions. Taken together, PSL1 functions as a PG that modifies cell wall biosynthesis, plant development and drought tolerance in rice.

MORE FLORET1 Encodes a MYB Transcription Factor That Regulates Spikelet Development in Rice.

Rice (Oryza sativa) spikelets have a unique inflorescence structure, and the mechanisms regulating their development are not yet fully understood. Moreover, approaches to manipulate spikelet development have the potential to increase grain yield. In this study, we identified and characterized a recessive spikelet mutant, namely more floret1 (mof1). The mof1 mutant has a delayed transition from the spikelet to the floral meristem, inducing the formation of extra lemma-like and palea-like organs. In addition, the main body of the palea was reduced, and the sterile lemma was enlarged and partially acquired hull (lemma and/or palea) identity. We used map-based cloning to identify the MOF1 locus and confirmed our identification by complementation and by generating new mof1 alleles using CRISPR-Cas9 gene editing. MOF1 encodes a MYB domain protein with the typical ethylene response factor-associated amphiphilic repression motifs, is expressed in all organs and tissues, and has a strong repression effect. MOF1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs to possibly repress the expression of downstream target genes. Taken together, our results reveal that MOF1 plays an important role in the regulation of organ identity and spikelet determinacy in rice.

A Strigolactone Biosynthesis Gene Contributed to the Green Revolution in Rice.

Plant architecture is a complex agronomic trait and a major factor of crop yield, which is affected by several important hormones. Strigolactones (SLs) are identified as a new class hormoneinhibiting branching in many plant species and have been shown to be involved in various developmental processes. Genetical and chemical modulation of the SL pathway is recognized as a promising approach to modify plant architecture. However, whether and how the genes involved in the SL pathway could be utilized in breeding still remain elusive. Here, we demonstrate that a partial loss-of-function allele of the SL biosynthesis gene, HIGH TILLERING AND DWARF 1/DWARF17 (HTD1/D17), which encodes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), increases tiller number and improves grain yield in rice. We found that the HTD1 gene had been widely utilized and co-selected with Semidwarf 1 (SD1), both contributing to the improvement of plant architecture in modern rice varieties since the Green Revolution in the 1960s. Understanding how phytohormone pathway genes regulate plant architecture and how they have been utilized and selected in breeding will lay the foundation for developing the rational approaches toward improving crop yield.