Estimating Key Phenological Dates of Multiple Rice Accessions Using Unmanned Aerial Vehicle-Based Plant Height Dynamics for Breeding

Efficient and high-quality estimation of key phenological dates in rice is of great significance in breeding work. Plant height(PH) dynamics are valuable for estimating phenological dates. However, research on estimating the key phenological dates of multiple rice accessions based on PH dynamics has been limited. In 2022, field traits were collected using unmanned aerial vehicle(UAV)-based images across 435 plots, including 364 rice varieties. PH, dates of initial heading(IH) and full heading(FH), and panicle initiation(PI), and growth period after transplanting(GPAT) were collected during the rice growth stage. PHs were extracted using a digital surface model(DSM) and fitted using Fourier and logistic models. Machine learning algorithms, including multiple linear regression, random forest(RF), support vector regression, least absolute shrinkage and selection operator, and elastic net regression, were employed to estimate phenological dates. Results indicated that the optimal percentile of the DSM for extracting rice PH was the 95th(R^(2) = 0.934, RMSE = 0.056 m). The Fourier model provided a better fit for PH dynamics compared with the logistic models. Additionally, curve features(CF) and GPAT were significantly associated with PI, IH, and FH. The combination of CF and GPAT outperformed the use of CF alone, with RF demonstrating the best performance among the algorithms. Specifically, the combination of CF extracted from the logistic models, GPAT, and RF yielded the best performance for estimating PI(R^(2) = 0.834, RMSE = 4.344 d), IH(R^(2) = 0.877, RMSE = 2.721 d), and FH(R^(2) = 0.883, RMSE = 2.694 d). Overall, UAV-based rice PH dynamics combined with machine learning effectively estimated the key phenological dates of multiple rice accessions, providing a novel approach for investigating key phenological dates in breeding work.

Molecular Mechanism of Rice Necrotic Lesion for Optimized Yield and Disease Resistance

How to balance rice resistance and yield is an important issue in rice breeding.Plants with mutated necrotic lesion genes often have persistent broad-spectrum resistance,but this broad-spectrum resistance usually comes at the expense of yield.Currently,many necrotic lesion mutants in rice have been identified,and these genes are involved in disease resistance pathways.This review provides a detailed introduction to the characteristics,classification,and molecular mechanisms of necrotic lesion formation.Additionally,we review the molecular regulatory pathways of genes involved in rice disease resistance.Concurrently,we summarize the relationship between resistance and yield in rice using newly developed gene editing methods.We discuss a rational and precise breeding strategy to better utilize molecular design technology for breeding disease-resistant and high-yield rice varieties.

Oriented Generation of Novel Thermo-Sensitive Genic Male Sterile Lines with Improved Grain Shape and Outcrossing Rate in Early-Season Rice

Two-line hybrid rice with excellent quality is preferred in the Chinese market.However,there is a trade-off between reducing costs for hybrid seed production and lowering the outcrossing rate of the sterile line,which is largely determined by the stigma exsertion rate(SER).In this study,we constructed mutants of male sterility lines with improved grain length(GL)and SER in three elite early-season indica rice varieties through targeted manipulation of the TMS5 and GS3 genes using CRISPR/Cas9-mediated multiplex systems.We obtained a series of marker-free gs3 single mutants and gs3tms5 double mutants with significantly higher SER,longer grains,and increased 1000-grain weight compared with the wild type(WT).Importantly,the typically thermo-sensitive genic male sterile(TGMS)trait with a higher SER was observed in gs3tms5 mutants,and their F1 hybrids exhibited remarkable improvements in grain shape and yield-related traits.Our findings provided an efficient method to generate new valuable TGMS germplasm with improved SER through the mutagenesis of GS3 and TMS5 synergistically,and demonstrated that GS3 had pleiotropic effects on grain size,SER,and grain quality in early-season indica rice.

Identification of the BTA8 gene reveals the contribution of natural variation to tiller angle in rice

Plant architecture is a collection of major agronomic traits that determines rice grain production,and it is mainly influenced by tillering,tiller angle,plant height and panicle morphology(Wang and Li 2006).Tiller angle is one of the critical components that determines rice plant architecture,which in turn influences grain yield mainly due to its large impact on plant density(Wang et al.2022).

A 9.5-kb deletion in the 1st intron of OsMADS51 enhances temperature sensitivity in rice

Temperature is an important environmental factor affecting heading date of rice.Despite its importance,genes responsible for temperature-sensitive heading in rice have remained elusive.Our previous study identified a quantitative trait locus qHd1 which advances heading date under high temperatures.A 9.5-kb insertion was found in the first intron of OsMADS51 in indica variety Zhenshan 97(ZS97).However,the function of this natural variant in controlling temperature sensitivity has not been verified.In this study,we used CRISPR/Cas9 to knock out the 9.5-kb insertion in ZS97.Experiments conducted under cotrolled conditions in phytotrons confirmed that deletion increased temperature sensitivity and advanced heading by downregulating the expression level of OsMADS51.One-hybrid assays in yeast,ChIP-quantitative polymerase chain reaction,electrophoretic mobility shift,and luciferase-based transient transactivation assays collectively confirmed that OsMADS51 affects heading date by regulation of heading date gene Ehd1.We further determined that the long non-coding RNA HEATINR is generated from the first intron of OsMADS51,offering an explanation for how the 9.5-kb insertion affects temperature sensitivity.We also found that OsMADS51 was strongly selected in early/late-season rice varieties in South China,possibly accounting for their strong temperature sensitivity.These insights not only advance our understanding of the molecular mechanisms underlying the temperature-responsive regulation of heading date in rice but also provide a valuable genetic target for molecular breeding.

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

Recent advances in molecular biology research of a rice pest, the brown planthopper

The brown planthopper, Nilaparvata lugens St?l, has become a major threat in tropical Asian and China since the rice green revolution of the 1960 s. Currently, insecticide application remains the primary choice for controlling this rice insect pest, but heavy use of insecticides poses dangerous risks to beneficial natural enemies and pollinators, and stimulates N. lugens reproductivity, and has caused a resurgence of the pest in the major rice-planting regions throughout Asia. Achieving the long-lasting goal of sustainable management of N. lugens requires understanding of the molecular basis of outbreaks of the pest and the development of environment-friendly pest-control strategies. Here, we review the recent molecular advances in N. lugens research on the aspects of its endosymbionts, virus transmission, insecticide resistance, and interaction between N. lugens and rice plants. We also put forward further research directions that may shed some lights on management of the rice pest.

OsbZIP01 Affects Plant Growth and Development by Regulating OsSD1 in Rice

As the‘Green Revolution’gene,SD1(encoding GA20ox2),has been widely applied to improve yield in rice breeding.However,research on its transcriptional regulation is limited.Here,we identified a transcription factor OsbZIP01,which can suppress the expression of SD1 and regulate gibberellin(GA)biosynthesis in rice.Knockout mutants of OsbZIP01 exhibited increased plant height,while the overexpression lines showed a semi-dwarf phenotype and diminished germination rate.Furthermore,the semi-dwarf phenotype of OE-bZIP01,was caused by the reduced internode length,which was accompanied by a thin stem width.The predominant expression of OsbZIP01 was observed in leaves and sheaths.OsbZIP01 protein was localized in the nucleus and showed transcriptional repression activity.In addition,OsbZIP01 could directly bind to the promoter of the OsSD1 gene,and inhibit its transcription.The semi-dwarf phenotype of OE-bZIP01 could be rescued by exogenous GA_(3).Meanwhile,the bzip01 sd1 double mutant showed a shorter shoot length compared with the wild type,indicating that OsbZIP01 regulated plant growth mainly through the GA biosynthesis pathway.Collectively,OsbZIP01 negatively regulates GA biosynthesis by restraining SD1 transcription,thereby affecting plant growth and development.

Genetic and environmental control of rice tillering

Increasing tiller number is a target of high-yield rice breeding. Identification of tiller-defect mutants and their corresponding genes is helpful for clarifying the molecular mechanism of rice tillering. Summarizing research progress on the two processes of rice tiller formation, namely the formation and growth of axillary meristem, this paper reviews the effects of genetic factors, endogenous hormones, and exogenous environment on rice tillering, finding that multiple molecular mechanisms and signal pathways regulating rice tillering cooperate rice tillering, and discusses future research objectives and application of its regulatory mechanism. Elucidation of theis mechanism will be helpful for breeding high-yielding rice cultivars with ideal plant type via molecular design breeding.

Disruption of LEAF LESION MIMIC 4 affects ABA synthesis and ROS accumulation in rice

Lesion mimic mutants(LMMs) are advantageous materials for studying programmed cell death(PCD).Although some rice LMM genes have been cloned, the diversity of functions of these genes indicates that the mechanism of cell death regulation in LMMs needs further study. In this study, we identified a rice light-dependent leaf lesion mimic mutant 4(llm4) that showed abnormal chloroplast structure, photoinhibition, reduced photosynthetic protein levels, massive accumulation of reactive oxygen species(ROS), and PCD. Map-based cloning and complementation testing revealed that LLM4 encodes zeaxanthin epoxidase(ZEP), an enzyme involved in the xanthophyll cycle, which functions in plant photoprotection,ROS scavenging, and carotenoid and abscisic acid(ABA) biosynthesis. The ABA content was decreased,and the contents of 24 carotenoids differed between the llm4 mutant and the wild type(WT). The llm4mutant showed reduced dormancy and greater sensitive to ABA than the WT. We concluded that the mutation of LLM4 resulted in the failure of xanthophyll cycle, in turn causing ROS accumulation. The excessive ROS accumulation damaged chloroplast structure and induced PCD, leading eventually to the formation of lesion mimics.

Effect of GW8 Gene Editing on Appearance Quality of Erect-Panicle Type (dep1) Japonica Rice

Most high-yielding japonica rice varieties in China carry dep1,a multi-effective regulator of plant architecture and grain shape,resulting in erect panicle with short and round grain shape.However,its appearance quality needs to be improved since long-grain rice is favored by the market.GW8 is a dominant gene regulating grain shape,and its loss-of-function genotype leads to elongated grains with a better quality in appearance.

Additive Effects of QTLs/Genes on Rice Grain Size Traits Revealed by Genetic Comparisons

Grain size is controlled by many QTLs and/or genes.Despite intensive study,the genetic interactions between these QTLs/genes remain largely unclear.We previously found that the japonica large-grain rice cultivar TD70 with 1000-grain weight of 68.6 g carries superior alleles at the loci GW2,GS3,qGL3,GS5 and GW8,all of which are known to control grain size,while the indica rice variety Kasalath with 1000-grain weight of 19.1 g has normal alleles at these loci.

Transgenic cry1Ab/vip3H+epsps Rice with Insect and Herbicide Resistance Acted No Adverse Impacts on the Population Growth of a Non-Target Herbivore, the White-Backed Planthopper, Under Laboratory and Field Conditions

Numerous Bt rice lines expressing Cry protein derived from Bacillus thuringiensis Berliner （Bt） have been developed since 1989. However, the potential risks posed by Bt rice on non-target organisms still remain debate. The white-backed planthopper （WBPH）, Sogatella furcifera （Horváth）, is one of the most economically important insect pests of rice in Asian countries and also one of the main non-target herbivores of transgenic rice. In the current study, impacts of transgenic cry1Ab/vip3H＋epsps rice （G6H1） with both insect and herbicide resistance on WBPH were evaluated to ascertain whether this transgenic rice line had potential risks for this sap-sucking pest under laboratory and ifeld conditions. The laboratory results showed that no signiifcant difference in egg developmental duration, nymphal survival rate and female fecundity was found for WBPH between G6H1 and its non-transgenic isoline （XS110）. However, the development duration of nymphs was signiifcantly shorter and female longevity signiifcantly longer when WBPH fed on G6H1 by comparison with those on its control. To verify the results found in laboratory, a 3-yr ifeld trial was conducted to monitor WBPH population using both the vacuum-suction machine and beat plate methods. Although the seasonal density of WBPH nymphs and total density of nymphs and adults were not signiifcantly affected by transgenic rice regardless of the sampling methods, the seasonal density of WBPH adults in transgenic rice plots was slightly lower than that in the control when using the vacuum-suction machine. Based on these results both from laboratory and ifeld, it is clear that our tested transgenic rice line will not lead higher population of WBPH. However, long-term ifeld experiments to monitor the population dynamics of WPBH at large scale need to be conducted to conifrm the present conclusions in future.

Gapless Genome Assembly of ZH8015 and Preliminary Multi-Omics Analysis to Investigate ZH8015's Responses Against Brown Planthopper Infestation

Accurate genomic information is essential for advancing genetic breeding research in specific rice varieties.This study presented a gapless genome assembly of the indica rice cultivar Zhonghui 8015(ZH8015)using Pac Bio HiFi,Hi-C,and ONT(Oxford Nanopore Technologies)ultra-long sequencing technologies,annotating 43037 gene structures.Subsequently,utilizing this genome along with transcriptomic and metabolomic techniques,we explored ZH8015's response to brown planthopper(BPH)infestation.Continuous transcriptomic sampling indicated significant changes in gene expression levels around 48 h after BPH feeding.Enrichment analysis revealed particularly significant alterations in genes related to reactive oxygen species scavenging and cell wall formation.Metabolomic results demonstrated marked increases in levels of several monosaccharides,which are components of the cell wall and dramatic changes in flavonoid contents.Omics association analysis identified differentially expressed genes associated with key metabolites,shedding light on ZH8015's response to BPH infestation.In summary,this study constructed a reliable genome sequence resource for ZH8015,and the preliminary multi-omics results will guide future insect-resistant breeding research.

OsbZIP53 Negatively Regulates Immunity Response by Involving in Reactive Oxygen Species and Salicylic Acid Metabolism in Rice

The basic region/leucine zipper(bZIP)transcription factors play important roles in plant development and responses to abiotic and biotic stresses.OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5-RNAi transgenic plants.To further investigate the biological functions of OsbZIP53,we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants.Comprehensive analysis of phenotypical,physiological,and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants,but also regulated the immune response through the salicylic acid pathway.Specifically,disrupting OsbZIP53 increased H2O2 accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs(Osrboh genes)and weakened H2O2 degradation by directly targeting OsMYBS1.In addition,the growth of osbzip53 mutants was seriously impaired,while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type,suggesting that OsbZIP53 has a balancing effect on rice immune response and growth.

Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

The morphological development of rice(Oryza sativa L.)leaves is closely related to plant architecture,physiological activities,and resistance.However,it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment.In this study,a drought-sensitive,roll-enhanced,and narrow-leaf mutant(renl1)was induced from a semi-rolled leaf mutant(srl1)by ethyl methane sulfonate(EMS),which was obtained from Nipponbare(NPB)through EMS.Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase,allelic to NRL1/OsCLSD4.The RENL1 mutation resulted in reduced vascular bundles,vesicular cells,cellulose,and hemicellulose contents in cell walls,diminishing the water-holding capacity of leaves.In addition,the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species(ROS)was decreased,leading to an increase in ROS after drought stress.Meanwhile,genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components.Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance,and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance,thereby realizing simultaneous improvement of multiple traits in rice.

Rice AGL1 determines grain size and sterile lemma identity

The grass spikelet is a unique inflorescence structure that determines grain size.Although many genetic factors have been well characterized for grain size and glume development,the underlying molecular mechanisms in rice are far from established.Here,we isolated rice gene,AGL1 that controlled grain size and determines the fate of the sterile lemma.Loss of function of AGL1 produced larger grains and reduced the size of the sterile lemma.Larger grains in the agl1 mutant were caused by a larger number of cells that were longer and wider than in the wild type.The sterile lemma in the mutant spikelet was converted to a rudimentary glume-like organ.Our findings showed that the AGL1(also named LAX1)protein positively regulated G1 expression,and negatively regulated NSG1 expression,thereby affecting the fate of the sterile lemma.Taken together,our results revealed that AGL1 played a key role in negative regulation of grain size by controlling cell proliferation and expansion,and supported the opinion that rudimentary glume and sterile lemma in rice are homologous organs.

A polygalacturonase gene OsPG1 modulates water homeostasis in rice

A dynamic plant architecture is the basis of plant adaptation to changing environments.Although many genes regulating leaf rolling have been identified,genes directly associated with water homeostasis are largely unknown.Here,we isolated a rice mutant,dynamic leaf rolling 1(dlr1),characterized by‘leaf unfolding in the morning-leaf rolling at noon-leaf unfolding in the evening’during a sunny day.Water content was decreased in rolled leaves and water sprayed on leaves caused reopening,indicating that in vivo water deficiency induced the leaf rolling.Map-based cloning and expression tests demonstrated that an A1400G single base mutation in Oryza sativa Polygalacturonase 1(OsPG1)/PHOTO-SENSITIVE LEAF ROLLING 1(PSL1)was responsible for the dynamic leaf rolling phenotype in the dlr1 mutant.OsPG1 encodes a polygalacturonase,one of the main enzymes that degrade demethylesterified homogalacturonans in plant cell walls.OsPG1 was constitutively expressed in various tissues and was enriched in stomata.Mutants of the OsPG1 gene exhibited defects in stomatal closure and decreased stomatal density,leading to reduced transpiration and excessive water loss under specific conditions,but had normal root development.Further analysis revealed that mutation of OsPG1 led to reduced pectinase activity in the leaves and increased demethylesterified homogalacturonans in guard cells.Our findings reveal a mechanism by which OsPG1 modulates water homeostasis to control dynamic leaf rolling,providing insights for plants to adapt to environmental variation.

Effects of Nitrogen-Regulating Gene AreA on Growth,Pathogenicity,and Fumonisin Synthesis of Fusarium proliferatum

Rice spikelet rot disease not only threatens rice yields but also poses risks to humans and animals due to the production of the category 2B carcinogen fumonisins by the pathogen Fusarium proliferatum.Nitrogen(N)metabolism is known to have a significant influence on fungal growth and the synthesis of secondary metabolites.AreA is a global N regulatory gene belonging to the GATA transcription factor family.In this study,we observed that theΔAreA mutant exhibited a notable reduction in growth rate and conidium production.Pathogenicity experiments revealed thatΔAreA had almost lost its ability to infect rice spikelets.

Single-cell transcriptome sequencing reveals the mechanism regulating rice plumule development

Seed plumules comprise multiple developing tissues and are key sites for above-ground plant organ morphogenesis.Here,the spatial expression of genes in developing rice seed plumules was characterized by single-cell transcriptome sequencing in Zhongjiazao 17,a popular Chinese indica rice cultivar.Of 15 cell clusters,13 were assigned to cell types using marker genes and cluster-specific genes.Marker genes of multiple cell types were expressed in several clusters,suggesting a complex developmental system.Some genes for signaling by phytohormones such as abscisic acid were highly expressed in specific clusters.Various cis-elements in the promoters of genes specifically expressed in cell clusters were calculated,and some key hormone-related motifs were frequent in certain clusters.Spatial expression patterns of genes involved in rapid seed germination,seedling growth,and development were identified.These findings enhanced our understanding of cellular diversity and specialization within plumules of rice,a monocotyledonous model crop.