Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.

Development of herbicide resistance genes and their application in rice

Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years,there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate,acetolactate synthase inhibitor herbicides,acetyl-Co A carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes,and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes,by artificial mutagenesis and mutant screening,and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems,and discusses the future prospects for the development of herbicideresistant rice.

Innovation and development of the third-generation hybrid rice technology

The breeding and large-scale application of hybrid rice contribute significantly to the food supply worldwide.Currently,hybrid seed production uses cytoplasmic male sterile(CMS)lines or photoperiod/thermo-sensitive genic male sterile(PTGMS)lines as female parent.Despite huge successes,both systems have intrinsic problems.CMS systems are mainly restricted by the narrow restorer resources that make it difficult to breed superior hybrids,while PTGMS systems are limited by conditional sterility of the male sterile lines that makes the propagation of both PTGMS seeds and hybrid seeds vulnerable to unpredictable climate changes.Recessive nuclear male sterile(NMS)lines insensitive to environmental conditions are widely distributed and are ideal for hybrid rice breeding and production,but the lack of effective ways to propagate the pure NMS lines in a large scale renders it impossible to use them for hybrid rice production.The development of"the third-generation hybrid rice technology"enables efficient propagation of the pure NMS lines in commercial scale.This paper discusses the establishment of"the thirdgeneration hybrid rice technology"and further innovations.This new technology breaks the limitations of CMS and PTGMS systems and will bring a big leap forward in hybrid rice production.

Trp_(548)Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides

Herbicide resistance in crop plants is valuable for integrated weed management in agriculture. Herbicide resistant rice, in particular, is important to management of weedy rice, a close relative of cultivated rice and a noxious weed prevalent in rice fields that remains challenging to farmers worldwide. Herbicide resistant plants can be obtained through transgenic approach or by mutagenesis of regular plant and screening of mutants with elevated resistance to herbicide. In this study, we conducted ethyl methyl sulfonate mutagenesis(EMS) to elite indica cultivar Huanghuazhan(HHZ) and screened for mutants resistant to imazapic, a herbicide that can inhibit the acetolactate synthase(ALS) in plants. We obtained three mutants of Os ALS gene that have not been reported previously in rice. One of the mutants, with Trp_(548) changed to Met(W_(548)M), was analyzed in more details in this study. This mutation had no negative effect on the plant physiology and morphology as well as rice yield. Compared with the imidazolinone-resistant mutant S_(627)N(Ser_(627) changed to Asn) that has been deployed for Clearfield rice development, W_(548)M mutant showed high levels of resistance to a broad spectrum of five families of ALSinhibiting herbicides, in addition to a higher level of resistance to herbicides of the imidazolinone family.The herbicide-resistance was stably inherited by crossing into other rice lines. Thus, the W_(548)M mutation provides a valuable resource for breeding of herbicide resistant rice and weed management.

Generation of a series of mutant lines resistant to imidazolinone by screening an EMS-based mutant library in common wheat

The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective at small doses,and exhibit broad-spectrum herbicidal action in the field.Therefore,the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields.In the present study,352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis.Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated,and mutation in one of three Ta ALS homolog genes conferred IMI resistance,and such a mutation is a dominant trait.Further analysis showed that taals-d exhibited the greatest IMI resistance,whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants.In terms of IMI resistance,the taals triple mutant was stronger than the taals double mutants,and the taals double mutants were stronger than the single mutants,indicating a dose-dependent effect of the Ta ALS mutation on IMI resistance in wheat.Biochemical analysis indicated that the mutation in Ta ALS increased the tolerance of Ta ALS to inhibition by IMI.Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance,and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.

A functional characterization of TaMs1 orthologs in Poaceae plants

TaMs1 encodes a non-specific lipid transfer protein(nsLTP) and is required for pollen development in wheat. Although MS1 is a Poaceae-specific gene, the roles of MS1 genes in other Poaceae plants are unknown, especially in rice and maize. Here, we identified one ortholog in rice(OsLTPg29) and two orthologs in maize(ZmLTPg11 and ZmLTPx2). Similar to TaMs1, both OsLTPg29 and ZmLTPg11 genes are specifically expressed in the microsporocytes, and both OsLTPg29 and ZmLTPg11 proteins showed lipid-binding ability to phosphatidic acid and several phosphoinositides. To determine their roles in pollen development, we created osltpg29 mutants and zmltpg11 zmltpx2 double mutants by CRISPR/Cas9.osltpg29, not zmltpg11 zmltpx2, is defective in pollen development, and only OsLTPg29, not ZmLTPg11,can rescue the male sterility of tams1 mutant. Our results demonstrate that the biological function of MS1 in pollen development differs in the evolution of Poaceae plants.

Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels

In addition to DNA sequence information, site-specific histone modifications are another important determinant of gene expression in a eukaryotic organism. We selected four modification sites in common histones that are known to significantly impact chromatin function and generated monoclonal or polyclonal antibodies that recognize each of those site-specific modifications. We used these antibodies to demonstrate that the site-specific histone modification levels remain relatively constant in different organs of the same organism. We also compared the levels of selected histone modifications among several representative organisms and found that site-specific modifications are highly variable among different organisms, providing new insight into the evolutionary divergence of specific histone modifications.

Green light mediates atypical photomorphogenesis by dual modulation of Arabidopsis phytochromes B and A

Although green light(GL)is located in the middle of the visible light spectrum and regulates a series of plant developmental processes,the mechanism by which it regulates seedling development is largely unknown.In this study,we demonstrated that GL promotes atypical photomorphogenesis in Arabidopsis thaliana via the dual regulations of phytochrome B(phyB)and phyA.Although the Pr-to-Pfr conversion rates of phyB and phyA under GL were lower than those under red light(RL)in a fluence rate-dependent and time-dependent manner,long-term treatment with GL induced high Pfr/Pr ratios of phyB and phyA.Moreover,GL induced the formation of numerous small phyB photobodies in the nucleus,resulting in atypical photomorphogenesis,with smaller cotyledon opening angles and longer hypocotyls in seedlings compared to RL.The abundance of phyA significantly decreased after short-and long-term GL treatments.We determined that four major PHYTOCHROME-INTERACTING FACTORs(PIFs:PIF1,PIF3,PIF4,and PIF5)act downstream of phyB in GL-mediated cotyledon opening.In addition,GL plays opposite roles in regulating different PIFs.For example,under continuous GL,the protein levels of all PIFs decreased,whereas the transcript levels of PIF4 and PIF5 strongly increased compared with dark treatment.Taken together,our work provides a detailed molecular framework for understanding the role of the antagonistic regulations of phyB and phyA in GL-mediated atypical photomorphogenesis.

Light-stabilized GIL1 suppresses PIN3 activity to inhibit hypocotyl gravitropism

Light and gravity coordinately regulate the directional growth of plants.Arabidopsis Gravitropic in the Light 1(GIL1)inhibits the negative gravitropism of hypocotyls in red and far-red light,but the underlying molecular mechanisms remain elusive.Our study found that GIL1 is a plasma membrane-localized protein.In endodermal cells of the upper part of hypocotyls,GIL1 controls the negative gravitropism of hypocotyls.GIL1 directly interacts with PIN3 and inhibits the auxin transport activity of PIN3.Mutation of PIN3 suppresses the abnormal gravitropic response of gil1 mutant.The GIL1 protein is unstable in darkness but it is stabilized by red and far-red light.Together,our data suggest that light-stabilized GIL1 inhibits the negative gravitropism of hypocotyls by suppressing the activity of the auxin transporter PIN3,thereby enhancing the emergence of young seedlings from the soil.

Increased long-distance and homo-trans interactions related to H3K27me3 in Arabidopsis hybrids

In plants,the genome structure of hybrids changes compared with their parents,but the effects of these changes in hybrids remain elusive.Comparing reciprocal crosses between Col×C24 and C24×Col in Arabidopsis using high-throughput chromosome conformation capture assay(Hi-C)analysis,we found that hybrid three-dimensional(3D)chromatin organization had more long-distance interactions relative to parents,and this was mainly located in promoter regions and enriched in genes with heterosis-related pathways.The interactions between euchromatin and heterochromatin were increased,and the compartment strength decreased in hybrids.In compartment domain(CD)boundaries,the distal interactions were more in hybrids than their parents.In the hybrids of CURLY LEAF(clf)mutants clfCol×clfC24and clfC24×clfCol,the heterosis phenotype was damaged,and the long-distance interactions in hybrids were fewer than in their parents with lower H3K27me3.ChIP-seq data revealed higher levels of H3K27me3 in the region adjacent to the CD boundary and the same interactional homo-trans sites in the wild-type(WT)hybrids,which may have led to more long-distance interactions.In addition,the differentially expressed genes(DEGs)located in the boundaries of CDs and loop regions changed obviously in WT,and the functional enrichment for DEGs was different between WT and clf in the longdistance interactions and loop regions.Our findings may therefore propose a new epigenetic explanation of heterosis in the Arabidopsis hybrids and provide new insights into crop breeding and yield increase.

Single-nucleus RNA and ATAC sequencing analyses provide molecular insights into early pod development of peanut fruit

Peanut(Arachis hypogaea L.)is an important leguminous oil and economic crop that produces flowers aboveground and fruits belowground.Subterranean fruit-pod development,which significantly affects peanut production,involves complex molecular mechanisms that likely require the coordinated regulation of multiple genes in different tissues.To investigate the molecular mechanisms that underlie peanut fruitpod development,we characterized the anatomical features of early fruit-pod development and integrated single-nucleus RNA-sequencing(snRNA-seq)and single-nucleus assay for transposase-accessible chromatin with sequencing(snATAC-seq)data at the single-cell level.We identified distinct cell types,such as meristem,embryo,vascular tissue,cuticular layer,and stele cells within the shell wall.These specific cell types were used to examine potential molecular changes unique to each cell type during pivotal stages of fruit-pod development.snRNA-seq analyses of differentially expressed genes revealed cell-type-specific insights that were not previously obtainable from transcriptome analyses of bulk RNA.For instance,we identified MADS-box genes that contributes to the formation of parenchyma cells and gravity-related genes that are present in the vascular cells,indicating an essential role for the vascular cells in peg gravitropism.Overall,our single-nucleus analysis provides comprehensive and novel information on specific cell types,gene expression,and chromatin accessibility during the early stages of fruit-pod development.This information will enhance our understanding of the mechanisms that underlie fruit-pod development in peanut and contribute to efforts aimed at improving peanut production.

Two haplotype-resolved,gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit

Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value,especially because of the high content of vitamin C in its fruit.In this study,we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var.‘Donghong’(DH)and Actinidia latifolia‘Kuoye’(KY),with total lengths of 608327852 and 640561626 bp for 29 chromosomes,respectively.With a burst of structural variants involving inversion,translocations,and duplications within 8.39 million years,the metabolite content of DH and KY exhibited differences in saccharides,lignans,and vitamins.A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha,both of which have ultra-high vitamin C content.With each assembly phased into two complete haplotypes,we identified allelic variations between two sets of haplotypes,leading to protein sequence variations in 26494 and 27773 gene loci and allele-specific expression of 4687 and 12238 homozygous gene pairs.Synchronized metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents;free fatty acids and flavonols accumulated in the early stages,but sugar substances and amino acids accumulated in the late stages.The AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in fruit.Compared with wild varieties and other Actinidia species,AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content,providing a possible explanation on why red-flesh kiwifruits are sweeter.Collectively,these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.

Conserved H3K27me3-associated chromatin looping mediates physical interactions of gene clusters in plants

Higher-order chromatin organization is essential for transcriptional regulation,genome stability maintenance,and other genome functions.Increasing evidence has revealed significant differences in 3D chromatin organization between plants and animals.However,the extent,pattern,and rules of chromatin organization in plants are still unclear.In this study,we systematically identified and characterized long-range chromatin loops in the Arabidopsis 3D genome.We identified hundreds of long-range cis chromatin loops and found their anchor regions are closely associated with H3K27me3 epigenetic modifications.Furthermore,we demonstrated that these chromatin loops are dependent on Polycomb group(PcG)proteins,suggesting that the Polycomb repressive complex2(PRC2)complex is essential for establishing and maintaining these novel loops.Although most of these PcG-medicated chromatin loops are stable,many of these loops are tissue-specific or dynamically regulated by different treatments.Interestingly,tandemly arrayed gene clusters and metabolic gene clusters are enriched in anchor regions.Long-range H3K27me3-marked chromatin interactions are associated with the coregulation of specific gene clusters.Finally,we also identified H3K27me3-associated chromatin loops associated with gene clusters in Oryza sativa and Glycine max,indicating that these long-range chromatin loops are conserved in plants.Our results provide novel insights into genome evolution and transcriptional coregulation in plants.

A High-Density SNP Genotyping Array for Rice Biology and Molecular Breeding

A high-density single nucleotide polymorphism （SNP） array is critically important for geneticists and molecu- lar breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the devel- opment of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for dif- ferent objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies （GWAS） analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding

Watermelon,Citrullus lanatus,is the world's third largest fruit crop.Reference genomes with gaps and a narrow genetic base hinder functional genomics and genetic improvement of watermelon.Here,we report the assembly of a telomere-to-telomere gap-free genome of the elite watermelon inbred line G42 by incorporating high-coverage and accurate long-read sequencing data with multiple assembly strategies.All 11 chromosomes have been assembled into single-contig pseudomolecules without gaps,representing the highest completeness and assembly quality to date.The G42 reference genome is 369321829 bp in length and contains 24205 predicted protein-coding genes,with all 22 telomeres and 11 centromeres characterized.Furthermore,we established a pollen-EMS mutagenesis protocol and obtained over 200000M1 seeds from G42.In a sampling pool,48 monogenic phenotypic mutations,selected from 223M1and 78 M2 mutants with morphological changes,were confirmed.The average mutation density was 1 SNP/1.69Mband1 indel/4.55 Mb per M1 plant and 1SNP/1.08Mb and 1 indel/6.25 Mb per M2 plant.Taking advantage of the gap-free G42 genome,8039 mutations from 32 plants sampled from M1 and M2 families were identified with 100%accuracy,whereas only 25% of the randomly selected mutations identified using the 97103v2 reference genome could be confirmed.Using this library and the gap-free genome,two genes responsible for elongated fruit shape and male sterility(CiMs1)were identified,both caused by a single basechange from G to A.The validated gap-free genome and its EMS mutation library provide invaluable resources for functional genomics and genetic improvement of watermelon.

Origin and Evolution of Core Components Responsible for Monitoring Light Environment Changes during Plant Terrestrialization

Light serves as the source of energy as well as an information signal for photosynthetic plants. During evolution, plants have acquired the ability to monitor environmental light radiation and adjust their developmental patterns to optimally utilize light energy for photosynthesis. The mechanisms of light perception and signal transduction have been comprehensively studied in past decades, mostly in a few model plants, including Arabidopsis thaliana. However, systematic analyses of the origin and evolution of core components involved in light perception and signaling are still lacking. In this study, we took advantage of the recently sequenced genomes and transcriptomes covering all the main Archaeplastida clades in the public domain to identify orthologous genes of core components involved in light perception and signaling and to reconstruct their evolutionary history. Our analyses suggested that acclimation to different distribution of light quality in new environments led to the origination of specific light signaling pathways in plants. The UVR8 (UV Resistance Locus 8) signaling pathway originated during the movement of plants from the deeper sea to shallow water and enabled plants to deal with ultraviolet B light (UV-B). After acquisition of UV-B adaptation, origination of the phytochrome signaling pathway helped plants to colonize water surface where red light became the prominent light energy source. The seedling emergence pathway, which is mediated by a combination of light and phytohormone signals that orchestrate plant growth pattern transitions, originated before the emergence of seed plants. Although cryptochromes and some key components of E3 ubiquitin ligase systems already existed before the divergence of the plant and animal kingdoms, the coevolution and optimization of light perception and downstream signal transduction components, including key transcription factors and E3 ubiquitin ligase systems, are evident during plant terrestrialization.

Gibberellin Signal Transduction in Rice

In the past decade, significant knowledge has accumulated regarding gibberellin （GA） signal transductlon In rice as a result of studies using multiple approaches, particularly molecular genetics. The present review highlights the recent developments In the identification of GA signaling pathway components, the discovery of GA-Induced destructlon of GA signaling repressor （DELLA protein）, and the possible mechanism underlying the regulation of GA- responsive gene expression in rice.

Genomic basis for light control of plant development

Light is one of the key environmental signals regulating plant growth and development.Therefore,understanding the mechanisms by which light controls plant development has long been of great interest to plant biologists.Traditional genetic and molecular approaches have successfully identified key regulatory factors in light signaling,but recent genomic studies have revealed massive reprogramming of plant transcriptomes by light,identified binding sites across the entire genome of several pivotal transcription factors in light signaling,and discovered the involvement of epigenetic regulation in light-regulated gene expression.This review summarizes the key genomic work conducted in the last decade which provides new insights into light control of plant development.

Development of the “Third-Generation” Hybrid Rice in China

Rice is a major cereal crop for China. The development of the ‘‘three-line" hybrid rice system based on cytoplasmic male sterility in the 1970 s(first-generation) and the ‘‘two-line" hybrid rice system based on photoperiod-and thermo-sensitive genic male-sterile lines(second-generation)in the 1980 s has contributed significantly to rice yield increase and food security in China. Here we describe the development and implementation of the ‘‘third-generation" hybrid rice breeding system that is based on a transgenic approach to propagate and utilize stable recessive nuclear male sterile lines, and as such, the male sterile line and hybrid rice produced using such a system is nontransgenic. Such a system should overcome the intrinsic problems of the ‘‘first-generation" and‘‘second-generation" hybrid rice systems and hold great promise to further boost production of hybrid rice and other crops.

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol)Ⅲpromoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p)and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.