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    Towards a better recording of microtubule cytoskeletal spatial organization and dynamics in plant cells
    Weiwei Liu, Chaofeng Wang, Guangda Wang, Yinping Ma, Juan Tian, Yanjun Yu, Li Dong and Zhaosheng Kong
    J Integr Plant Biol 2019, 61 (4): 388-393.  
    doi: 10.1111/jipb.12721
    Abstract (Browse 301)  |   Save
    Numerous fluorescent marker lines are currently available to visualize microtubule (MT) architecture and dynamics in living plant cells, such as markers expressing p35S::GFP-MBD or p35S::GFP-TUB6. However, these MT marker lines display obvious defects that affect plant growth or produce unstable fluorescent signals. Here, a series of new marker lines were developed, including the pTUB6::VisGreen-TUB6-expressing line in which TUB6 is under the control of its endogenous regulatory elements and eGFP is replaced with VisGreen, a brighter fluorescent protein. Moreover, two different markers were combined into one expression vector and developed two dual-marker lines. These marker lines produce bright, stable fluorescent signals in various tissues, and greatly shorten the screening process for generating dual-marker lines. These new marker lines provide a novel resource for MT research.
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    Application and future perspective of CRISPR/Cas9 genome editing in fruit crops
    Junhui Zhou, Dongdong Li, Guoming Wang, Fuxi Wang, Merixia Kunjal, Dirk Joldersma and Zhongchi Liu
    J Integr Plant Biol 2020, 62 (3): 269-286.  
    doi: 10.1111/jipb.12793
    Abstract (Browse 419)  |   Save

    Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9‐base‐editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene‐free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer‐friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.

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    Re-evaluation of physical interaction between plant peroxisomes and other organelles using live-cell imaging techniques
    Kazusato Oikawa, Makoto Hayashi, Yasuko Hayashi and Mikio Nishimura
    J Integr Plant Biol 2019, 61 (7): 836-852.  
    doi: 10.1111/jipb.12805
    Abstract (Browse 235)  |   Save
    environmental conditions. Plant organelles, with various functions, migrate along actin filaments and contact other types of organelles, leading to physical interactions at a specific site called the membrane contact site. Recent studies have revealed the importance of physical interactions in maintaining efficient metabolite flow between organelles. In this review, we first summarize peroxisome function under different environmental conditions and growth stages to understand organelle interactions. We then discuss current knowledge regarding the interactions between peroxisome and other organelles, i.e., the oil bodies, chloroplast, and mitochondria from the perspective of metabolic and physiological regulation, with reference to various organelle interactions and techniques for estimating organelle interactions occurring in plant cells.
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    Split Nano luciferase complementation for probing protein-protein interactions in plant cells
    Feng-Zhu Wang, Nannan Zhang, Yan-Jun Guo, Ben-Qiang Gong and Jian-Feng Li
    J Integr Plant Biol 2020, 62 (8): 1065-1079.  
    DOI: 10.1111/jipb.12891
    Abstract (Browse 440)  |   Save

    Deciphering protein‐protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual‐use system enabling the detection of PPIs through the Nluc‐based SLC and co‐immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA‐ or FLAG‐tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium‐transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc‐based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.

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    Large‐scale identification of expression quantitative trait loci in Arabidopsis reveals novel candidate regulators of immune responses and other processes
    Xingang Wang, Min Ren, Danni Liu, Dabao Zhang, Cuijun Zhang, Zhaobo Lang, Alberto P. Macho, Min Zhang and Jian‐Kang Zhu
    J Integr Plant Biol 2020, 62 (10): 1469-1484.  
    doi: 10.1111/jipb.12930
    Abstract (Browse 377)  |   Save
    The extensive phenotypic diversity within natural populations of Arabidopsis is associated with differences in gene expression. Transcript levels can be considered as inheritable quantitative traits, and used to map expression quantitative trait loci (eQTL) in genome‐wide association studies (GWASs). In order to identify putative genetic determinants for variations in gene expression, we used publicly available genomic and transcript variation data from 665 Arabidopsis accessions and applied the single nucleotide polymorphism‐set (Sequence) Kernel Association Test (SKAT) method for the identification of eQTL. Moreover, we used the penalized orthogonal‐components regression (POCRE) method to increase the power of statistical tests. Then, gene annotations were used as test units to identify genes that are associated with natural variations in transcript accumulation, which correspond to candidate regulators, some of which may have a broad impact on gene expression. Besides increasing the chances to identify real associations, the analysis using POCRE and SKAT significantly reduced the computational cost required to analyze large datasets. As a proof of concept, we used this approach to identify eQTL that represent novel candidate regulators of immune responses. The versatility of this approach allows its application to any process that is subjected to natural variation among Arabidopsis accessions.
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    Developing a new model system for potato genetics by androgenesis
    Miru Du, Ting Wang, Qun Lian, Xiaojie Zhang, Guohui Xin, Yuanyuan Pu, Glenn J. Bryan and Jianjian Qi
    J Integr Plant Biol 2021, 63 (4): 628-633.  
    doi: 10.1111/jipb.13018
    Abstract (Browse 382)  |   Save
    High heterozygosity and tetrasomic inheritance complicate studies of asexually propagated polyploids, such as potato. Reverse genetics approaches, especially mutant library construction, can be an ideal choice if a proper mutagenesis genotype is available. Here, we aimed to generate a model system for potato research using anther cultures of Solanum verrucosum, a self‐compatible diploid potato with strong late blight resistance. Six of the 23 regenerants obtained (SVA4, SVA7, SVA22, SVA23, SVA32, and SVA33) were diploids, and their homozygosity was estimated to be >99.99% with 22 polymorphic InDel makers. Two lines—SVA4 and SVA32—had reduced stature (plant height ≤80 cm), high seed yield (>1,000 seeds/plant), and good tuber set (>30 tubers/plant). We further confirmed the full homozygosity of SVA4 and SVA32 using whole‐genome resequencing. These two regenerants possess all the characteristics of a model plant: diploidy, 100% homozygosity, self‐compatibility, and amenability to transgenesis. Thus, we have successfully generated two lines, SVA4 and SVA32, which can potentially be used for mutagenesis and as model plants to rejuvenate current methods of conducting potato research.
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    Multiplex and optimization of dCas9‐TV‐mediated gene activation in plants
    Xiangyu Xiong, Jieping Liang, Zhenxiang Li, Ben‐Qiang Gong and Jian‐Feng Li
    J Integr Plant Biol 2021, 63 (4): 634-645.  
    doi: 10.1111/jipb.13023
    Abstract (Browse 403)  |   Save
    Synthetic gene activators consisting of nuclease‐dead Cas9 (dCas9) for single‐guide RNA (sgRNA)‐directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9‐TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9‐TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA‐sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000‐fold in rice. Moreover, the yeast GCN4 coiled coil‐mediated dCas9‐TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self‐amplification loop for dCas9‐TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9‐TV‐refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9‐TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9‐TADs in plants.
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    Comparative transcriptomics reveals hidden issues in the plant response to arthropod herbivores
    M. Estrella Santamaria, Alejandro Garcia, Ana Arnaiz, Irene Rosa‐Diaz, Gara Romero‐Hernandez, IsabelDiaz and Manuel Martinez
    J Integr Plant Biol 2021, 63 (2): 312-326.  
    doi: 10.1111/jipb.13026
    Abstract (Browse 311)  |   Save
    Plants experience different abiotic/biotic stresses, which trigger their molecular machinery to cope with them. Besides general mechanisms prompted by many stresses, specific mechanisms have been introduced to optimize the response to individual threats. However, these key mechanisms are difficult to identify. Here, we introduce an in‐depth species‐specific transcriptomic analysis and conduct an extensive meta‐analysis of the responses to related species to gain more knowledge about plant responses. The spider mite Tetranychus urticae was used as the individual species, several arthropod herbivores as the related species for meta‐analysis, and Arabidopsis thaliana plants as the common host. The analysis of the transcriptomic data showed typical common responses to herbivory, such as jasmonate signaling or glucosinolate biosynthesis. Also, a specific set of genes likely involved in the particularities of the Arabidopsis‐spider mite interaction was discovered. The new findings have determined a prominent role in this interaction of the jasmonate‐induced pathways leading to the biosynthesis of anthocyanins and tocopherols. Therefore, tandem individual/general transcriptomic profiling has been revealed as an effective method to identify novel relevant processes and specificities in the plant response to environmental stresses.
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    Three‐dimensional reconstruction and comparison of vacuolar membranes in response to viral infection
    Xueting Wang, Juncai Ma, Xuejiao Jin, Ning Yue, Peng Gao, Keith Ka Ki Mai, Xian‐Bing Wang, Dawei Li, Byung‐Ho Kang and Yongliang Zhang
    J Integr Plant Biol 2021, 63 (2): 353-364.  
    doi: 10.1111/jipb.13027
    Abstract (Browse 289)  |   Save
    The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three‐dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV‐AC), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck‐like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV‐AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.
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    Benefiting others and self: Production of vitamins in plants
    Yufei Li, Chenkun Yang, Hasan Ahmad, Mohamed Maher, Chuanying Fang and Jie Luo
    J Integr Plant Biol 2021, 63 (1): 210-227.  
    doi: 10.1111/jipb.13047
    Abstract (Browse 272)  |   Save
    Vitamins maintain growth and development in humans, animals, and plants. Because plants serve as essential producers of vitamins, increasing the vitamin contents in plants has become a goal of crop breeding worldwide. Here, we begin with a summary of the functions of vitamins. We then review the achievements to date in elucidating the molecular mechanisms underlying how vitamins are synthesized, transported, and regulated in plants. We also stress the exploration of variation in vitamins by the use of forward genetic approaches, such as quantitative trait locus mapping and genome‐wide association studies. Overall, we conclude that exploring the diversity of vitamins could provide new insights into plant metabolism and crop breeding.
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    The plant cell wall: Biosynthesis, construction, and functions
    Baocai Zhang, Yihong Gao, Lanjun Zhang and Yihua Zhou
    J Integr Plant Biol 2021, 63 (1): 251-272.  
    doi: 10.1111/jipb.13055
    Abstract (Browse 483)  |   Save
    The plant cell wall is composed of multiple biopolymers, representing one of the most complex structural networks in nature. Hundreds of genes are involved in building such a natural masterpiece. However, the plant cell wall is the least understood cellular structure in plants. Due to great progress in plant functional genomics, many achievements have been made in uncovering cell wall biosynthesis, assembly, and architecture, as well as cell wall regulation and signaling. Such information has significantly advanced our understanding of the roles of the cell wall in many biological and physiological processes and has enhanced our utilization of cell wall materials. The use of cutting‐edge technologies such as single‐molecule imaging, nuclear magnetic resonance spectroscopy, and atomic force microscopy has provided much insight into the plant cell wall as an intricate nanoscale network, opening up unprecedented possibilities for cell wall research. In this review, we summarize the major advances made in understanding the cell wall in this era of functional genomics, including the latest findings on the biosynthesis, construction, and functions of the cell wall.
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    The CBP/p300 histone acetyltransferases function as plant‐specific MEDIATOR subunits in Arabidopsis
    Jing Guo, Long Wei, Shan‐Shan Chen, Xue‐Wei Cai, Yin‐Na Su, Lin Li, She Chen and Xin‐Jian He
    J Integr Plant Biol 2021, 63 (4): 755-771.  
    DOI: 10.1111/jipb.13052
    Abstract (Browse 288)  |   Save
    In eukaryotes, MEDIATOR is a conserved multi‐subunit complex that links transcription factors and RNA polymerase II and that thereby facilitates transcriptional initiation. Although the composition of MEDIATOR has been well studied in yeast and mammals, relatively little is known about the composition of MEDIATOR in plants. By affinity purification followed by mass spectrometry, we identified 28 conserved MEDIATOR subunits in Arabidopsis thaliana, including putative MEDIATOR subunits that were not previously validated. Our results indicated that MED34, MED35, MED36, and MED37 are not Arabidopsis MEDIATOR subunits, as previously proposed. Our results also revealed that two homologous CBP/p300 histone acetyltransferases, HAC1 and HAC5 (HAC1/5) are in fact plant‐specific MEDIATOR subunits. The MEDIATOR subunits MED8 and MED25 (MED8/25) are partially responsible for the association of MEDIATOR with HAC1/5, MED8/25 and HAC1/5 co‐regulate gene expression and thereby affect flowering time and floral development. Our in vitro observations indicated that MED8 and HAC1 form liquid‐like droplets by phase separation, and our in vivo observations indicated that these droplets co‐localize in the nuclear bodies at a subset of nuclei. The formation of liquid‐like droplets is required for MED8 to interact with RNA polymerase II. In summary, we have identified all of the components of Arabidopsis MEDIATOR and revealed the mechanism underlying the link of histone acetylation and transcriptional regulation.
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    Genome-wide profiling of circular RNAs, alternative splicing, and R-loops in stem-differentiating xylem of Populus trichocarpa
    Xuqing Liu, Yubang Gao, Jiakai Liao, Miao Miao, Kai Chen, Feihu Xi, Wentao Wei, Huihui Wang, Yongsheng Wang, Xi Xu, Anireddy S.N. Reddy and Lianfeng Gu
    J Integr Plant Biol 2021, 63 (7): 1294-1308.  
    DOI: 10.1111/jipb.13081
    Abstract (Browse 278)  |   Save
    Circular RNAs (circRNAs) are a recently discovered type of non-coding RNA derived from pre-mRNAs. R-loops consist of a DNA:RNA hybrid and the associated single-stranded DNA. In Arabidopsis thaliana, circRNA:DNA R-loops regulate alternative splicing (AS) of SEPALLATA3 (SEP3). However, the occurrence and functions of circRNAs and R-loops in Populus trichocarpa are largely unexplored. Here, we performed circRNA-enriched sequencing in the stem-differentiating xylem (SDX) of P. trichocarpa and identified 2,742 distinct circRNAs, including circ-CESA4, circ-IRX7, and circ-GUX1, which are generated from genes involved in cellulose, and hemicellulose biosynthesis, respectively. To investigate the roles of circRNAs in modulating alternative splicing (AS), we detected 7,836 AS events using PacBio Iso-Seq and identified 634 circRNAs that overlapped with 699 AS events. Furthermore, using DNA:RNA hybrid immunoprecipitation followed by sequencing (DRIP-seq), we identified 8,932 R-loop peaks that overlapped with 181 circRNAs and 672 AS events. Notably, several SDX-related circRNAs overlapped with R-loop peaks, pointing to their possible roles in modulating AS in SDX. Indeed, overexpressing circ-IRX7 increased the levels of R-loop structures and decreased the frequency of intron retention in linear IRX7 transcripts. This study provides a valuable R-loop atlas resource and uncovers the interplay between circRNAs and AS in SDX of P. trichocarpa.
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    MP3RNA-seq: Massively parallel 3′ end RNA sequencing for high-throughput gene expression profiling and genotyping
    Jian Chen, Xiangbo Zhang, Fei Yi, Xiang Gao, Weibin Song, Haiming Zhao and Jinsheng Lai
    J Integr Plant Biol 2021, 63 (7): 1227-1239.  
    doi: 10.1111/jipb.13077
    Abstract (Browse 290)  |   Save
    Transcriptome deep sequencing (RNA-seq) has become a routine method for global gene expression profiling. However, its application to large-scale experiments remains limited by cost and labor constraints. Here we describe a massively parallel 3′ end RNA-seq (MP3RNA-seq) method that introduces unique sample barcodes during reverse transcription to permit sample pooling immediately following this initial step. MP3RNA-seq allows for handling of hundreds of samples in a single experiment, at a cost of about $6 per sample for library construction and sequencing. MP3RNA-seq is effective for not only high-throughput gene expression profiling, but also genotyping. To demonstrate its utility, we applied MP3RNA-seq to 477 double haploid lines of maize. We identified 19,429 genes expressed in at least 50% of the lines and 35,836 high-quality single nucleotide polymorphisms for genotyping analysis. Armed with these data, we performed expression and agronomic trait quantitative trait locus (QTL) mapping and identified 25,797 expression QTLs for 15,335 genes and 21 QTLs for plant height, ear height, and relative ear height. We conclude that MP3RNA-seq is highly reproducible, accurate, and sensitive for high-throughput gene expression profiling and genotyping, and should be generally applicable to most eukaryotic species.
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    Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize
    Yuanyuan Yan, Jinjie Zhu, Xiantao Qi, Beijiu Cheng, Changlin Liu and Chuanxiao Xie
    J Integr Plant Biol 2021, 63 (9): 1671-1680.  
    doi: 10.1111/jipb.13086
    Abstract (Browse 415)  |   Save
    Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.
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    Exploring the diversity of plant proteome
    Yanmei Chen, Yi Wang, Jun Yang, Wenbin Zhou and Shaojun Dai
    J Integr Plant Biol 2021, 63 (7): 1197-1210.  
    DOI: 10.1111/jipb.13087
    Abstract (Browse 410)  |   Save
    The tremendous functional, spatial, and temporal diversity of the plant proteome is regulated by multiple factors that continuously modify protein abundance, modifications, interactions, localization, and activity to meet the dynamic needs of plants. Dissecting the proteome complexity and its underlying genetic variation is attracting increasing research attention. Mass spectrometry (MS)-based proteomics has become a powerful approach in the global study of protein functions and their relationships on a systems level. Here, we review recent breakthroughs and strategies adopted to unravel the diversity of the proteome, with a specific focus on the methods used to analyze posttranslational modifications (PTMs), protein localization, and the organization of proteins into functional modules. We also consider PTM crosstalk and multiple PTMs temporally regulating the life cycle of proteins. Finally, we discuss recent quantitative studies using MS to measure protein turnover rates and examine future directions in the study of the plant proteome.
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    Efficient generation of homozygous substitutions in rice in one generation utilizing an rABE8e base editor
    Chuang Wei, Chong Wang, Meng Jia, Hong‐Xuan Guo, Peng‐Yu Luo, Mu‐Gui Wang, Jian‐Kang Zhu and Hui Zhang
    J Integr Plant Biol 2021, 63 (9): 1595-1599.  
    doi: 10.1111/jipb.13089
    Abstract (Browse 450)  |   Save
    A new deaminase, TadA8e, was recently evolved in the laboratory. TadA8e catalyzes DNA deamination over 1,000 times faster than ABE7.10. We developed a high-efficiency adenine base editor, rABE8e (rice ABE8e), combining monomeric TadA8e, bis-bpNLS and codon optimization. rABE8e had substantially increased editing efficiencies at NG-protospacer adjacent motif (PAM) and NGG-PAM target sequences compared with ABEmax. For most targets, rABE8e exhibited nearly 100% editing efficiency and high homozygous substitution rates in the specific editing window, especially at Positions A5 and A6. The ability to rapidly generate plant materials with homozygous base substitutions will benefit gene function research and precision molecular breeding.
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    Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses
    Liang Zhang, Tao Wang, Guangyang Wang, Aoyue Bi, Misganaw Wassie, Yan Xie, Liwen Cao, Huawei Xu, Jinmin Fu, Liang Chen, Yang Zhao and Tao Hu
    J Integr Plant Biol 2021, 63 (8): 1410-1415.  
    doi: 10.1111/jipb.13101
    Abstract (Browse 267)  |   Save
    Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.
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    Natural variation in linalool metabolites: One genetic locus, many functions?
    Jun He, Rayko Halitschke, Ian T. Baldwin and Meredith C. Schuman
    J Integr Plant Biol 2021, 63 (8): 1416-1421.  
    doi: 10.1111/jipb.13104
    Abstract (Browse 193)  |   Save
    The ubiquitous volatile linalool is metabolized in plants to nonvolatile derivatives. We studied Nicotiana attenuata plants which naturally vary in (S)-(+)-linalool contents, and lines engineered to produce either (R)-(-)- or (S)-(+)-linalool. Only (S)-(+)-linalool production was associated with slower growth of a generalist herbivore, and a large fraction was present as nonvolatile derivatives. We found that variation in volatile linalool and its nonvolatile glycosides mapped to the same genetic locus which harbored the biosynthetic gene, NaLIS, but that free linalool varied more in environmental responses. This study reveals how (S)-(+)-linalool and conjugates differ in their regulation and possible functions in resistance.
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    LjaFGD: Lonicera japonica functional genomics database
    Qiaoqiao Xiao, Zhongqiu Li, Mengmeng Qu, Wenying Xu, Zhen Su and Jiaotong Yang
    J Integr Plant Biol 2021, 63 (8): 1422-1436.  
    DOI: 10.1111/jipb.13112
    Abstract (Browse 230)  |   Save
    Lonicera japonica Thunb., a traditional Chinese herb, has been used for treating human diseases for thousands of years. Recently, the genome of L. japonica has been decoded, providing valuable information for research into gene function. However, no comprehensive database for gene functional analysis and mining is available for L. japonica. We therefore constructed LjaFGD (www.gzybioinformatics.cn/LjaFGD and bioinformatics.cau.edu.cn/LjaFGD), a database for analyzing and comparing gene function in L. japonica. We constructed a gene co-expression network based on 77 RNA-seq samples, and then annotated genes of L. japonica by alignment against protein sequences from public databases. We also introduced several tools for gene functional analysis, including Blast, motif analysis, gene set enrichment analysis, heatmap analysis, and JBrowse. Our co-expression network revealed that MYB and WRKY transcription factor family genes were co-expressed with genes encoding key enzymes in the biosynthesis of chlorogenic acid and luteolin in L. japonica. We used flavonol synthase 1 (LjFLS1) as an example to show the reliability and applicability of our database. LjaFGD and its various associated tools will provide researchers with an accessible platform for retrieving functional information on L. japonica genes to further biological discovery.
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    Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display
    Shi‐Jian Song, Gyeong‐Im Shin, Jinyong Noh, Jiho Lee, Deok‐Hwan Kim, Gyeongryul Ryu, Gyeongik Ahn, Hyungmin Jeon, Hai‐Ping Diao, Youngmin Park, Min Gab Kim, Woe‐Yeon Kim, Young‐Jin Kim, Eun‐Ju Sohn, Chang Seon Song and Inhwan Hwang
    J Integr Plant Biol 2021, 63 (8): 1505-1520.  
    DOI: 10.1111/jipb.13141
    Abstract (Browse 271)  |   Save
    Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.
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    Development of an efficient plant dual cytosine and adenine editor
    Rongfang Xu, Fanna Kong, Ruiying Qin, Juan Li, Xiaoshuang Liu and Pengcheng Wei
    J Integr Plant Biol 2021, 63 (9): 1600-1605.  
    doi: 10.1111/jipb.13146
    Abstract (Browse 357)  |   Save
    An enhanced CDA-like (eCDAL) was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editor version 1(pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.
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    Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus
    Hui Li, Xinren Dai, Xiong Huang, Mengxuan Xu, Qiao Wang, Xiaojing Yan, Ronald R. Sederoff and Quanzi Li
    J Integr Plant Biol 2021, 63 (11): 1906-1921.  
    DOI: 10.1111/jipb.13159
    Abstract (Browse 1095)  |   Save
    High-throughput single-cell RNA sequencing (scRNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba×Populus glandulosa. The scRNA-seq profiled 9,798 cells, which were grouped into 12 clusters. Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations, we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type (cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.
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