An Arabidopsis single-nucleus atlas decodes leaf senescence and nutrient allocation

Cell, Год журнала: 2025, Номер 188(11), С. 2856 - 2871.e16

Опубликована: Апрель 11, 2025

Язык: Английский

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A positive feedback loop of cytokinin signaling ensures efficient de novo shoot regeneration in Arabidopsis

New Phytologist, Год журнала: 2025, Номер unknown

Опубликована: Янв. 29, 2025

Plants possess a remarkable ability to regenerate tissues, which enables the healing of wounds and induction de novo organogenesis. In vitro plant tissue culture techniques are based on regenerative capacity plants facilitate reprogramming differentiated somatic cells into new organ or even an entire (Sugimoto et al., 2010). Differentiated tissues used as explants generate pluripotent cell mass, called callus, auxin-rich callus-inducing medium (CIM) (Ikeuchi 2013; Zhai & Xu, 2021; Yin 2024). Subsequently, callus undergoes shoot regeneration cytokinin-rich shoot-inducing (SIM) (Che 2007). A particular emphasis has been placed organogenesis because low rate frequently limits in many species (Ijaz 2012; Zimik Arumugam, 2017). Consistent with fact that during involves conversion from meristem (Meng 2017; Ogura 2023), key regulators apical (SAM) establishment implicated 2016; Eshed Williams, Mathew Prasad, 2021). The PLETHORA 3 (PLT3), PLT5, PLT7 genes, expressed whole process regeneration, play role progenitor formation. Upon transferring SIM, they specifically promote promeristem formation by activating CUP-SHAPED COTYLEDON 1 (CUC1) CUC2 (Kareem 2015). CUC1 proteins involved promoting SHOOT MERISTEMLESS (STM) expression polarizing PIN-FORMED (PIN1) localization initiate development (Hibara 2003; Bilsborough 2011; Kamiuchi 2014; Kareem also activates XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 9 (XTH9) encoding wall-loosening enzyme nonprogenitor contributes establishing polarity for (Varapparambath 2022). Additionally, main cytokinin regulatory axis is linked stem callus. Type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), positive signaling, directly WUSCHEL (WUS), unequivocally regulates niche Zhang Accordingly, mutations type-B ARRs lead impaired By contrast, type-A ARRs, negative repress (Buechel APETALA2 (AP2)/ETHYLENE FACTOR (ERF)-type transcription factor gene ENHANCER OF REGENERATION (ESR1)/DORNROSCHEN (DRN) diverse aspects including wound-induced (Iwase particular, culture, ESR1 induced response promotes (Banno 2001; Iwase Ectopic substantially enhances whereas esr1 mutants display reduced calli Despite importance its modes action remain unclear. this study, we report stimulates signaling ensures efficient regeneration. ARR ultimately activate WUS. Notably, bind promoter expression, feedback loop signaling. Collectively, ESR1–type-B module acts crucial player strongly responses maximize efficiency. Arabidopsis thaliana ecotype Columbia-0 was all experiments. arr1 arr12 (CS6981), arr12-1 (CS6978), ARR1-Ypet (CS71599), ARR12-Ypet (CS71601) seeds were obtained Biological Resource Center. drn-1/esr1-2 ProESR1:ESR1-GFP gifts Dr John Chandler (Chandler 2007) Akira 2017), respectively. For generation 35S:ARR1-MYC 35S:MYC-ESR1 transgenic plants, (35S:ARR1-10×MYC) constructs cloned pGWB520 (Invitrogen) Myc-pBA plasmids, respectively, transformed via Agrobacterium-mediated transformation. grown under long-day (LD; 16 h : 8 h, light dark) conditions white fluorescent (150 μmol photons m−2 s−1) at 22–23°C. To induce formation, leaf 2-wk-old hypocotyl root 7-d-old plants. Tissue CIM (MS medium, vitamins, 3% w/v sucrose, 0.1 μg ml−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 kinetin) incubated 22°C dark indicated time period. preincubated 7 d transferred SIM 0.9 l−1 indole-3-acetic (IAA), 2.5 2-isopentenyladenine (iPA)) 25°C continuous conditions. Total RNA extracted materials using TRI reagent (TaKaRa Bio, Tokyo, Japan) according manufacturer's recommendations. First-strand complementary DNA synthetized Moloney Murine Leukemia Virus reverse transcriptase (Enzynomics, Seoul, South Korea) protocol. Reverse quantitative polymerase chain reaction (RT-qPCR) analyses performed TOPreal SYBR Green qPCR High-ROX PreMIX (Enzynomics). Primers listed Supporting Information Table S1. abundance PCR products each primer pair normalized relative EUKARYOTIC TRANSLATION INITIATION 4A1 (eIF4A) (At3g13920). comparative ΔΔCt method evaluate quantity amplified product sample. threshold cycle (Ct) automatically determined analysis software default parameters. Specificity RT-qPCR reactions melt curve products. chromatin immunoprecipitation (ChIP) assay previously reported (Lee Transgenic samples containing epitope-tagged cross-linked 1% formaldehyde. ground fine powder liquid nitrogen then sonicated. sonicated complexes precipitated Pierce Anti-c-Myc Magnetic Beads (88842; Invitrogen). Precipitated purified Purification Kit (Cosmogenetech, Korea). specific fragments precipitate quantified qPCR, values eIF4a (Table S2). Reporter effector plasmids constructed perform transient assays protoplasts. construct reporter and/or genic sequences ARR1, ARR12, ESR1, WUS separately modified pCAMBIA1305 vector minimal 35S sequence GUS coding sequence. cDNAs pBA002 plasmid CaMV promoter. Protoplast isolation previous some modifications (Jeong Ten-day-old seedlings soaked 20 ml solution 2% (w/v) viscozyme, cellulase, pectinase, 10 mM MES (pH 5.7), 0.47 M d-mannitol, CaCl2 6 room temperature. sieved through 70 μm nylon mesh (Carolina Biologicals, Burlington, NC, USA) spun 100 g min Recombinant co-transformed protoplasts polyethylene glycol (PEG)-mediated activity measured after incubation 23°C. 35S:luciferase (LUC) co-transformed, along internal control. LUC Luciferase Assay System (Promega). Protoplasts isolated transfected described above. One milliliter (1 × 107 ml−1) DNA. After incubation, harvested lysed 400 μl IP buffer (50 Tris–HCl pH 7.5, 150 NaCl, 0.1% NP-40, 1× protease inhibitor cocktail). lysate rotated 12 rpm 4°C ensure complete lysis. Cell debris removed centrifugation 000 4°C. resulting supernatant (400 μl) carefully tube immediately 30 Anti-MYC magnetic beads Invitrogen) 2 gentle rotation rpm. collected stand washed three times buffer. Immunoprecipitated eluted boiling 45 2× sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) loading min. subjected SDS–PAGE subsequently immunodetected anti-MYC (05-724; Millipore) anti-GFP antibody (ab290; Abcam, Cambridge, UK). Etiolated ProESR1:ESR1-GFP, ARR1-Ypet, fixed 4% formaldehyde cleared ClearSee (031-25151; Wako Chemicals, Osaka, Japan). walls stained Calcofluor White (4404-43-7; MP Santa Ana, CA, USA). Fluorescence signals observed confocal microscope (ECLIPSE Ti2; Nikon, GFP YFP excited wavelength 488 nm laser power 6.5, emitted detected 502/41 nm. 405 0.1, 429/74 determine molecular components associated two-step process, assembled list genes primarily respond known normal growth. Among others, particularly interested mechanism remains unclear, despite biological importance. validate results studies, examined whether responds cytokinin. Indeed, increased exogenous treatment showed negligible auxin young (Fig. 1a). agreement result, while negligibly influenced transiently upon 1b). Confocal imaging initially inner layers but later mainly outer 1c). Given layer gives rise SAM (Ogura spatial suggests estimate impact tested ESR1-deficient esr1-2 mutant 2007; Leaf SIM. number regenerated shoots counted quantify capability tissues. produced almost no shoots, wild-type (WT) approximately four five per 21 (DAS) 1d,e), consistent result Similar 1d,e). However, it should be noted did not participate proliferation, exhibited clear alterations compared WT S1). further support these results, explant-derived 35S:ESR1 ectopic albeit S2; Banno These observations suggest essential regulator culture. We next asked what regulated 4 DAS, when highly As plays cytokinin-dependent profiles, core well organogenesis, such CUC1, CUC2, KIP-RELATED PROTEIN (KRP1), STM, TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL (TCP3) (Daimon Liu Meng Yang 2020; Wu revealed couple selectively altered SIM: ARR1 downregulated regardless origin explants, other mutation (Figs 2a, S3). It notable upregulated (Matsuo 2009), unchanged 2a), may explain indirectly affects CUC expression. WOX5 gene, pluripotency acquisition, uninfluenced S4). Time-course transcript accumulation ARR12 2–4 their 2b). Consistently, WUS, target DAS 2b), indicating ARR–WUS module. binds promoters whose 2a). Chromatin immunoprecipitation-quantitative (ChIP-qPCR) S5) loci 2c,d). direct binding loci, conducted carrying ESR1-binding genomic fused co-transfected expressing 2e). co-expression significantly activity, control 2f), loci. Although WOUND INDUCED DEDIFFERENTIATION (WIND1) factor, responsible cellular seems likely SIM-induced would fully WIND1, patterns WIND1 varied considerably S6). While persisted activation occurred 1a,b), factor(s) studies co-expressed Fig. 1b), especially S7). Furthermore, stages overlapped 3a). support, emergence severely double mutants, 3b,c), raising possibility interdependent process. thus analyzed found gradually calli, completely diminished 3d). Next, cis-element presence motifs 3e; Zubo verify locus, ChIP-qPCR Direct 3f). Similarly, shown associate start site S8). supported 3g,h). Altogether, indicate regulate other, form responses. genetic relationship between crossed 35S:ARR12 investigated capacity. one can S9), comparable respective S10), supporting function same pathway addition, enhanced 4a,b), 4c). compromised background 4d–f), both required robust close linkage interact S11). addition mutual transcriptional activation, locus S12, S13) (see Discussion section). Taken together, our study demonstrates thereby accelerate progression 4g). Cytokinin perceived sensor histidine kinases, HISTIDINE KINASE2 (AHK2), AHK3, AHK4, AHKs allows autophosphorylation receiver domains (Liu 2019). phosphoryl group PHOSPHOTRANSFER PROTEINs (AHPs) translocate nucleus phosphorylation-dependent manner (To Kieber, 2008). This growth development, (Leibfried 2005; Riefler 2006; Gordon 2009). example, circuit determines growth, receptors size (Higuchi 2004; Kurakawa pivotal maintenance critical provoked emergence. factors establish exhibits defects rescued cytokinin-responsive protein loss-of-function exhibit although position network elusive. Here, demonstrate forms ARRs. reinforces responses, thus, 2023). showing strengthens stimuli-responsive signal transduction (Pandey 2018; Ohashi-Ito 2019), constituted maximizes evidenced interdependence full They complex synergistically important specifying region, where bind. Binding S13), arr had relatively lower S13). account loss overexpressing 4a,b). suspected specification dependent types. niches formed, stage incubation. Considering constitutive throughout sometimes represses (Temman type-specific controversial effect Temman appears related Wound depend trigger reentry (Dewitte Ikeuchi wound-responsive reprogramming, WIND1-induced 2011). regulation reprograming. integrative wounding have severe considering protoplast (Xu 2021), ESR1–ARR anticipated wide range Based conservation across Larriba 2022), knowledge widely applied improve efficiency various applications, hurdle crop thank (Cologne Biocenter, Germany) providing (Center Sustainable Science, seeds. work Basic Science Research (NRF-2022R1I1A1A01071792 KL NRF-2022R1A2B5B02001266 PJS) Laboratory (NRF-2022R1A4A3024451) programs National Foundation Korea, New Breeding Technologies Development Program (RS-2024-00322275) Rural Administration. None declared. PJS conceived designed study. KL, HY O-SP wrote manuscript. All authors read approved contributed equally work. data contained within article S1–S13; Tables S1, S1 Callus esr1-2. S2 35S:MYC-ESR1. S3 S4 calli. S5 Protein S6 Expression S7 S8 locus. S9 Positive ARR12. S10 De 35S:ARR12. S11 Interactions S12 S13 Requirement analysis. assays. Please note: Wiley content functionality any supplied authors. Any queries (other than missing material) directed Phytologist Central Office. publisher information content) corresponding author article. neutral regard jurisdictional claims maps institutional affiliations.

Язык: Английский

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Enhancing genetic transformation efficiency in cucurbit crops through AtGRF5 overexpression: Mechanistic insights and applications

Journal of Integrative Plant Biology, Год журнала: 2025, Номер unknown

Опубликована: Апрель 11, 2025

ABSTRACT Transgenic and gene‐editing technologies are essential for gene functional analysis crop improvement. However, the pleiotropic effects unknown mechanisms of morphogenic genes have hindered their broader application. In this study, we employed one‐step de novo shoot organogenesis (DNSO) method, demonstrated that overexpression Arabidopsis thanalia GROWTH‐REGULATING FACTOR 5 ( AtGRF5 ) significantly enhanced genetic transformation efficiency in cucurbit crops by promoting callus proliferation increasing dense cells during regeneration. High‐resolution time‐series transcriptomics single‐cell RNA sequencing revealed induced auxin‐related expanded stem cell populations cucumber DNSO. Using DNA‐affinity purification (DAP‐seq) combination with spatiotemporal differential expression analysis, identified CsIAA19 as a key downstream target AtGRF5, its modulation playing pivotal role Rescuing ‐overexpressing explant reversed To address growth defects caused overexpression, developed an abscisic acid‐inducible system, improving across diverse genotypes while minimizing effects. summary, research provides mechanistic insights into ‐mediated offers practical solution to overcome challenges modification.

Язык: Английский

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Development of an inducible DNA barcoding system to understand lineage changes in Arabidopsis regeneration

Developmental Cell, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 1, 2024

Язык: Английский

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The China national GeneBank sequence archive (CNSA) 2024 update

Horticulture Research, Год журнала: 2025, Номер 12(5)

Опубликована: Фев. 4, 2025

The China National GeneBank Sequence Archive (CNSA) is an open and freely accessible curated data repository built for archiving, sharing, reutilizing of multiomics data. remarkable advancement in sequencing technologies has triggered a paradigm shift life science research. However, it also poses tremendous challenges the research community management reusability. With dramatic advance like spatial transcriptome sequencing, brings unprecedented explosion sequence new requirements archiving. CNSA was established 2017 as one fundamental infrastructures to offer archiving worldwide community. Here, we present state-of-the-art enhancements encompassing dramatical increase varied types data, latest features services implemented well consistent efforts supporting global cooperation biodiversity preservation utilization. provides public open-sharing relevant metadata including genome, transcriptome, metabolism, proteome from single-cell (also resolved) level individual population level, further analyzed results. As 2024, archived >16.3 petabytes provided curation, preservation, open-share service >1581 publications >560 institutions. It plays pivotal role scientific projects such 10 000 Plant Genomes Project. So far, been recommended by various academic publishers Cell, Elsevier, Oxford University Press. at https://db.cngb.org/cnsa/.

Язык: Английский

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Recent progress in single-cell transcriptomic studies in plants

Plant Biotechnology Reports, Год журнала: 2025, Номер unknown

Опубликована: Апрель 3, 2025

Язык: Английский

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Single-Cell Transcriptome Reveals the Cellular Response to PEG-Induced Stress in Wheat Leaves

Journal of Agricultural and Food Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Апрель 27, 2025

Drought is a major factor limiting the production and yield of wheat bread (Triticum aestivum). Therefore, investigating drought-related response mechanism an urgent priority. Here, single-cell transcriptome drought-nonsusceptible susceptible seedlings subjected to PEG-induced stress was systematically analyzed study at cellular level. We identified five cell types using known marker genes constructed leaf atlas. On this foundation, several potential specific for each type were identified, which provide reference further annotation. Moreover, we heterogeneity in mechanisms regulatory networks among types. Specifically, drought mesophyll cells correlated with photosynthetic pathway. Pseudotime trajectory analysis revealed transition epidermal from their normal function defense under stress. also characterized associated response. Notably, two transcription factors (TraesCS1D02G223600 TraesCS1D02G072900) as master regulators most Our provides detailed insights into The gene resources obtained our could be applied breed better crop plants improved tolerance.

Язык: Английский

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All roads lead to dome: Multicellular dynamics during de novo meristem establishment in shoot regeneration

Current Opinion in Plant Biology, Год журнала: 2025, Номер 85, С. 102733 - 102733

Опубликована: Май 3, 2025

Язык: Английский

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Mitigating Heat Stress Response in CRISPR/Cas‐Mediated Edited Crops by Altering the Expression Pattern of Noncoding DNA

Опубликована: Май 9, 2025

Язык: Английский

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Single-cell views of fate reprogramming in de novo organogenesis

Journal of Plant Research, Год журнала: 2025, Номер unknown

Опубликована: Май 14, 2025

Язык: Английский

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