Biology subjects

Zhu, J.-K.

Publications and source records attributed to Zhu, J.-K..

15 recordsLinked to original sources

bioRxiv2026

Reassessing the contribution of the histone H3 tail to KRAB-DNMT3L-mediated epigenetic silencing

Neumann et al. introduced CHARM, a compact epigenetic silencer in which a histone H3 tail fused to DNMT3L was proposed to recruit and stimulate endogenous DNMT3A, enabling durable gene repression without a fused DNMT3A catalytic domain. Here, we evaluated the contribution of the H3 tail in independent reporter and endogenous-gene contexts. In an SNRPN reporter system, a KRAB-DNMT3L-dCas9 construct lacking the H3 tail displayed silencing kinetics comparable to CRISPRcharm Kv2, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this silencing. Similarly, after transient delivery of editor mRNAs to HEK293T cells, CRISPRcharm Kv2 did not consistently outperform the corresponding H3-tail-free construct at three endogenous loci, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this activity. These observations suggest that the engineered H3 tail does not confer a general functional advantage within the KRAB-DNMT3L-dCas9 architecture under the conditions tested.

synthetic biology↗

bioRxiv2026

Sex-dimorphic immune trajectories across the canine lifespan and early-life signatures of geroprotective interventions

Canine models are invaluable for translational geroscience, but widespread gonadectomy in companion dogs obscures natural sex-specific aging trajectories. Here, we characterize the age-associated hematologic and serum cytokine profiles of 80 intact laboratory Beagles spanning 1 to 11 years. Contrary to classical linear models of immunosenescence and inflammaging, we reveal that canine immune aging is highly dynamic and sexually dimorphic. While absolute leukocyte counts declined continuously, cytokine remodeling was sex-specific: intact males exhibited extensive age-dependent inflammatory restructuring, whereas females demonstrated restricted fluctuations. To establish baseline geroprotective signatures, we evaluated 24 young beagles following a 90-day intervention with rapamycin, canagliflozin, or dietary restriction. Rapamycin induced robust immune modulation, while canagliflozin prompted early, sex-biased weight loss and narrower cytokine responses. Together, our findings demonstrate that canine immune aging involves complex, sexually dimorphic remodeling. By utilizing an intact cohort to isolate natural dimorphic trajectories, we provide a crucial foundation for developing precision, sex-optimized geroprotectors.

physiology↗

bioRxiv2026

Rapid sensing and relaying of cellular hyperosmotic-stress signals via RAF-SnRK2 core condensates

Hyperosmolarity caused by drought, high salinity, or cold stress inhibits plant growth and crop productivity1,2. A conserved protein-kinase cascade of cytosolic B-RAFs and SnRK2s is rapidly activated upon osmotic stresses to initiate downstream adaptive responses, which represents one of the fastest known responses to osmotic stress in plants3-8. How the kinase cascade is activated by osmotic stress is unknown. Here, we show that Arabidopsis B4 subgroup RAFs have intrinsically disordered regions and directly sense both ionic and non-ionic hyperosmolarity by reversible condensation. B4-RAFs recruit and co-condense with subclass-I SnRK2s to phosphorylate and turn on SnRK2s, evading the non-condensable inhibitory A-clade PP2C phosphatases. This straightforward osmosensing and relaying module can be fully reconstituted in E. coli by co-expressing three components or in solution in a test tube using recombinant proteins. Our findings identify B-RAFs as the chief cellular osmosensors that detect low water potential by co-condensation, forming a signal hub with SnRK2s to orchestrate adaptive responses in plants, and represent an evolutionarily conserved osmosensing mechanism across kingdoms.

plant biology↗

bioRxiv2025

Identification of a putative RBOHD-FERONIA-CRK10-PIP2;6 plasma membrane complex that interacts with phyB to regulate ROS production in Arabidopsis thaliana

O_LIReactive oxygen species (ROS) regulate plant growth, development, and responses to the environment. ROS production by the RESPIRATORY BURST OXIDASE PROTEIN D (RBOHD) protein was recently shown to be regulated by PHYTOCHROME B (phyB), and phyB was found to be phosphorylated by FERONIA, highlighting the possibility that these three proteins interact to regulate ROS levels during stress. C_LIO_LIImmunoprecipitation and proximity labelling, followed by split-luciferase and functional validation assays, were used to study the interactions between FERONIA, phyB, and RBOHD during excess light (EL) stress in Arabidopsis thaliana. C_LIO_LIWe reveal that phyB and FERONIA interact with RBOHD, that phosphorylation of phyB by FERONIA, as well as the kinase activity of FERONIA, are required for RBOHD-driven ROS production in response to EL stress, and that CYSTEINE-RICH RECEPTOR LIKE KINASE 10 (CRK10) and PLASMA MEMBRANE INTRINSIC PROTEIN 2;6 (PIP2;6) interact with RBOHD and phyB and are also required for EL-driven RBOHD ROS production. C_LIO_LIOur findings uncover the existence of a putative plasma membrane complex between FERONIA, RBOHD, CRK10, and PIP2;6 that interacts with phyB to regulate ROS production in Arabidopsis in response to stress. This complex could play a canonical role in the integration and regulation of multiple signaling pathways in plants. C_LI Plain Language SummaryWe identified a complex between several different proteins at the plasma membrane that interacts with the light and temperature receptor protein phytochrome B to regulate reactive oxygen species formation during stress in plants. This complex could be involved in the regulation and integration of multiple abiotic and biotic signals in plants. tenten

plant biology↗

bioRxiv2023

Syntaxin of plants 32 regulates pollen wall development and pollen tube cell wall integrity via controlling secretory pathway

Pollen tubes (PTs) elongate in a polar way to deliver sperm cells to the ovule. Pollen wall development and PT cell wall integrity (CWI) maintenance are critical for PT growth and double fertilization. Pollen wall development mainly relies on secretion of exine precursors in tapetum. RALF4/19-ANX/BUPS-MRI and RALF4/19-LRX-AUN are two distinct signaling pathways but converge to fine-tune CWI during PT growth. Here, we discovered that atsyp32+/-, AtSYP32 RNAi and AtSYP3132 RNAi lines were male sterile. The tapetum development in these lines were disturbed, and the pollen wall structure was impaired resulting in pollen grain and tube bursting and less PTs navigated to micropyles. Strikingly, there were numerous ectopic secretory vesicles retained in pollen cytoplasm, and the abundance or distribution of polysaccharides and AGPs altered significantly in PTs of the mutants and RNAi lines. AtSYP32 interacted with the vesicle transport regulators SEC31B, SEC22 and BET12, the PT CWI regulators RALF19 and LRX11, and the XyG xylosyltransferase XXT5, in the Golgi apparatus. Transcription of some genes related to pollen wall biosynthesis and PT CWI maintenance were seriously affected by AtSYP32 downregulation. Our findings illustrate that AtSYP32 plays essential roles in pollen wall development and PT CWI maintenance via controlling secretory pathway. IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSPollen wall is the most complex cell wall. Pollen wall development mainly relies on secretion of precursors of exine and pollen coat in tapetal cells. Pollen tubes (PTs) grow in a polar way to deliver sperm cells to the ovule. Maintenance of PT cell wall integrity (CWI) is critical for PT elongation and double fertilization. RALF4/19 ligands interact with BUPS-ANX receptors, signaling it in an autocrine manner to maintain CWI during PT elongation. RALF4/19-LRX-AUN pathway is distinct with RALF4/19-ANX/BUPS-MRI pathway but they converge to fine-tune CWI during PT growth. Biosynthesis of PT cell wall involves multiple subcellular compartments and vesicle transport pathways. Golgi apparatus acts as a hub in vesicle trafficking. Golgi-syntaxin AtSYP31 and AtSYP32 regulate pollen development by controlling intra-Golgi transport and Golgi morphology QuestionWhat is AtSYP32 role in pollen wall and tapetum development? Who are the AtSYP32 partners that regulate secretion of cell wall biosynthesis materials? FindingsWe found that no homozygote progeny was obtained from self-pollinated atsyp32+/- alleles due to pollen sterile. The tapetum development and degeneration in atsyp32+/- mutants was severely delayed, and the pollen wall and PT wall structure were impaired. Strikingly, there were numerous ectopic secretory vesicles retained in pollen cytoplasm in atsyp32+/- mutants, and the abundance or distribution of PT wall polysaccharides and AGPs altered obviously. AtSYP32 interacted with the vesicle transport regulators SEC31B, SEC22 and BET12, the PT CWI regulators RALF19 and LRX11, and XyG xylosyltransferase XXT5, in the Golgi. All these highlight that AtSYP32 regulates pollen wall development and maintenance of PT CWI via controlling secretory pathway. Next stepsThe biological significances and the molecular mechanisms of AtSYP32 interacting with XXT5, RALF19 and LRX11 are elusive but thought-provoking. We are going to clarify the mechanisms.

molecular biology↗

bioRxiv2022

Perturbation of mitochondrial Ca2+ homeostasis activates cross-compartmental proteostatic response in Arabidopsis

Mitochondrial Ca2+ (mtCa2+) homeostasis is essential to mitochondrial functions. However, how mtCa2+ homeostasis is achieved and the consequences of impaired mtCa2+ homeostasis in plants is poorly understood. Here, we demonstrate a critical role for mitochondrial Ca2+ uniporter (MCU) in the control of mtCa2+ uptake for mtCa2+ homeostasis in planta by characterizing MCU mutants and overexpressed plants. Impaired MCU-controlled mtCa2+ homeostasis (iMUCH) in gain-of-function and loss-of-function MCU plants causes the misregulation of mitochondrial gene expression that triggers mitonuclear protein imbalance. Transcriptome integrated with proteomics analysis reveal activation of multiple compartmental UPR gene expression and decrease of cytosolic translation with selective repression of ribosome and RNA modification protein synthesis upon iMUCH. Intriguingly, TOR signalling is not involved in cytosolic translational response to iMUCH, but the reduction of eIF phosphorylation is evident under iMUCH induced mitochondrial stress. Thus, our study unveils the essential functions of MCU proteins for mtCa2+ homeostasis, and the involvement of MCU-controlled mtCa2+ homeostasis in mitochondrial stress dependent regulation of protein synthesis for cellular proteostasis that is connected to plant growth and stress resistance.

plant biology↗

bioRxiv2022

Root twisting drives halotropism via stress-induced microtubule reorientation

Plants have evolved signaling mechanisms that guide growth away from adverse environments that can cause yield losses. Root halotropism is a sodium-specific negative tropism that is crucial for surviving and thriving under high salinity. Although root halotropism was discovered some years ago, the underlying molecular and cellular mechanisms remain unknown. Here, we show that abscisic acid (ABA)-mediated root twisting determines halotropism in Arabidopsis. An ABA-activated SnRK2 protein kinase (SnRK2.6) phosphorylates the microtubule-associated protein SP2L at Ser406, which induces a change in the anisotropic cell expansion at the root transition zone that located between the apical meristem and basal elongation zone, and is required for root twisting during halotropsim. Salt stress triggers SP2L-mediated cortical microtubule reorientation in cells at the transition zone, which guides cellulose microfibril patterns. Our findings outline the cellular and molecular mechanisms of root halotropism and indicate that anisotropic cell expansion through MT-reorientation and microfibril deposition have a central role in mediating tropic responses.

plant biology↗

bioRxiv2021

A H3K9me2-Binding Protein AGDP3 Limits DNA Methylation and Transcriptional Gene Silencing in Arabidopsis

DNA methylation is critical for tuning gene expression to prevent potentially deleterious gene-silencing. The Arabidopsis DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic loci. However, the mechanism recruiting ROS1 to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor required for ROS1-mediated DNA demethylation, and targets ROS1 to specific loci. We found that AGDP3 could bind to the H3K9me2 mark by its AGD12 cassette. The crystal structure of the AGDP3 AGD12 in complex with an H3K9me2 peptide reveals the molecular basis for the specific recognition, that the dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. Interestingly, a histidine residue located in the methylysine binding aromatic cage enables AGDP3 pH-dependent H3K9me2 binding capacity. Considering the intracellular pH correlates with the histone acetylation status, our results provide the molecular mechanism for the regulation of ROS1 DNA demethylase by the gene silencing H3K9me2 mark and the potential crosstalk with active histone acetylation mark.

plant biology↗

bioRxiv2020

Coupling of H3K27me3 recognition with transcriptional repression through the BAH-PHD-CPL2 complex in Arabidopsis

Histone 3 Lys 27 trimethylation (H3K27me3)-mediated epigenetic silencing plays a critical role in multiple biological processes. However, the H3K27me3 recognition and transcriptional repression mechanisms are only partially understood. Here, we report a new mechanism for H3K27me3 recognition and transcriptional repression. Our structural and biochemical data showed that the BAH domain protein AIPP3 and the PHD proteins AIPP2 and PAIPP2 cooperate to read H3K27me3 and unmodified H3K4 histone marks, respectively, in Arabidopsis. The BAH-PHD bivalent histone reader complex silences a substantial subset of H3K27me3-enriched loci, including a number of development and stress response-related genes such as the RNA silencing effector gene ARGONAUTE 5 (AGO5) and We found that the BAH-PHD module associates with CPL2, a plant-specific Pol II carboxyl terminal domain (CTD) phosphatase, to form the BAH-PHD-CPL2 complex (BPC) for transcriptional repression. The BPC complex represses transcription through CPL2-mediated CTD dephosphorylation, thereby causing inhibition of Pol II release from the transcriptional start site. Our work reveals a mechanism coupling H3K27me3 recognition with transcriptional repression through the alteration of Pol II phosphorylation states, thereby contributing to our understanding of the mechanism of H3K27me3-dependent silencing.

plant biology↗

bioRxiv2020

Latent defense response to non-pathogenic microbial factors impairs plant-rhizobacteria mutualism

Unlike pathogens that trigger plant defense responses, commensal or beneficial microbes are compatible with plants and do not elicit a defense response. An assumption underlying the compatibility is that plants are inert in mounting a defense response to non-pathogenic microbial factors. However, the mechanisms underlying this inertness in defense are unknown. Here a forward genetic screen led to the isolation of an Arabidopsis mutant displaying a new type of immunity which we named as latent defense response (LDR) to a beneficial rhizobacterium. The mutant, known as gp1 for Growth-Promotion 1, is impaired in rhizobacteria-induced plant growth-promotion due to disrupted oleic acid homeostasis and consequent activation of defense responses. Several bacterial volatile compounds trigger LDR in gp1 but not wild type plants. GP1 dysfunction strongly represses colonization of the beneficial rhizobacterium and alters root-associated microbiota. Our findings reveal a hidden layer of plant defense, LDR, which is suppressed by GP1 to allow mutualistic association between plants and beneficial rhizobacteria. One Sentence SummaryA hidden layer of host immunity against non-pathogenic microbes leads to plant incompatibility with beneficial rhizobacteria.

plant biology↗

bioRxiv2020

The SUMO E3 Ligase SIZ1 Positively Regulates Active DNA Demethylation by Stabilizing ROS1 in Arabidopsis thaliana

The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis. Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Using a methylation-sensitive PCR (CHOP-PCR)-based forward genetic screen for Arabidopsis DNA hypermethylation mutants, we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation. Dysfunction of SIZ1 leads to hyper-methylation at approximately one thousand genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared to that in wild type plants, the protein level of ROS1 is significantly decreased, even though there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1 positively regulates active DNA demethylation by promoting the stability of ROS1 protein through SUMOylation. Short SummaryThe 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) is indispensable for proper DNA methylation landscape in Arabidopsis. Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Here, we show that SIZ1-mediated SUMOylation of ROS1 enhances its stability and positively regulates active DNA demethylation.

plant biology↗

bioRxiv2020

A novel protein complex that regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo.Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

plant biology↗

bioRxiv2019

A virus-encoded protein suppresses methylation of the viral genome through its interaction with AGO4 in the Cajal body

In plants, establishment of de novo DNA methylation is regulated by the RNA-directed DNA methylation (RdDM) pathway. RdDM machinery is known to concentrate in the Cajal body, but the biological significance of this localization has remained elusive. Here, we show that the anti-viral methylation of the Tomato yellow leaf curl virus (TYLCV) genome occurs specifically in the Cajal body of Nicotiana benthamiana cells. Methylation of the viral genome is countered by a virus-encoded protein, V2, which interacts with the central RdDM component AGO4, interfering with its binding to the viral DNA; Cajal body localization of the V2-AGO4 interaction is required for the viral protein to exert this function. Taken together, our results draw a long sought-after functional connection between RdDM, the Cajal body, and anti-viral DNA methylation, paving the way for a deeper understanding of DNA methylation and anti-viral defences in plants.

plant biology↗

bioRxiv2019

Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS

Cell-fate in eukaryotes is regulated by MAP Kinases (MAPKs) that translate external cues to cellular responses. In plants, two MAPKs, MPK3/6, regulate diverse processes of development, environmental response, and immunity. Yet, the mechanism bridging these shared signaling components with a specific target remains unresolved. Focusing on the development of stomata, epidermal valves for gas exchange and transpiration, we report here that the bHLH protein SCREAM functions as a scaffold by recruiting MPK3/6 to downregulate SPEECHLESS, a transcription factor initiating stomatal cell lineages. SCREAM directly binds with MPK3/6 through an evolutionarily-conserved yet unconventional bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 at the 2.75[A] resolution unraveled bipartite binding of SCREAM with MPK6, that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism directly linking extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell-fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.

plant biology↗

bioRxiv2017

A plant receptor-like kinase promotes cell-to-cell spread of RNAi and is targeted by a virus

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an anti-viral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus has the ability to inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homologue, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signalling components have been co-opted to play multiple functions in plants.

plant biology↗