Jian-Kang Zhu

Shanghai Institutes for Biological Sciences, Shanghai, Shanghai Shi, China

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Publications (84)664.07 Total impact

  • Pengcheng Wang · Jian-Kang Zhu ·
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    ABSTRACT: The in-gel protein kinase assay is a powerful method to measure the protein phosphorylation activity of specific protein kinases. Any protein substrate can be embedded in polyacrylamide gels where they can be phosphorylated by protein kinases that are separated in the gel under denaturing conditions and then renatured. The kinase activity can be visualized in situ in the gels by autoradiography. This method has been used to compare the activities of protein kinases in parallel samples or to identify their potential substrates. Here, we describe in detail an in-gel kinase assay to measure the activity of some protein kinases in plants.
    Methods in molecular biology (Clifton, N.J.) 11/2015; 1363:189-197. DOI:10.1007/978-1-4939-3115-6_15 · 1.29 Impact Factor
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    ABSTRACT: The C-REPEAT-BINDING FACTOR (CBF) pathway has important roles in plant responses to cold stress. How the CBF genes themselves are activated after cold acclimation remains poorly understood. In this study, we characterized cold tolerance of null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4), which encodes a protein that associates with RNA polymerases Pol V and Pol II, and is required for RNA-directed DNA methylation (RdDM) in Arabidopsis. The results showed that dysfunction of RDM4 reduced cold tolerance, as evidenced by decreased survival and increased electrolyte leakage. Mutation of RDM4 resulted in extensive transcriptomic reprogramming. CBFs and CBF regulon genes were down-regulated in rdm4 but not nrpe1 (the largest subunit of PolV) mutants, suggesting that the role of RDM4 in cold stress responses is independent of the RdDM pathway. Overexpression of RDM4 constitutively increased the expression of CBFs and regulon genes and decreased cold-induced membrane injury. A great proportion of genes affected by rdm4 overlapped with those affected by CBFs. Chromatin immunoprecipitation results suggested that RDM4 is important for Pol II occupancy at the promoters of CBF2 and CBF3. We present evidence of a considerable role for RDM4 in regulating gene expression at low temperature, including the CBF pathway in Arabidopsis.
    New Phytologist 11/2015; DOI:10.1111/nph.13727 · 7.67 Impact Factor
  • Chizuko Yamamuro · Jian-Kang Zhu · Zhenbiao Yang ·
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    ABSTRACT: The action of phytohormones in plants requires the spatiotemporal regulation of their accumulation and responses at various levels. Recent studies reveal an emerging relationship between the function of phytohormones and epigenetic modifications. In particular, evidence suggests that auxin biosynthesis, transport, and signal transduction is modulated by microRNAs and epigenetic factors such as histone modification, chromatin remodeling, and DNA methylation. Furthermore, some phytohormones have been shown to affect epigenetic modifications. These findings are shedding light on the mode of action of phytohormones and opening up a new avenue of research on phytohormones as well as revealing mechanisms regulating the epigenetic modifications.
    Molecular Plant 11/2015; DOI:10.1016/j.molp.2015.10.008 · 6.34 Impact Factor
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    ABSTRACT: The phytohormone abscisic acid (ABA) is important for growth, development and stress responses in plants. Recent research has identified ABA receptors and signalling components that regulate seed germination and stomatal closure. However, proteins that regulate ABA signalling remain poorly understood. Here we use a forward-genetic screen to identify rbm25-1 and rbm25-2, two Arabidopsis mutants with increased sensitivity to growth inhibition by ABA. Using RNA-seq, we found that RBM25 controls the splicing of many pre-mRNAs. The protein phosphatase 2C HAB1, a critical component in ABA signalling, shows a dramatic defect in pre-mRNA splicing in rbm25 mutants. Ectopic expression of a HAB1 complementary DNA derived from wild-type mRNAs partially suppresses the rbm25-2 mutant phenotype. We suggest that RNA splicing is of particular importance for plant response to ABA and that the splicing factor RBM25 has a critical role in this response.
    Nature Communications 09/2015; 6:8139. DOI:10.1038/ncomms9139 · 11.47 Impact Factor
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    ABSTRACT: The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.
    Plant Biotechnology Journal 09/2015; DOI:10.1111/pbi.12468 · 5.75 Impact Factor
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    ABSTRACT: The newly developed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations and corrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA (sgRNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sgRNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications. © 2015, Science China Press and Springer-Verlag Berlin Heidelberg.
    07/2015; 60(15). DOI:10.1007/s11434-015-0848-2
  • Chunzhao Zhao · Zhaobo Lang · Jian-Kang Zhu ·
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    ABSTRACT: CBF transcription factors, which play important roles in cold acclimation, regulate the expression of approximately 170 cold-responsive genes, termed the CBF regulon. Recent work by Park et al. showed that CBF regulon genes and other cold-responsive genes are regulated by a complex network that involves many early cold-induced transcription factors. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Trends in Plant Science 06/2015; 20(8). DOI:10.1016/j.tplants.2015.06.001 · 12.93 Impact Factor
  • Pengcheng Wang · Jian-Kang Zhu · Zhaobo Lang ·
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    ABSTRACT: Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.
    Plant signaling & behavior 05/2015; 10(6). DOI:10.1080/15592324.2015.1031939
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    ABSTRACT: Paramutation is an epigenetic phenomenon that has been observed in a number of multicellular organisms. The epigenetically silenced state of paramutated alleles is not only meiotically stable but also "infectious" to active homologous alleles. The molecular mechanism of paramutation remains unclear, but components involved in RNA-directed DNA methylation (RdDM) are required. Here, we report a multi-copy pRD29A-LUC transgene in Arabidopsis thaliana that behaves like a paramutation locus. The silent state of LUC is induced by mutations in the DNA glycosylase gene ROS1. The silent alleles of LUC are not only meiotically stable but also able to transform active LUC alleles into silent ones, in the absence of ros1 mutations. Maintaining silencing at the LUC gene requires action of multiple pathways besides RdDM. Our study identified specific factors that are involved in the paramutation-like phenomenon and established a model system for the study of paramutation in Arabidopsis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 05/2015; 107(8). DOI:10.1016/j.celrep.2015.04.034 · 8.36 Impact Factor
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    ABSTRACT: "Drying without dying" is an essential trait in land plant evolution. Unraveling how a unique group of angiosperms, the Resurrection Plants, survive desiccation of their leaves and roots has been hampered by the lack of a foundational genome perspective. Here we report the ∼1,691-Mb sequenced genome of Boea hygrometrica, an important resurrection plant model. The sequence revealed evidence for two historical genome-wide duplication events, a compliment of 49,374 protein-coding genes, 29.15% of which are unique (orphan) to Boea and 20% of which (9,888) significantly respond to desiccation at the transcript level. Expansion of early light-inducible protein (ELIP) and 5S rRNA genes highlights the importance of the protection of the photosynthetic apparatus during drying and the rapid resumption of protein synthesis in the resurrection capability of Boea. Transcriptome analysis reveals extensive alternative splicing of transcripts and a focus on cellular protection strategies. The lack of desiccation tolerance-specific genome organizational features suggests the resurrection phenotype evolved mainly by an alteration in the control of dehydration response genes.
    Proceedings of the National Academy of Sciences 05/2015; 112(18):201505811. DOI:10.1073/pnas.1505811112 · 9.67 Impact Factor
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    ABSTRACT: CRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALEN-induced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and ~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
    Plant Biotechnology Journal 04/2015; DOI:10.1111/pbi.12372 · 5.75 Impact Factor
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    Ying Zhang · Pengcheng Wang · Wanchen Shao · Jian-Kang Zhu · Juan Dong ·
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    ABSTRACT: Cell polarization is linked to fate determination during asymmetric division of plant stem cells, but the underlying molecular mechanisms remain unknown. In Arabidopsis, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) is polarized to control stomatal asymmetric division. A mitogen-activated protein kinase (MAPK) cascade determines terminal stomatal fate by promoting the degradation of the lineage determinant SPEECHLESS (SPCH). Here, we demonstrate that a positive-feedback loop between BASL and the MAPK pathway constitutes a polarity module at the cortex. Cortical localization of BASL requires phosphorylation mediated by MPK3/6. Phosphorylated BASL functions as a scaffold and recruits the MAPKKK YODA and MPK3/6 to spatially concentrate signaling at the cortex. Activated MPK3/6 reinforces the feedback loop by phosphorylating BASL and inhibits stomatal fate by phosphorylating SPCH. Polarization of the BASL-MAPK signaling feedback module represents a mechanism connecting cell polarity to fate differentiation during asymmetric stem cell division in plants. Copyright © 2015 Elsevier Inc. All rights reserved.
    Developmental Cell 04/2015; DOI:10.1016/j.devcel.2015.02.022 · 9.71 Impact Factor
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    ABSTRACT: During the transition from water to land, plants had to cope with the loss of water through transpiration, the inevitable result of photosynthetic CO2 fixation on land [1, 2]. Control of transpiration became possible through the development of a new cell type: guard cells, which form stomata. In vascular plants, stomatal regulation is mediated by the stress hormone ABA, which triggers the opening of the SnR kinase OST1-activated anion channel SLAC1 [3, 4]. To understand the evolution of this regulatory circuit, we cloned both ABA-signaling elements, SLAC1 and OST1, from a charophyte alga, a liverwort, and a moss, and functionally analyzed the channel-kinase interactions. We were able to show that the emergence of stomata in the last common ancestor of mosses and vascular plants coincided with the origin of SLAC1-type channels capable of using the ancient ABA drought signaling kinase OST1 for regulation of stomatal closure.
    Current Biology 03/2015; · 9.57 Impact Factor
  • Xiangqiang Zhan · Jian-Kang Zhu · Zhaobo Lang ·
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    ABSTRACT: Cold temperatures trigger the ICE1-CBF-COR transcriptional cascade in plants, which reprograms gene expression to increase freezing tolerance. In this issue of Developmental Cell, Ding et al. (2015) report that cold stress activates the protein kinase OST1 to phosphorylate and thereby stabilize and stimulate ICE1. This enhances plant tolerance to freezing temperatures. Copyright © 2015 Elsevier Inc. All rights reserved.
    Developmental Cell 02/2015; 32(3):257-8. DOI:10.1016/j.devcel.2015.01.004 · 9.71 Impact Factor
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    ABSTRACT: Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for regulation of active DNA demethylation is not well understood. By using an Arabidopsis line expressing the ProRD29A:LUC and Pro35S:NPTII transgenes, we isolated an mbd7 (Methyl-CpG-binding domain protein 7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 physically interacts with ROS5/IDM2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The gypsy type LTR retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis. Copyright © 2015, American Society of Plant Biologists.
    Plant physiology 01/2015; 167(3). DOI:10.1104/pp.114.252106 · 6.84 Impact Factor
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    Jian-Kang Zhu · Yang Zhao · Zhulong Chan ·
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    ABSTRACT: Embodiments of the present invention relate generally to drought tolerant transgenic plants and methods of creating the drought tolerant transgenic plants. In one embodiment, plants are transformed such that a PYL polypeptide is overexpressed in the plant. In an embodiment, the plant is transformed with a cassette or vector that comprises a polynucleotide encoding for one or more of the PYL polypeptides, which may be under the control of an inducible or constitutive promoter. In embodiments, overexpression of PYL13 results in plants having increased drought tolerance, such as a decreased transpiration rate, a decreased stomatal conductance, an increased photosynthetic rate, accelerated stress responsive gene expression, and increased water use efficiency, and/or an increased survival rate compared to a control plant. In some embodiments, other PYL proteins having the single point mutations identified in PYL13 are expressed in the plant.
    Ref. No: US 2015-0074844, Year: 01/2015
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    ABSTRACT: The phytohormone abscisic acid (ABA) plays important roles in plant development and adaptation to environmental stress. ABA induces the production of nitric oxide (NO) in guard cells, but how NO regulates ABA signaling is not understood. Here, we show that NO negatively regulates ABA signaling in guard cells by inhibiting open stomata 1 (OST1)/sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6) through S-nitrosylation. We found that SnRK2.6 is S-nitrosylated at cysteine 137, a residue adjacent to the kinase catalytic site. Dysfunction in the S-nitrosoglutathione (GSNO) reductase (GSNOR) gene in the gsnor1-3 mutant causes NO overaccumulation in guard cells, constitutive S-nitrosylation of SnRK2.6, and impairment of ABA-induced stomatal closure. Intro-duction of the Cys137 to Ser mutated SnRK2.6 into the gsnor1-3/ ost1-3 double-mutant partially suppressed the effect of gsnor1-3 on ABA-induced stomatal closure. A cysteine residue correspond-ing to Cys137 of SnRK2.6 is present in several yeast and human protein kinases and can be S-nitrosylated, suggesting that the S-nitrosylation may be an evolutionarily conserved mechanism for protein kinase regulation. NO | ABA | drought | GSNOR | stomata
    Proceedings of the National Academy of Sciences 12/2014; 112(2). DOI:10.1073/pnas.1423481112 · 9.67 Impact Factor
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    ABSTRACT: Due to its ability to be rapidly generated and propagated over long distances, H2O2 is an important second messenger for biotic and abiotic stress signaling in plants. In response to low water potential and high salt concentrations sensed in the roots of plants, the stress hormone abscisic acid (ABA) activates NADPH oxidase to generate H2O2, which is propagated in guard cells in leaves to induce stomatal closure and prevent water loss from transpiration. Using a reconstituted system, we demonstrate that H2O2 reversibly prevents the protein phosphatase HAB1, a key component of the core ABA-signaling pathway, from inhibiting its main target in guard cells, SnRK2.6/OST1 kinase. We have identified HAB1 C186 and C274 as H2O2-sensitive thiols and demonstrate that their oxidation inhibits both HAB1 catalytic activity and its ability to physically associate with SnRK2.6 by formation of intermolecular dimers.
    PLoS ONE 12/2014; 9(12):e113643. DOI:10.1371/journal.pone.0113643 · 3.23 Impact Factor
  • Ting Ban · Jian-Kang Zhu · Karsten Melcher · H Eric Xu ·
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    ABSTRACT: RNA-binding proteins play crucial roles in RNA processing and function as regulators of gene expression. Recent studies have defined the structural basis for RNA recognition by diverse RNA-binding motifs. While many RNA-binding proteins recognize RNA sequence non-specifically by associating with 5' or 3' RNA ends, sequence-specific recognition by RNA-binding proteins is typically achieved by combining multiple modular domains to form complex binding surfaces. In this review, we present examples of structures from different classes of RNA-binding proteins, identify the mechanisms utilized by them to target specific RNAs, and describe structural principles of how protein-protein interactions affect RNA recognition specificity. We also highlight the structural mechanism of sequence-dependent and -independent interactions in the Cas9-RNA-DNA complex.
    Cellular and Molecular Life Sciences CMLS 11/2014; 72(6). DOI:10.1007/s00018-014-1779-9 · 5.81 Impact Factor

Publication Stats

2k Citations
664.07 Total Impact Points


  • 2012-2015
    • Shanghai Institutes for Biological Sciences
      Shanghai, Shanghai Shi, China
  • 2011-2015
    • Purdue University
      • Department of Horticulture and Landscape Architecture
      ウェストラファイエット, Indiana, United States
  • 2013
    • Tsinghua University
      • School of Life Sciences
      Peping, Beijing, China
  • 2009-2013
    • University of California, Riverside
      • • Institute for Integrative Genome Biology
      • • Department of Botany and Plant Sciences
      Riverside, California, United States
  • 2007
    • Maria Curie-Sklodowska University in Lublin
      Lyublin, Lublin Voivodeship, Poland