Lei Zhu

China Agricultural University, Beijing, Beijing Shi, China

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Publications (12)79.83 Total impact

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    ABSTRACT: For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca2+-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.
    The Plant Cell 03/2013; · 9.25 Impact Factor
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    ABSTRACT: BACKGROUND: Ordered cortical microtubule (MT) arrays play a critical role in the spatial control of cell division and expansion and are essential for plant growth, morphogenesis, and development. Various developmental, hormonal, and mechanical signals and a large number of MT-associated proteins are known to impact cortical MT organization, but the underlying mechanisms remain poorly understood. Our previous studies show that auxin signaling, which is mediated by the ROP6 Rho GTPase and its effector RIC1, promotes the ordering of cortical MTs in pavement cells, but it is unknown how RIC1 controls the organization of cortical MTs into well-ordered arrays. RESULTS: Our genetic screens identified the conserved MT-severing protein katanin (KTN1) as a downstream component of the ROP6-RIC1 signaling pathway leading to well-ordered arrangement of cortical MTs. KTN1 and RIC1 proteins displayed overlapping localization. In vivo and in vitro studies showed that RIC1 physically interacts with and promotes the MT-severing activity of KTN1. Live-cell imaging reveals a role for RIC1 in promoting detachment of branched MTs that is known to rely on KTN1. CONCLUSION: We have demonstrated that a Rho GTPase signaling pathway regulates katanin-mediated MT severing in plant cells and uncovered an explicit regulatory mechanism underpinning the alignment and ordering of cortical MTs in plants. Our findings provide new insights into regulatory mechanisms underlying growth stimuli such as auxin promote the organization of cortical MTs into parallel arrays in plants.
    Current biology: CB 02/2013; · 10.99 Impact Factor
  • Lei Zhu, Ying Fu
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    ABSTRACT: Proteins generally interact with some other proteins to achieve their cellular functions. Fluorescence resonance energy transfer (FRET) microscopy provides a powerful technique to elucidate such interactions in vivo. FRET occurs when two properly chosen fluorophores are sufficiently close (less than 10 nm). Aided by multiple colored fluorescent proteins (FPs), FRET microscopy has been widely used in live cells for detection of protein-protein interaction and in some cases protein activity in a real-time in vivo manner, which contributes to the understanding of the mechanisms for the regulation of many cellular activities, such as signal transduction pathways. Here, we describe a convenient and fast FRET imaging microscopy involving transiently expressed proteins fused with an FRET pair of fluorescent proteins (e.g., cyn fluorescent protein and yellow fluorescent protein). We describe an example of the FRET-based assay used to analyze ROP GTPase activity in live plant cells.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 876:145-52. · 1.29 Impact Factor
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    ABSTRACT: The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation.
    The Plant Cell 12/2011; 23(12):4411-27. · 9.25 Impact Factor
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    ABSTRACT: Plasmodesmata (PD) are the communication channels which allow the trafficking of macromolecules between neighboring cells. Such cell-to-cell movement of macromolecules is regulated during plant growth and development; however, little is known about the regulation mechanism of PD size exclusion limit (SEL). Plant viral movement proteins (MPs) enhance the invasion of viruses from cell to cell by increasing the SEL of the PD and are therefore a powerful means for the study of the plasmodesmal regulation mechanisms. In a recent study, we reported that the actin cytoskeleton is involved in the increase of the PD SEL induced by MPs. Microinjection experiments demonstrated that actin depolymerization was required for the Cucumber mosaic virus (CMV) MP-induced increase in the PD SEL. In vitro experiments showed that CMV MP severs actin filaments (F-actin). Furthermore, through the analyses of two CMV MP mutants, we demonstrated that the F-actin severing ability of CMV MP was required to increase the PD SEL. These results are similar to what has been found in Tobacco mosaic virus MP. Thus, our data suggests that actin dynamics may participate in the regulations of the PD SEL.
    Plant signaling & behavior 12/2010; 5(12):1663-5.
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    ABSTRACT: Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.
    The Plant Cell 08/2010; 22(8):2710-26. · 9.25 Impact Factor
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    ABSTRACT: Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)-stabilizing agent phalloidin but not by treatment with the F-actin-destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins.
    The Plant Cell 04/2010; 22(4):1373-87. · 9.25 Impact Factor
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    ABSTRACT: Tubulin-folding cofactors play important roles in regulating plant development. Arabidopsis tubulin-folding cofactor B (AtTFC B) is an Arabidopsis homolog of mammalian tubulin-folding cofactor B, whose biological function in plant development remains poorly understood. Here we report that the homozygous attfc b (-/-) allele caused embryonic lethality. Embryogenesis was arrested at early embryo stage and the cells contained one or multiple nuclei. Plants carrying a heterozygous attfc b (+/-) allele exhibited enlarged mesophyll cells and leaf epidermal cells with bulged nuclei. Flow cytometry analysis showed increased ploidy in the leaves of the attfc b (+/-) mutant, as well as increased levels of Cdc2A and CycB1;1. In addition, immunofluorescence assay showed increased numbers of spindles and phragmoplasts in the attfc b (+/-) mutant. These results suggest that AtTFC B plays an important role in plant cell division.
    Frontiers in bioscience (Elite edition) 01/2010; 2:752-63.
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    ABSTRACT: Cortical microtubules (MTs) participate in the spatial control of cell expansion and division that is required for plant growth and morphogenesis. Well-ordered transverse cortical MTs promote cell elongation and restrict radial cell expansion. The molecular mechanism controlling their ordering is poorly understood. We report the first known signaling pathway that promotes the organization of cortical MTs into parallel arrays oriented perpendicular to the axis of cell elongation in plants. Well-ordered MTs locally restrict cell expansion to promote indentation formation in the jigsaw-puzzle-shaped pavement cells of Arabidopsis leaves. Deleting ROP6, a Rho-family GTPase, randomized cortical MTs and released the localized restriction of cell expansion, whereas ROP6 overexpression enhanced MT ordering, turning the jigsaw-puzzle appearance of cells into a cylindrical shape. ROP6 directly binds and activates MT-associated RIC1 to achieve the MT ordering. The ROP6-RIC1 pathway also affects MT ordering of hypocotyl cells, showing a broad role for this pathway in the spatial regulation of cell expansion.
    Current biology: CB 10/2009; 19(21):1827-32. · 10.99 Impact Factor
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    ABSTRACT: We reported previously that the protein SB401 from Solanum berthaultii binds to and bundles both microtubules and F-actin. In the current study, we investigated the regulation of SB401 activity by its phosphorylation. Our experimental results showed that the phosphorylation of SB401 by casein kinase II (CKII) downregulates the activities of SB401, namely the bundling of microtubules and enhancement of the polymerization of tubulin. However, phosphorylation of SB401 had no observable effect on its bundling of F-actin. Further investigation using extract of potato pollen indicated that a CKII-like kinase may exist in potato pollen. Antibodies against CKII alpha recognized specifically a major band from the pollen extract and the pollen extract was able to phosphorylate the SB401 protein in vitro. The CKII-like kinase showed a similar ability to downregulate the bundling of microtubules. Our experiments demonstrated that phosphorylation plays an important role in the regulation of SB401 activity. We propose that this phosphorylation may regulate the effects of SB401 on microtubules and the actin cytoskeleton.
    Journal of Integrative Plant Biology 04/2009; 51(3):235-42. · 3.75 Impact Factor
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    ABSTRACT: Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.
    The Plant Cell 04/2007; 19(3):877-89. · 9.25 Impact Factor
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    ABSTRACT: Complete cellulose synthesis is required to form functional cell walls and to facilitate proper cell expansion during plant growth. AtCESA2 is a member of the cellulose synthase A family in Arabidopsis (Arabidopsis thaliana) that participates in cell wall formation. By analysis of transgenic seedlings, we demonstrated that AtCESA2 was expressed in all organs, except root hairs. The atcesa2 mutant was devoid of AtCESA2 expression, leading to the stunted growth of hypocotyls in seedlings and greatly reduced seed production in mature plants. These observations were attributed to alterations in cell size as a result of reduced cellulose synthesis in the mutant. The orientation of microtubules was also altered in the atcesa2 mutant, which was clearly observed in hypocotyls and petioles. Complementary expression of AtCESA2 in atcesa2 could rescue the mutant phenotypes. Together, we conclude that disruption of cellulose synthesis results in altered orientation of microtubules and eventually leads to abnormal plant growth. We also demonstrated that the zinc finger-like domain of AtCESA2 could homodimerize, possibly contributing to rosette assemblies of cellulose synthase A within plasma membranes.
    Plant physiology 02/2007; 143(1):213-24. · 6.56 Impact Factor

Publication Stats

246 Citations
79.83 Total Impact Points

Institutions

  • 2007–2013
    • China Agricultural University
      • China State Key Laboratory of Plant Physiology and Biochemistry
      Beijing, Beijing Shi, China