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The Subcellular Localization and Blue-Light- Induced Movement of Phototropin 1-GFP in Etiolated Seedlings of Arabidopsis thalianaw

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Molecular Plant 2008年第1期1卷 103-117页

作者： Ying-Lang Wan William Eisinger David Ehrhardt Ulrich Kubitscheck frantisek baluska Winslow Briggs Dept Plant Cell Biology Institute of Cellular and Molecular Botany University of Bonn Kirschallee 1 D-53115 Bonn Germany Dept Biology Santa Clara University Santa Clara CA 95053 Dept Plant Biology Carnegie Institution of Washington Stanford CA 94305 Institute for Physical and Theoretical Chemistry Wegelerstr. 12 D-53115 Bonn Germany

Phototropin 1 （photl） is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOTI：：GFP （Sakamoto and Briggs, 2002）, we investigated the pattern of cellular and subcellular localization of photl in 3-4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOTT：GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOTI：：GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescenco a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOTT：GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced photl relocalization modulates blue-light-activated signal transduction.

关键词： Phototropin 1 (phot1) PHOT1::GFP Arabidopsis thalianaw

Dynamics and roles of phragmoplast microfilaments in cell plate formation during cytokinesis of tobacco BY-2 cells

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Chinese Science Bulletin 2009年第12期54卷 2051-2061页

作者： ZHANG Yan ZHANG WenJie baluska frantisek MENZEL Diedrik REN HaiYuni Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education Beijing Normal University Beijing 100875 China Institute of Cellular and Molecular Botany University of Bonn D-53115 Bonn Germany

The phragmoplast is a special apparatus that functions in establishing a cell plate in dividing plant cells. It is known that microfilaments (MFs) are involved in constituting phragmoplast structure, but the dynamic distribution and role of phragmoplast MFs are far from being understood. In this study, the precise structure and dynamics of MFs during the initiation and the late lateral expansion of the phragmoplast were observed by using a tobacco BY-2 cell line stably expressing the microfilament reporter construct GFP-fABD2. Three-dimensional imaging showed that the phragmoplast MFs were initiated by two populations of MFs emerging between the reconstituting daughter nuclei at anaphase, which migrated to the mid-zone and gave rise to two layers of microfilament arrays. FM4-64 stained vesicles accumulated and fused with the cell plate between the two populations of MFs. The two layers of microfilament arrays of phragmoplast with ends overlapped always surrounded the centrifugally expanding cell plate. Partial disruption of MFs at metaphase by low concentration of latrunculin B resulted in the inhibition of the cell plate consolidation and the blockage of cell plate lateral expansion, whereas high concentration of latrunculin B restrained the progression of the cell cycle. Treating the cell after the initiation of phragmoplast led to the cease of the expansion of the cell plate. Our observations provide new insights into the precise structure and dynamics of phragmoplast MFs during cytokinesis and suggest that dynamic phragmoplast MFs are important in cell plate formation.

关键词：植物细胞板形微丝烟草胞质成膜结构绿色荧光蛋白 MFs

Aluminium toxicity targets PIN2 in Arabidopsis root apices: Effects on PIN2 endocytosis, vesicular recycling, and polar auxin transport

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Chinese Science Bulletin 2008年第16期53卷 2480-2487页

作者： SHEN Hong HOU NingYan Markus SCHLICHT WAN YingLang Stefano MANCUSO frantisek baluska College of Resources and Environment South China Agricultural University Guangzhou 510642 China Institute of Cellular and Molecular Botany Bonn University Bonn D-53115 Germany Electrophysiology Laboratory Department of Horticulture University of Florence Viale delle Idee 30 1-50019 Florence Italy

The most obvious symptom of Al toxicity is the inhibition of root growth. However, the mechanism of Al-inhibiting root growth remains to be elucidated. In this study, auxin transport and vesicle movement of an auxin-efflux carrier (PIN2) were investigated in Arabidopsis roots in response to Al stress. Results indicated that Al inhibited the apical transport of auxin in root tips of Arabidopsis significantly. The severe inhibition was localized in the cells of transition zone, where the concentration of auxin was only 34% that of the control. Brefeldin A (BFA), an inhibitor of vesicle transport, induced the dot-like structure of PIN2 vesicle significantly. Al decreased the size of dot-like structure of PIN2 vesicles. Re- sults of real-time RT-PCR and Western-blotting analysis showed that Al increased the transcript level of PIN2 and the accumulation of PIN2 protein in horizontal direction of plasma membrane, but decreased its distribution in endosomes, suggesting that Al inhibited the transport of PIN2 vesicles from plasma membrane to endosomes. Results of cytoskeleton-depolymering drugs indicated that it was via the pathway of disruption of actin microfilaments that Al inhibited the transport of PIN2 vesicles. Exposed to Al stress, the cells of elongation zone had less Al uptake and less transport frequency of vesicles than cells of transition zone. Taken together, our results suggested that Al inhibited root growth mainly by modulating the transport of PIN2 vesicles between plasma membrane and endosomes, thus block- ing auxin transport and root growth.

关键词：铝毒性胞吞作用生长素

Spatiotemporal Dynamics of the BRI1 Receptor and its Regulation by Membrane Microdomains in Living Arabidopsis Cells

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Molecular Plant 2015年第9期8卷 1334-1349页

作者： Li Wang Hong Li Xueqin Lv Tong Chen Ruili Li Yiqun xuei Jianjun Jiang Biao Jin frantisek baluska Jozef Samaj Xuelu Wang Jinxing Lin Key Laboratory of Ptant Resources Institute of Botany Chinese Academy of Sciences Beijing 100093 China College of Horticulture and Plant Protection Yangzhou University Yangzhou 225009 China College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 100083 China State Key Laboratory of Genetic Engineering and Institute of Plant Biology School of Life Sciences Fudan University Shanghai 200433 China Institute of Cellular and Molecular Botany University of Bonn Kirschallee 1 D-53115 Bonn Germany Department of Cell Biology Palacky University OIomouc Olomouc 78371 Czech Republic College of Life Science and Technology Huazhong Agricultural University Wuhan 430070 China These authors contributed equally to the article.

The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRII. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFIotl-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFIotl amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bril-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.

关键词： BRI1 BR signaling endocytosis membrane microdomains spatiotemporal dynamics

Arabidopsis Blue Light Receptor Phototropin 1 Undergoes Blue Light-Induced Activation in Membrane Microdomains

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Molecular Plant 2018年第6期11卷 846-859页

作者： Yiqun Xue Jingjing Xing Yinglang Wan Xueqin Lv Lusheng Fan Yongdeng Zhang Kai Song Li Wang Xiaohua Wang Xin Deng frantisek baluska John M. Christie Jinxing Lin Institute of Botany Chinese Academy of Sciences Beijing 100093 China University of Chinese Academy of Sciences Beijing 100049 China Department of Botany and Plant Sciences University of California Riverside CA 92521 USA Department of Cell Biology Yale University School of Medicine New Haven CT 06520 USA Institute of Cellular and Molecular Botany University of Bonn 53115 Bonn Germany Institute of Molecular Cell and Systems Biology University of Glasgow Bower Building Glasgow G12 8QQ UK College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 100083 China

Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.

关键词： photl VA-TIRFM spatiotemporal dynamics blue light signaling membrane microdomains

Arabidopsis Synaptotagmin 2 Participates in Pollen Germination and Tube Growth and Is Delivered to Plasma Membrane via Conventional Secretion

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Molecular Plant 2015年第12期8卷 1737-1750页

作者： Hui Wang Shengcheng Han Wei Siao Chunqing Song Yun Xiang Xiaorong Wu Pengyu Cheng Hongjuan Li JAn Jasik Karol Micieta JAn Turna Boris Voigtt frantisek baluska Jin Liu Yingdian Wang Heping Zhao Beijing Key Laboratory of Gene Resource and Molecular Development College of Life Science Beijing Normal University Beijing 100875 China Department of Plant Cell Biology IZMB University of Bonn Kirschallee 153115 Bonn Germany School of Life Science Lanzhou University Lanzhou 730000 China Comenius University Science Park Comenius University Bratislava Mlynska dolina 842 15 Bratislava 4 Slovakia Department of Botany Faculty of Natural Sciences Comenius University Revova 39 811 02 Bratislava 1 SIovakia Department of Botany Faculty of Natural Sciences Comenius University Revova 39 811 02 Bratislava 1 Slovakia Institute of Botany Slovak Academy of Sciences Dubravska cesta 9 SK-84523 Bratislava Slovak Republic These authors contributed equally to this article. present address: Office of Teaching Affairs Handan University Handan 056005 China

Arabidopsis synaptotagmin 2 （SYT2） has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen ofArabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmManl-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

关键词： synaptotagmins pollen germination calcium-dependent phospholipid binding C2 domains conventional secretion

Dynamic spatial reorganization of BSK1 complexes in the plasma membrane underpins signal-specific activation for growth and immunity

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Molecular Plant 2021年第4期14卷 588-603页

作者： Bodan Su Xi Zhang Li Li Sammar Abbas Meng Yu Yaning Cui frantisek baluska Inhwan Hwang Xiaoyi Shan Jinxing Lin Beijing Advanced Innovation Center for Tree Breeding by Molecular Design Beijing Forestry UniversityBeijing 100083China College of Biological Sciences&Biotechnology Beijing Forestry UniversityBeijing 100083China lnstitute of Cellular and Molecular Botany University of BonnBonn53115Germany 々Division of Integrative Biosciences and Biotechnology Pohang University of Science and TechnologyPohang37673Korea Present address:School of Life Sciences Tsinghua UniversityBeijing 100084China

Growth and immunity are opposing processes that compete for cellular resources,and proper resource allocation is crucial for plant ***1 plays a key role in the regulation of both growth and immunity by associating with BRI1 and FLS2,***,it remains unclear how two antagonistic signals co-opt BSK1 to induce signal-specific *** we show that the dynamic spatial reorganiization of BSK1 within the plasma membrane underlies the mechanism of signal-specific activation for growth or *** BSK1 localizes to membrane rafts as *** BSK1-associated FLS2 and BRI1,flg22 or exogenous brassinosteroid(BR)treatment did not decrease BSK1 levels at the plasma membrane(PM)but rather induced BSK1 multimerization and dissociation from FLS2/BSK1 or BRI1/BSK1,***,flg22-activated BSK1 translocated from membrane rafts to non-membrane-raft regions,whereas BR-activated BSK1 remained in membrane *** applied together with flg22,BR suppressed various flg22-induced BSK1 activities such as BSK1 dissociation from FLS2/BSK1,BSK1 interaction with MAPKKK5,and BSK translocation together with *** together,this study provides a unique insight into how the precise control of BSK1 spatiotemporal organization regulates the signaling specificity to balance plant growth and immunity.

关键词： BSK1 dynamic spatiotemporal organization membrane rafts growth and immunity

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铝抑制拟南芥根尖PIN2循环和囊泡运输

科学通报 2008年第15期53卷 1785-1792页

作者：沈宏侯凌艳 SCHLICHT Markus WAN Ying Lang MANCUSO Stefano baluska frantisek 华南农业大学资源环境学院广州510642 Institute of Cellular and Molecular Botany University of BonnBonn D-53115Germany Electrophysiology Laboratory Department of HorticultureUniversity of FlorenceFlorence 1-50019Italy

铝对植物毒害作用最明显的症状是迅速抑制根尖生长.然而,铝抑制根尖生长的机制并不清楚.本文研究了铝对生长素和生长素运输载体(PIN2)囊泡运输的影响.结果表明,铝抑制拟南芥根尖生长素运输,其中过渡区生长素抑制率最高,达66%.布雷菲尔... 详细信息

铝对植物毒害作用最明显的症状是迅速抑制根尖生长.然而,铝抑制根尖生长的机制并不清楚.本文研究了铝对生长素和生长素运输载体(PIN2)囊泡运输的影响.结果表明,铝抑制拟南芥根尖生长素运输,其中过渡区生长素抑制率最高,达66%.布雷菲尔德菌素(Brefeldin A,BFA,一种囊泡运输抑制剂)明显诱导PIN2囊泡在细胞内形成点状结构,铝处理降低点状结构的大小,表明铝抑制PIN2囊泡在细胞内的运输.实时定量PCR和蛋白印迹反应发现,铝增加PIN2基因的转录表达,促进PIN2蛋白在细胞膜水平方向累积.细胞骨架解聚药物处理表明,铝抑制PIN2囊泡的运输,主要通过破坏肌球蛋白微丝来完成.铝处理下,拟南芥根尖伸长区细胞比过渡区具有较少的铝吸收和较低的囊泡运输频率.上述结果表明,通过调节生长素运输载体(PIN2)在质膜与胞内移动,阻碍生长素的运输,铝抑制了拟南芥根尖的生长.

关键词：铝处理 PIN2 拟南芥细胞骨架生长素运输