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The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis

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Journal of Integrative Plant Biology 2022年第10期64卷 1916-1934页

作者： Yi Yang Fang Liu Le Liu Mingyue Zhu Jinfeng Yuan Yan‐Xia Mai Jun‐Jie Zou Jie Le Yonghong Wang Klaus Palme Xugang Li Yong Wang Long Wang State Key Laboratory of Crop Biology College of Life SciencesShandong Agricultural UniversityDaizong Street 61Tai'an 271018China Shandong Provincial Key Laboratory of Biophysics Institute of BiophysicsDezhou UniversityDezhou 253023China Sino‐German Joint Research Center on Agricultural Biology College of Life SciencesShandong Agricultural UniversityDaizong Street 61Tai'an 271018China Faculty of Biology Institute of Biology II/Molecular Plant PhysiologyAlbert‐Ludwigs‐University of FreiburgSchänzlestrasse 1Freiburg D‐79104Germany National Key Laboratory of Plant Molecular Genetics(NKLPMG) CAS Center for Excellence in Molecular Plant SciencesInstitute of Plant Physiology and Ecology(SIPPE)Chinese Academy of Sciences(CAS)Shanghai 200032China Biotechnology Research Institute Chinese Academy of Agricultural SciencesBeijing 100081China Key Laboratory of Plant Molecular Physiology CAS Center for Excellence in Molecular Plant SciencesInstitute of BotanyChinese Academy of SciencesBeijing 100093China University of Chinese Academy of Sciences Beijing 100049China

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin.This response depends on the concerted activities of the auxin transporters such as pin-FORMED(pin)proteins for auxin efflux and AUXIN RESISTANT 1(AUX1)for auxin influx.However,how the auxin gradient is established remains elusive.Here we identified a new mutant with a short root,strong auxin distribution in the lateral root cap and an impaired gravitropic response.The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor(URI).At URI interacted with prefoldin 2(PFD2)and PFD6,twoβ-type PFD members that modulate actin and tubulin patterning in roots.The auxin reporter DR5_(rev):GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips.Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter pin2 is disrupted in aturi-1 roots as well as in pfd mutants.We propose that At URI cooperates with PFDs to recycle pin2and modulate auxin distribution.

关键词： Arabidopsis auxin cytoskeleton pin2 prefoldin(PFD) unconventional prefoldin RPB5 interactor(URI)

Overexpression of the Lcpin2 and Ltpin2 Gene in Arabidopsis thaliana Promotes Root Elongation

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Phyton-International Journal of Experimental Botany 2023年第8期92卷 2383-2397页

作者： Zijian Cao Guoxia Xue Lingfeng Hu Haoxian Qu Shuang Liang Jisen Shi Jinhui Chen Zhaodong Hao Key Laboratory of Forest Genetics&Biotechnology of Ministry of Education Co-Innovation Center for Sustainable Forestry in Southern ChinaNanjing Forestry UniversityNanjing210037China

The auxin polar transporter,pin-FORMED 2(pin2)plays an important role in root development.However,it remains unclear whether pin2 genes form two Liriodendron species,L.chinense(Lcpin2)and L.tulipifera(Ltpin2),are both involved in root development and whether and to what extent these two genes diverge in function.Here,we cloned and overexpressed Lcpin2 and Ltpin2 in Arabidopsis thaliana wild-type(WT)and Atpin2 mutant.Phylogenetic and sequence analysis showed a small degree of differentiation between these two Liriodendron pin2 genes.Tissue-speciﬁc gene expression analysis indicated that both Liriodendron pin2 genes were highly expressed in roots,implying a potential role in root development.Finally,heterologous overexpression of Lcpin2 and Ltpin2 in Arabidopsis both signiﬁcantly increased the root length compared to wild-type and empty vector.Furthermore,the root length defect in Atpin2 was complemented both by Lcpin2 and Ltpin2.However,heterologous overexpression of Lcpin2 and Ltpin2 cannot rescue the defect in root gravitropism of Atpin2 mutants.Taken together,ourﬁndings unravel pin2 genes from the magnoliids plant Liriodendron were functionally conserved with Atpin2 in the dicotyledonous plant Arabidopsis in regard to the regulation of root length,but not root gravitropism.This study also provides a potential target for genetic improvement of the root system in these valuable forest trees Liriodendron.

关键词： Liriodendron pin2 auxin polar transport root development

pin FORMED 2 Modulates the Transport of Arsenite in Arabidopsis thaliana

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Plant Communications 2020年第3期1卷 42-56页

作者： Mohammad Arif Ashraf Kana Umetsu Olena Ponomarenko Michiko Saito Mohammad Aslam Olga Antipova Natalia Dolgova Cheyenne D.Kiani Susan Nehzati Keitaro Tanoi Katsuyuki Minegishi Kotaro Nagatsu Takehiro Kamiya Toru Fujiwara Christian Luschnig Karen Tanino Ingrid Pickering Graham N.George Abidur Rahman United Graduate School of Agricultural Sciences Iwate UniversityMoriokaIwateJapan Department of Plant Bio Sciences Faculty of AgricultureIwate UniversityMoriokaIwateJapan Molecular and Environmental Science Research Group Department of Geological SciencesUniversity of SaskatchewanSaskatoonSKCanada Argonne National Lab Advanced Photon SourceXSD-MICLemontILUSA Isotope Facility for Agricultural Education and Research Graduate School of Agricultural and Life SciencesThe University of TokyoBunkyo-kuTokyoJapan Department of Radiopharmaceuticals Development National Institute of Radiological SciencesNational Institutes for Quantum and Radiological Science and TechnologyInageChibaJapan Department of Applied Biological Chemistry Graduate School of Agricultural and Life SciencesThe University of TokyoBunkyo-kuTokyoJapan Department of Applied Genetics and Cell Biology University of Natural Resources and Life SciencesVienna(BOKU)Muthgasse 181180 WienAustria Department of Plant Sciences College of Agriculture and BioresourcesUniversity of SaskatchewanSaskatoonSKCanada Agri-Innovation Center Iwate UniversityMoriokaIwateJapan

Arsenic contamination is a major environmental issue,as it may lead to serious health hazard.The reduced trivalent formof inorganic arsenic,arsenite,is in generalmore toxic to plants comparedwith the fully oxidized pentavalent arsenate.Theuptakeof arsenite inplants hasbeenshown tobemediatedthrough a large subfamily of plant aquaglyceroporins,nodulin 26-like intrinsic proteins(NIPs).However,the efflux mechanisms,as well as themechanismof arsenite-induced root growth inhibition,remain poorly understood.Usingmolecular physiology,synchrotron imaging,and root transport assay approaches,we show that the cellular transport of trivalent arsenicals inArabidopsis thalianais stronglymodulatedbypinFORMED2(pin2)auxin efflux transporter.Root transport assay using radioactive arsenite,X-ray fluorescence imaging(XFI)coupled with X-ray absorption spectroscopy(XAS),and inductively coupled plasma mass spectrometry analysis revealed that pin2 plants accumulate higher concentrations of arsenite in roots comparedwith the wild-type.At the cellular level,arsenite specifically targets intracellular sorting of pin2 and thereby alters the cellular auxin homeostasis.Consistently,loss of pin2 function results in arsenite hypersensitivity in roots.XFI coupled with XAS further revealed that loss of pin2 function results in specific accumulation of arsenical species,but not the other metals such as iron,zinc,or calcium in the root tip.Collectively,these results suggest that pin2 likely functions as an arsenite efflux transporter for the distribution of arsenical species in planta.

关键词： auxin arsenite pin2 trafficking transport

Titanium Dioxide Nanoparticles Promote Root Growth by Interfering with Auxin Pathways in Arabidopsis thaliana

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Phyton-International Journal of Experimental Botany 2020年第4期89卷 883-891页

作者： Jiali Wei Ying Zou ping Li Xiaojun Yuan Shanghai Key Laboratory of Bio-Energy Crops Research Center for Natural ProductsPlant Science CenterSchool of Life SciencesShanghai UniversityShanghai200444China

TiO_(2) nanoparticles(nano-TiO_(2))are widely used in the world,and a considerable amount of nano-TiO_(2) is released into the environment,with toxic effects on organisms.In the various species of higher plants,growth,including seed germination,root elongation,and biomass accumulation,is affected by nano-TiO_(2).However,the underlying molecular mechanisms remain to be elucidated.In this study,we observed that nano-TiO_(2) promoted root elongation in a dose-dependent manner.Furthermore,we found that nano-TiO_(2) elevated auxin accumulation in the root tips of the auxin marker lines DII-VENUS and DR5::GUS,and,correspondingly,quantitative real-time PCR analysis revealed that nano-TiO_(2) increased the expression levels of auxin biosynthesis-and transport-related genes.GFP fluorescence observation using transgenic pin2-GFP indicated that nano-TiO_(2) promoted root growth by inducing pin2 accumulation.Thus,we propose that nano-TiO_(2) promote root growth in Arabidopsis thaliana by altering the expression levels of auxin biosynthesis-and transport-related genes.

关键词： Nanoparticles nano-TiO_(2) root length auxin pin2 auxin-related genes

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拟南芥pin2调控根向地性的功能位点分析

拟南芥PIN2调控根向地性的功能位点分析

作者：刘静兰州大学

学位级别：硕士

根尖向地性生长是植物根系构型在土层中快速建成的重要保障,有利于植物根系从土层中吸收水、无机盐和养分,从而促进植株地上部分的营养生长和生殖生长,是植物进行土壤固着生活不可或缺的生存适应机制之一。早期的生理学研究已经暗示,重... 详细信息

根尖向地性生长是植物根系构型在土层中快速建成的重要保障,有利于植物根系从土层中吸收水、无机盐和养分,从而促进植株地上部分的营养生长和生殖生长,是植物进行土壤固着生活不可或缺的生存适应机制之一。早期的生理学研究已经暗示,重力诱导的生长素不对称分布是引起根尖向地性弯曲的根本原因,其中生长素输出载体pin2(pin-FORMED 2)介导的生长素极性运输在根尖生长素不对称分布建立过程中起着至关重要的调控作用。此外,pin2蛋白的极性定位和输出活性对生长素极性运输有重要作用。研究表明,pin2蛋白上许多功能位点如磷酸化位点、泛素化位点和内吞分拣基序等能够直接或间接调控pin2蛋白的极性定位和活性。因此,深入挖掘pin2调控根尖向地性的功能位点具有十分重要的生物学意义。为探究pin2蛋白上未发现的功能位点。本研究利用EMS诱变技术,已获得S141和T146两个氨基酸位点突变的突变体agr375和agr498株系,它们的根尖向地性与pin2突变体相似,均失去向地性反应。随后,利用定点突变技术构建相应点突变转基因株系,运用遗传学和细胞生物学的研究方法,初步阐明了S141和T146两个位点在pin2介导的根尖向地性生长中的生物学功能及其分子机制。具体研究结果如下:(1)通过EMS诱变,多代繁殖筛选,回交和回补等方法,确定两个pin2点突变株系agr375和agr498,并通过测序确定点突变分别位于pin2蛋白的第141位(S突变为N;S141N)和146位(T突变为I;T146I)。(2)利用定点突变技术,以Pro pin2::pin2-GFP(简称pin2-GFP)表达载体为模板,构建Pro pin2::pin2-GFP(简称S141A)、Pro pin2::pin2-GFP(简称S141D)、Pro pin2::pin2-GFP(简称T146A)和Pro pin2::pin2-GFP(简称T146D)定点突变表达载体,通过拟南芥花序浸染法,将其导入pin2,并获得稳定遗传株系。遗传学分析表明,S141A和T146A能有效回补pin2突变体表型,而S141D不能回补pin2突变体表型,T146D部分回补pin2突变体表型。此外,S141D的根尖对重力刺激响应不敏感,而T146D能部分响应重力刺激。以上这些结果表明,141和146位点对pin2介导的根尖向地性生长具有重要的生物学功能。(3)利用生长素反应的分子标记DR5::3×NLS-VENUS分析点突变pin2-GFP的根尖生长素分布和根尖重力刺激后生长素分布情况。结果表明,S141D和T146D能够影响pin2介导的生长素极性运输。(4)细胞生物学分析表明,S141D的pin2极性定位正常,而T146D的pin2极性分布增强。同时,相比野生型,S141D和T146D的生长素在根尖的积累增加,暗示这两个点突变可能均通过影响pin2的生长素输出活性调控生长素的极性运输。FRAP实验表明,S141D和T146D均减弱了pin2在质膜上的迁移。Confocal活细胞成像分析pin2的内吞速率,发现S141D和T146D增强了SA(内吞抑制剂)对pin2-GFP内吞的抑制效果。以上结果表明,141和146位点都影响pin2的内吞。据上述实验结果,本研究初步获得以下结论:pin2蛋白第五个跨膜区141和146位点的突变通过影响pin2活性调控生长素的极性运输,从而影响拟南芥根的向地性生长;S141D和T146D能够增强pin2在质膜上的成簇聚合,从而抑制SA处理后pin2蛋白的内吞速率。本研究为探究pin2跨膜区功能位点和影响pin2生长素输出活性的机制提出了新的观点和见解。

关键词：拟南芥 pin2 生长素根尖向地性功能位点极性定位成簇内吞

植物生长调节剂对‘库尔勒香梨’石细胞与木质素合成的影响及pin2基因功能验证

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植物生长调节剂对‘库尔勒香梨’石细胞与木质素合成的影响及PIN2...

作者：张倩塔里木大学

学位级别：硕士

植物外源激素的含量与种类会影响植物的生长发育,影响植物器官的脱落。而到目前为止有关外源激素对‘库尔勒香梨’果实发育过程中的石细胞发育与形成的机理研究较少。目前,应用分子生物学手段研究梨脱萼果与宿萼果差异分子机理的工作也... 详细信息

植物外源激素的含量与种类会影响植物的生长发育,影响植物器官的脱落。而到目前为止有关外源激素对‘库尔勒香梨’果实发育过程中的石细胞发育与形成的机理研究较少。目前,应用分子生物学手段研究梨脱萼果与宿萼果差异分子机理的工作也已相继展开。因此,本文从‘库尔勒香梨’脱萼果和宿萼果萼洼发育过程中石细胞含量与分布、木质素相关合成酶、维管束发育等生理生化指标,同时通过实时定量的PCR技术,分析IAA和TIBA处理的‘库尔勒香梨’萼洼pin2基因表达,验证该基因在萼洼处的功能,为今后利用基因工程手段改善‘库尔勒香梨’果实品质提供科学依据。主要研究结果如下:(1)于‘库尔勒香梨’大蕾期外源喷施100mg/LTIBA可明显降低果实萼洼处的石细胞与木质素含量。大蕾期喷洒IAA对果实萼洼处石细胞与木质素含量无显著影响。梨果实萼洼处PAL、POD和CAD酶活性总体呈下降趋势;本研究所选择的相关酶基因中,与本文上述研究结果中酶活性存在一致性的只有Pb CAD,其相对表达量逐渐下降,说明Pb CAD基因表达量的下降导致CAD酶活性的下降,从而影响‘库尔勒香梨’萼洼处木质素含量与石细胞含量。(2)间苯三酚染色后发现,果实会呈现梗端与萼端的石细胞多,而越靠近胴部可被染色的石细胞数目越少。在36DAF后,被染色的石细胞逐渐减少。与IAA处理的果实相比,TIBA处理的染色范围较小。同样石蜡切片观察发现,经过TIBA处理的萼洼处石细胞团与CK1相比面积较小,石细胞分布稀疏,有效的降低了石细胞含量;经IAA处理的萼洼处石细胞团分布交为松散,但是石细胞团的面积增大,会导致库尔勒‘库尔勒香梨’口感变差。石细胞的细胞壁完整,但与对照相比,TIBA处理后145天的石细胞分布更分散,细胞壁间隙更大,细胞壁被抑制,细胞内物质逐渐外渗。(3)在番茄植株根中,POD、PAL、CAD三种酶活与野生型对照相比均有显著性增加;而在番茄植株中的茎与叶片中木质素相关合成酶虽有变化,但变化不显著;除此外本研究还发现,转Pb pin2基因的番茄植株中根、茎与叶片中的木质素含量同样会增加,并且在根部呈现显著增加。木质素含量的变化与木质素相关合成酶呈变化呈正向相关,因此本研究结果与上述酶活性得出结果一致,证明Pb pin2基因可能是通过促进POD、PAL、CAD三种酶活性,从而影响木质素含量。

关键词： ‘库尔勒香梨’ 植物生长调节剂石细胞木质素 pin2

UV-B辐射下UVR2对拟南芥根向重力性生长的影响

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UV-B辐射下UVR2对拟南芥根向重力性生长的影响

作者：叶青山西师范大学

学位级别：硕士

UV-B最初被认为是一种胁迫因子,随着研究的深入,人们发现UV-B作为光信号对植物生长发育具有重要调节作用。UV-B辐射的波长、强度和持续时间决定了其对植物产生各种生物学影响。UV-B辐射导致植物产生大量DNA光产物,其中最主要的是环丁烷... 详细信息

UV-B最初被认为是一种胁迫因子,随着研究的深入,人们发现UV-B作为光信号对植物生长发育具有重要调节作用。UV-B辐射的波长、强度和持续时间决定了其对植物产生各种生物学影响。UV-B辐射导致植物产生大量DNA光产物,其中最主要的是环丁烷嘧啶二聚体(Cyclobutane pyrimidine dimer,CPD)。这些损伤会影响植物DNA的代谢和功能。植物可以通过光解酶(Photolyase)来修复UV-B诱导的DNA损伤。拟南芥中存在专门修复CPD的活性光解酶即UVR2。本研究以哥伦比亚生态型拟南芥(Arabidopsis thaliana,Col-0)和uvr2突变体为材料,通过观察UV-B辐射后野生型与uvr2突变体表型差异、淀粉粒的积累、生长素的响应和分布以及生长素输出载体pin2的分布情况,探讨了UVR2对拟南芥主根向重力性生长的影响。主要研究结果如下:(1)5 kJ/m剂量的UV-B处理野生型和uvr2突变体,处理后的野生型主根能够恢复向重力性生长,但uvr2突变体主根仍保持弯曲,失去向重力性生长。(2)UV-B辐射下uvr2突变体根尖中淀粉粒合成相关基因(SS2、SS3和ADG1)的表达量明显低于野生型,与淀粉粒降解有关基因(AMY3)的表达量明显高于野生型,UV-B辐射抑制了uvr2突变体根尖淀粉粒的积累。(3)UV-B辐射下调了uvr2突变体主根中生长素生物合成相关基因的表达,导致主根中生长素的浓度降低,引起根尖生长素的不对称分布。(4)UV-B辐射导致uvr2突变体根尖辐射侧的pin2丰度低于非辐射侧,说明pin2参与UV-B辐射导致的uvr2突变体主根的向重力性生长。(5)UV-B辐射下UVH1可能与UVR2共同作用来调控主根的向重力性生长。综上所述,本研究表明,UV-B通过减少拟南芥uvr2突变体根尖淀粉粒的积累和生长素的响应,通过调节pin2的不对称分布和根尖生长素的极性分布介导主根永久失去向重力性,探讨了UV-B辐射下UVR2对于拟南芥主根向重力性生长的影响。本研究为完善植物根系向重力性生长机制提供一定的理论依据。

关键词： UV-B UVR2 向重力性生长生长素 pin2

The MADS transcription factor CmANR1 positively modulates root system development by directly regulating Cmpin2 in chrysanthemum

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Horticulture Research 2018年第1期5卷 202-217页

作者： Cui-Hui Sun Jian-Qiang Yu Xi Duan Jia-Hui Wang Quan-Yan Zhang Kai-Di Gu Da-Gang Hu Cheng-Shu Zheng National Key Laboratory of Crop Biology College of Horticulture Science and EngineeringShandong Agricultural UniversityTai-AnShandong271018China Shandong Agricultural and Engineering University Ji-NanShandongChina

Plant root systems are essential for many physiological processes,including water and nutrient absorption.MADS-box transcription factor(TF)genes have been characterized as the important regulators of root development in plants;however,the underlying mechanism is largely unknown,including chrysanthemum.Here,it was found that the overexpression of CmANR1,a chrysanthemum MADS-box TF gene,promoted both adventitious root(AR)and lateral root(LR)development in chrysanthemum.Whole transcriptome sequencing analysis revealed a series of differentially expressed unigenes(DEGs)in the roots of CmANR1-transgenic chrysanthemum plants compared to wild-type plants.Functional annotation of these DEGs by alignment with Gene Ontology(GO)terms and biochemical pathway Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis indicated that CmANR1 TF exhibited“DNA binding”and“catalytic”activity,as well as participated in“phytohormone signal transduction”.Both chromatin immunoprecipitation–polymerase chain reaction(ChIP-PCR)and gel electrophoresis mobility shift assays(EMSA)indicated the direct binding of Cmpin2 to the recognition site CArG-box motif by CmANR1.Finally,a firefly luciferase imaging assay demonstrated the transcriptional activation of Cmpin2 by CmANR1 in vivo.Overall,our results provide novel insights into the mechanisms of MADS-box TF CmANR1 modulation of both AR and LR development,which occurs by directly regulating auxin transport gene Cmpin2 in chrysanthemum.

关键词： pin2 Whole alignment

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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