Leukamenin E调节拟南芥幼苗根部生长素极性运输及根系生长发育

本文采用14种拟南芥转基因报告株系,研究了对映-贝壳杉烷二萜(leukamenin E)调节拟南芥根部生长素极性运输转运蛋白表达并影响根生长发育的机制。结果表明:leukamenin E浓度在20～40μmol·L~(-1)范围显著抑制拟南芥幼苗主根的生长,较低浓度leukamenin E(10μmol·L~(-1))促进侧根提前发生并增加侧根数量; 10～30μmol·L-1的leukamenin E在处理拟南芥幼苗48 h后可显著提高其根尖部生长素水平;进一步检测根部生长素极性运输转运蛋白表达,发现该浓度条件下leukamenin E促进PIN1在转录水平表达并增加其蛋白丰度,但在转录水平抑制PIN2表达并减少其蛋白丰度;同时,显著减少PIN3、PIN7和PIN4蛋白在小柱和静止中心及其周围细胞的丰度以及AUX1的定位丰度; Leukamenin E对PIN1丰度的上调以及对PIN2、PIN3、PIN4、PIN7和AUX1定位丰度的减少可导致生长素在根尖顶部的积累以及根部生长素浓度梯度的改变,引起根生长的抑制及侧根的提前发生。     

头孢霉素对拟南芥主根生长发育的影响

以拟南芥野生型和相关转基因株系为材料,设置0、50、100、200和400μg/mL头孢霉素处理,考察头孢霉素对主根伸长生长、根尖分生组织活性、生长素分布运输以及干细胞活性的影响,探究头孢霉素对拟南芥主根生长发育的毒性作用机制。结果显示:(1)头孢霉素能以浓度依赖的方式抑制拟南芥主根的生长,并抑制分生组织长度和CYCB1;1基因的表达,说明它能抑制根尖分生组织活性。(2)头孢霉素能降低根尖生长素报告基因DR5∷GUS、DR5∷GFP和生长素极性运输蛋白PIN1、PIN2、PIN3、PIN7和AUX1的表达,说明它能抑制根尖生长素的分布和极性运输。(3)头孢霉素能下调根尖静止中心标记系WOX5∷GFP、QC25和QC46的表达,以及SHR和SCR蛋白的表达,说明它能抑制根尖干细胞活性。研究表明,头孢霉素能通过抑制根尖分生组织活性、生长素的分布和极性运输以及干细胞活性,从而调节拟南芥主根的生长发育。     

磷脂酶Dα1与H2S参与冬凌草甲素对拟南芥的化感作用

以拟南芥哥伦比亚野生型(WT)、磷脂酶Dα1(PLDα1)缺失型突变体pldα1、D-/L-半胱氨酸脱巯基酶(D-/L-CDes)缺失型突变体d-cdes和l-cdes幼苗为试验材料,60 μmol·L^-1冬凌草甲素为处理浓度,研究了拟南芥响应二萜类化合物冬凌草甲素的化感作用中磷脂酶Dα1(PLDα1)与气体信号分子硫化氢(H₂S)的信号关系。结果表明: 冬凌草甲素显著提高了野生型拟南芥幼苗H₂S含量、PLD和D-/L-CDes酶的活性及其基因表达;冬凌草甲素处理下,pldα1突变体幼苗的D-CDes和L-CDes活性明显低于WT,外源添加磷脂酸(PA)后D-CDes和L-CDes活性显著提高,并高于WT;冬凌草甲素显著抑制4种株系根的生长,其中d-cdes和l-cdes对冬凌草甲素更加敏感,外施NaHS可以促进冬凌草甲素处理下4种株系根的生长及内源H₂S产生,外施PA只对冬凌草甲素处理下的WT、pldα1和l-cdes株系根的生长及内源H₂S产生有促进作用,而对d-cdes株系没有明显作用。说明PLDα1和H₂S在拟南芥响应冬凌草甲素过程中发挥作用,且PLDα1/PA位于D-CDes上游,参与调控拟南芥幼苗H₂S的产生及根生长的信号过程。     

氮掺杂石墨烯量子点对拟南芥主根生长方向的影响

石墨烯量子点(GQDs)在电化学生物传感器、生物成像和生物医学等领域具有巨大的应用潜力, 在公众和环境中的暴露程度也越来越高, 近年来其生物安全性备受关注。截至目前, 有关石墨烯量子点对植物生长发育影响的研究较少。该文从细胞和分子水平探究了氮掺杂石墨烯量子点(N-GQDs)处理对拟南芥(Arabidopsis thaliana)主根生长方向的影响。结果表明, N-GQDs能够被根摄取, 并通过维管束运输。50-100 mg∙L^-1 N-GQDs处理可改变主根的生长方向, 使其朝着远离培养基的方向发生弯曲。研究发现, N-GQDs处理导致根尖小柱细胞中淀粉粒的积累减少, 生长素外排载体PIN3的表达量降低, 小柱细胞中的PIN3重新定位到远离培养基一侧的细胞外侧膜(即朝向空气), 促进根尖生长素的不对称分布, 从而引发主根朝着远离培养基的方向弯曲生长, 以避开较高浓度的N-GQDs环境。研究结果为进一步阐明N-GQDs处理改变根生长方向的机制提供了重要线索, 同时也为N-GQDs的生物安全性评价提供参考依据。     

生长素输出载体PIN家族研究进展

生长素极性运输调控植物的生长发育。生长素极性运输主要依赖3类转运蛋白: AUX/LAX、PIN和ABCB蛋白家族。生长素在细胞间流动的方向与PIN蛋白在细胞上的极性定位密切相关。PIN蛋白由1个中心亲水环和2个由中心亲水环隔开的疏水区组成。中心亲水环上含多个磷酸化位点, 其为一些蛋白激酶的靶点。PIN蛋白受多方面调控, 包括转录调控、转录后修饰以及胞内循环与降解, 以响应内源和外源信号。目前, 利用全基因组测序方法在禾谷类作物水稻(Oryza sativa)、玉米(Zea mays)和高粱(Sorghum bicolor)中分别鉴定出12、15和11个PIN基因, 但仅有少数PIN基因的功能被报道。该文从蛋白结构、活性调控和功能验证等方面综述了PIN蛋白在拟南芥(Arabidopsis thaliana)和禾谷类作物中的研究进展, 以期为探究PIN蛋白家族介导的生长素极性运输过程提供新的思路与线索。     

铝胁迫对拟南芥根尖ＡｔＰＩＮ２ 蛋白表达活性的影响

铝胁迫能影响根尖生长素的运输,这与生长素运输载体密切相关,PIN2作为根尖生长素的运输蛋白,其独特的组织定位可能诱导PIN2蛋白参与了铝调节生长素的运输过程。该研究以拟南芥PIN2缺失突变体( pin2)、PIN2□∷□GFP融合体及其野生型( WT)为材料,应用激光扫描共聚焦显微技术,研究铝处理对拟南芥根尖生长素运输蛋白PIN2的表达活性、蛋白在组织及亚细胞水平分布及其对铝内置化作用的影响。结果表明：短期铝处理或低铝浓度能明显增加拟南芥根尖细胞PIN2蛋白表达活性,而长期铝处理或高铝浓度抑制其表达活性;以100μmol?L-1 AlCl3处理4 h的蛋白表达活性最高。蛋白印迹反应发现,铝处理促进PIN2蛋白在细胞膜上累积,减少胞内囊泡中PIN2蛋白的含量;囊泡运输抑制剂( BFA)能抑制铝诱导PIN2蛋白的分配。铝胁迫增加拟南芥根尖细胞H2 O2累积,pin2的H2 O2累积量大于WT,而相对根长小于WT。 Morin染色结果显示,pin2的铝内置化显著小于WT。上述研究表明,PIN2蛋白在100μmol?L-1 AlCl3处理条件下活性最高,细胞膜累积程度加强,铝内置化能力增强,从而调节根系的生长发育。该研究结果进一步为铝抑制生长素的运输机制提供了理论基础。     

铝胁迫对拟南芥根尖AtPIN2蛋白表达活性的影响

铝胁迫能影响根尖生长素的运输,这与生长素运输载体密切相关,PIN2作为根尖生长素的运输蛋白,其独特的组织定位可能诱导PIN2蛋白参与了铝调节生长素的运输过程。该研究以拟南芥PIN2缺失突变体(pin2)、PIN2□∷□GFP融合体及其野生型(WT)为材料,应用激光扫描共聚焦显微技术,研究铝处理对拟南芥根尖生长素运输蛋白PIN2的表达活性、蛋白在组织及亚细胞水平分布及其对铝内置化作用的影响。结果表明:短期铝处理或低铝浓度能明显增加拟南芥根尖细胞PIN2蛋白表达活性,而长期铝处理或高铝浓度抑制其表达活性;以100μmol·L-1Al Cl3处理4 h的蛋白表达活性最高。蛋白印迹反应发现,铝处理促进PIN2蛋白在细胞膜上累积,减少胞内囊泡中PIN2蛋白的含量;囊泡运输抑制剂(BFA)能抑制铝诱导PIN2蛋白的分配。铝胁迫增加拟南芥根尖细胞H2O2累积,pin2的H2O2累积量大于WT,而相对根长小于WT。Morin染色结果显示,pin2的铝内置化显著小于WT。上述研究表明,PIN2蛋白在100μmol·L-1Al Cl3处理条件下活性最高,细胞膜累积程度加强,铝内置化能力增强,从而调节根系的生长发育。该研究结果进一步为铝抑制生长素的运输机制提供了理论基础。     

Leukamenin E调节拟南芥幼苗生长发育的模式及其机制

研究对映-贝壳杉烷型二萜化合物Leukamenin E对拟南芥种子萌发、下胚轴伸长以及根生长发育的作用模式,并探讨植物激素生长素和乙烯可能介导Leukamenin E影响拟南芥主根生长、侧根和根毛发育的初步机制。结果表明:Leukamenin E浓度在10~160μmol·L~(-1)范围对拟南芥种子的萌发率无显著影响,但高浓度Leukamenin E(80~160μmol·L-1)显著抑制种子的萌发速率。Leukamenin E对拟南芥幼苗根生长的抑制作用明显高于对下胚轴的抑制效应。进一步研究表明,Leukamenin E通过阻滞根尖细胞有丝分裂和细胞伸长进而抑制主根的生长,并能促进侧根提前发生并影响其形成数量,同时减少根毛密度及降低根毛长度。Leukamenin E联合乙烯利(乙烯释放剂)处理可阻止乙烯利单独使用对拟南芥幼苗根毛生长的促进作用,与Ag+(乙烯竞争抑制剂)联合乙烯利的作用效果相一致,表明Leukamenin E可能通过干扰根细胞乙烯途径而抑制根毛发育。流动注射化学发光分析和酶联免疫检测的结果发现,Leukamenin E显著上调拟南芥幼苗根组织中生长素(IAA)水平,表明生长素可能作为主要因子介导了Leukamenin E对拟南芥幼苗根生长发育的调节作用。     

白桦MYB基因响应激素及盐旱处理的表达研究

转录因子参与到植物激素不同的信号通路中及非生物胁迫的调控中，为研究白桦MYB转录因子家族基因对不同激素信号及非生物胁迫的响应，本研究以野生型白桦（Betula platyphylla）种子为材料，分别用10 μmol·L^-1的茉莉酸甲酯（MeJA）、100 μmol·L^-1的乙烯利（ETH）、20 μmol·L^-1的脱落酸（ABA）、50 μmol·L^-1的细胞激动素（KT）、200 mmol·L^-1 NaCl、100 mmol·L^-1 Mannitol以及水为对照进行处理，对激素处理4周的白桦下胚轴进行表型观察及基因表达分析，结果显示：激素处理能够影响白桦下胚轴及胚根的生长，经过ETH处理的白桦下胚轴和根短于水处理的对照，KT处理过的白桦下胚轴和根长于水处理的对照；MeJA、Ethylene及ABA处理对大部分BpMYBs的表达存在负调控，而BpMYB2，8，12基因上调表达；KT处理后，只有BpMYB11下调表达，其他的BpMYBs基因上调表达；NaCl处理后，大部分基因表现出先下调后上调的表达模式，在48 h被高度诱导；Mannitol模拟干旱处理条件下，大部分基因表现出先微弱上调后下调的表达模式。这些结果说明白桦MYB家族基因能够响应激素、盐和旱的处理，在调控白桦下胚轴及胚根应答外界信号的发育中起作用。本研究为探索激素调控木本植物生长发育的分子机制研究提供了数据和材料。     

多胺生物合成抑制剂D-精氨酸对拟南芥幼苗根系生长的影晌

为探讨多胺生物合成抑制剂D-精氨酸（D-arginine,D-Arg）对拟南芥根系生长的影响,首先用腐胺（0．1mmol‘L-1）和D—Arg（1．0mmol·L-1）处理种子萌发后生长2d的拟南芥幼苗。腐胺（0．1mmol·L-1）显著促进主根伸长,D-Arg（1．0mmol-L-1）显著抑制主根伸长,并对主根根尖的细胞形态有明显影响。为了进一步了解D—Arg影响拟南芥主根生长的机理,采用浓度梯度D．Arg处理幼苗根系。实验结果表明,随着D-Arg浓度增加（0．2～1．0mmol·L-1）,拟南芥幼苗主根生长受抑制的程度越严重。微分干涉观察主根根尖发现,外源施加D—Arg,引起拟南芥主根根尖分生区的细胞数目减少,使拟南芥幼苗表现出主根的伸长生长变缓。当分生区数目较少时,出现主根几乎不再仲长的现象。由此推测,多胺生物合成抑制剂D-Arg对拟南芥幼苗根生长的抑制作用机制,是D-Arg影响了其根尖分生区的细胞分裂活动,使分生区细胞数目减少,从而引起分生区长度减小,最终导致拟南芥主根仲长生长受到抑制。     

BYPASS1-LIKE regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis

Auxin and auxin-mediated signaling pathways are known to regulate lateral root development. Although exocytic vesicle trafficking plays an important role in recycling the PIN-FORMED (PIN) auxin efflux carriers and in polar auxin transport during lateral root formation, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis thaliana. b1l mutants contained significantly more lateral roots than the wild type, primarily due to increased lateral root primordium initiation. Furthermore, the auxin signal was stronger in stage I lateral root primordia of b1l than in those of the wild type. Treatment with exogenous auxin and an auxin transport inhibitor indicated that the lateral root phenotype of b1l could be attributed to higher auxin levels and that B1L regulates auxin efflux. Indeed, compared to the wild type, C-terminally green fluorescent protein-tagged PIN1 and PIN3 accumulated at higher levels in b1l lateral root primordia. B1L interacted with the exocyst, and b1l showed defective PIN exocytosis. These observations indicate that B1L interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation in Arabidopsis.     

Clathrin light chains regulate hypocotyl elongation by affecting the polarization of the auxin transporter PIN3 in Arabidopsis

PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.     

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.     

多氯联苯促进毛白杨不定根分化的效应

多氯联苯是一种典型的持久性有机污染物。研究表明多氯联苯具有毒物兴奋效应, 但其影响植物生长发育的机制尚不清楚。以毛白杨(Populous tomentosa)组培苗为材料, 探讨3 mg·L^-1 Aroclor1254对不定根分化、植物激素水平、与生长素相关的P009g125900、P006g142600和P002g222700基因表达、与细胞分裂素相关的P005g2489和P005g2376基因表达的影响。结果显示, Aroclor1254可促进毛白杨组培苗不定根分化, 缩短不定根初根时间与分化率达100%的时间, 提高不定根数目; 在不定根诱导期, 用Aroclor1254单独诱导, IAA/(ZR+dhZR)比值与阳性对照无显著差异, P006g142600、P002g222700、P009g125900、P005g2489和P005g2376基因表达变化趋势与IBA单独诱导下各基因表达变化趋势一致。为验证Aroclor1254是否具有生长素效应, 以玉米(Zea mays)和转生长素报告基因DR5::GUS的拟南芥(Arabidopsis thaliana)为材料, 观察Aroclor1254对胚芽鞘生长及DR5::GUS基因表达的影响。结果显示, 一定浓度的Aroclor1254对胚芽鞘的生长无显著影响, 但可诱导生长素报告基因表达。以上结果表明, 多氯联苯类化合物Aroclor1254虽不属于植物生长调节剂, 但具有毒物兴奋效应, 在一定浓度下具有类似生长素的生物学活性。     

Jasmonate modulates endocytosis and plasma membrane accumulation of the Arabidopsis PIN2 protein

The subcellular distribution of the PIN-FORMED (PIN) family of auxin transporters plays a critical role in auxin gradient-mediated developmental processes, including lateral root formation and gravitropic growth. Here, we report two distinct aspects of CORONATINE INSENSITIVE 1 (COI1)- and AUXIN RESISTANT 1 (AXR1)-dependent methyl jasmonate (MeJA) effects on PIN2 subcellular distribution: at lower concentration (5 μM), MeJA inhibits PIN2 endocytosis, whereas, at higher concentration (50 μM), MeJA reduces PIN2 accumulation in the plasma membrane. We show that mutations of ASA1 (ANTHRANILATE SYNTHASE a1) and the TIR1/AFBs (TRANSPORT INHIBITOR RESPONSE 1/AUXIN-SIGNALING F-BOX PROTEINs) auxin receptor genes impair the inhibitory effect of 5 μM MeJA on PIN2 endocytosis, suggesting that a lower concentration of jasmonate inhibits PIN2 endocytosis through interaction with the auxin pathway. In contrast, mutations of ASA1 and the TIR1/AFBs auxin receptor genes enhance, rather than impair, the reduction effect of 50 μM MeJA on the plasma membrane accumulation of PIN2, suggesting that this action of jasmonate is independent of the auxin pathway. In addition to the MeJA effects on PIN2 endocytosis and plasma membrane residence, we also show that MeJA alters lateral auxin redistribution on gravi-stimulation, and therefore impairs the root gravitropic response. Our results highlight the importance of jasmonate-auxin interaction in the coordination of plant growth and the adaptation response.     

Transmembrane kinase 1-mediated auxin signal regulates membrane-associated clathrin in Arabidopsis roots

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins. Clathrin triskelia are composed of clathrin heavy chains (CHCs) and light chains (CLCs), and the phytohormone auxin differentially regulates membrane-associated CLCs and CHCs, modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2 (PIN2). However, the molecular mechanisms by which auxin regulates clathrin are still poorly understood. Transmembrane kinase (TMKs) family proteins are considered to contribute to auxin signaling and plant development; it remains unclear whether they are involved in PIN transport by CME. We assessed TMKs involvement in the regulation of clathrin by auxin, using genetic, pharmacological, and cytological approaches including live-cell imaging and immunofluorescence. In tmk1 mutant seedlings, auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis, leading to an impaired asymmetric distribution of PIN2 and therefore auxin. Furthermore, TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin. In summary, TMK1 is essential for auxin-regulated clathrin recruitment and CME. TMK1 therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.