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题名: 丛枝菌根增强植物铬耐性机理研究
作者: 伍松林
学位类别: 博士
答辩日期: 2015-11
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 陈保冬
关键词: 丛枝菌根 ; 真菌 ; ; 耐性 ; 重金属 ; 迁移转化 ; 同步辐射 X射线光谱
其他题名: Mechanisms underlying the enhanced plant chromium tolerance by arbuscular mycorrhizal symbiosis
学位专业: 生态学
中文摘要:   丛枝菌根在植物适应重金属胁迫中具有重要作用,因而在重金属污染土壤的植物修复及生态恢复中具有极高的潜在应用价值。然而,关于丛枝菌根对植物耐受铬的影响鲜有报道,相关机理研究更是匮乏。本论文证实了丛枝菌根对植物适应铬污染环境的积极作用,同时深入研究了铬在菌根共生界面的迁移转化过程,揭示了丛枝菌根增强植物铬耐性机制。论文主
要研究内容及结论如下:
    (1)丛枝菌根能够增强植物铬耐性
     模拟研究了不同程度土壤铬污染水平[Cr(VI)添加量: 0、5、10、20  mg kg-1]下,丛枝菌根真菌(Rhizophagus irregularis)对蒲公英(Taraxacum platypecidum Diels.)和狗牙根(Cynodon dactylon (Linn.) Pers.)生长及吸收累积铬的影响。结果发现,丛枝菌根显著促进了蒲公英和狗牙根的生长,但对植物磷和铬吸收分配的影响因植物种类而异。蒲公英是铬敏感性植物,其菌根依赖性较高,而狗牙根对铬的耐受性较强,菌根依赖性相对较低。丛枝菌根能够显著促进蒲公英吸收磷,同时降低其对铬的吸收和向地上部运输,从而缓解蒲公英铬毒害。相反,丛枝菌根并没有改善狗牙根磷营养,但能够降低其对铬的吸收,同时在高浓度铬污染下抑制铬自根系向地上部的运输,以此来缓解植物铬毒害。研究同时也发现,丛枝菌根能够改变根际土壤中铬的形态及生物有效性。
    (2)丛枝菌根根外菌丝在菌根固持铬中起着重要作用
      对比研究了不同磷添加水平(0、30、60、150 mg kg-1)和接种丛枝菌根真菌(Rhizophagus irregularis)对蒲公英(Taraxacum platypecidum Diels.)铬耐性的影响。发现添加磷处理并不 能达到接种丛枝菌根真菌对植物所产生的积极效果,丛枝菌根在促进磷吸收的同时增强了根系对铬的固持能力。基于同步辐射光源的微区 X射线荧光分析(SRμ-XRF)发现铬在非接种植物主根中主要分布于皮层和维管束部位,而在接种植物主根中则主要分布在皮层部位,表明丛枝菌根能够抑制铬经由木质部向植物地上部运输。研究进一步通过三分室植物培养装置探究了丛枝菌根根外菌丝对铬的吸收及转运作用,发现丛枝菌根根外菌丝能够吸收铬并转运到菌根根系中,但似乎并没有将铬进一步转运到植物地上部,而是更多的固持在根系,说明丛枝菌根根外菌丝在菌根固持铬中起着重要作用。
    (3)铬在丛枝菌根共生界面的微观行为过程研究
    利用丛枝菌根真菌与 Ri T-DNA转化胡萝卜根双重培养体系,结合透射电子显微镜-能谱分析(TEM-EDS)和基于同步辐射光源的  X射线近边吸收精细结构(XAFS)技术深入研究了丛枝菌根根外菌丝对铬的吸收、转运和转化作用。在排除其它微生物干扰的情况下,证实了丛枝菌根根外菌丝能够通过主动运输方式吸收铬并转运至菌根根系,但同时将70%以上铬固持在根外菌丝中。进一步利用  XAFS分析发现,丛枝菌根真菌能够将Cr(VI)还原成Cr(III),并以磷酸铬类似物的形式固持在菌丝表面,而吸收进去的铬(以 Cr(VI)或 Cr(III)形式吸收)很可能以组氨酸结合态及其类似物的形式在菌丝中转运。进一步通过场发射扫描电子显微镜-能谱分析(FE-SEM-EDS)、扫描透射 X射线显微分析(STXM)及XAFS分析共同证实这些以磷酸铬类似物为主的铬化合物(还有少量羧基或组氨酸结合态铬)主要分布于菌丝表面的胞外聚合物(EPS)中,表明 EPS在菌丝还原固持  Cr(VI)中具有重要作用。STXM分析同时发现, Cr(VI)胁迫下(组培情况)菌根根系中铬主要分布于丛枝、根内菌丝及细胞壁等部位,这一现象同时在土培试验蒺藜苜蓿(Medicago  truncatula)根系中再现,说明丛枝菌根真菌结构在菌根根系中起到铬的“区室化”作用,并由此降低铬自真菌结构经由共生界面向植物细胞的转运,从而减轻植物铬毒害。
    论文深入揭示了菌根介导的铬在植物-土壤系统中的生物地球化学过程,阐明了菌根通过直接作用缓解植物铬毒害的机制,为菌根在铬污染土壤生态修复中的应用提供了重要理论基础,同时也为推测其他金属在菌根界面的环境行为提供了借鉴。
英文摘要:   Arbuscular mycorrhizal fungi (AMF) play an  important role in plant resistance to heavy metal stress,  and therefore  are  potentially  valuable  for phytoremediation  or  ecological  restoration of metal contaminated soils.  However, few studies focus  on the role of  AMF in relieving chromium (Cr) phytotoxicity and the underlying mechanisms. The present study confirmed the importance of AMF in alleviation of Cr phytotoxicity, and investigated the underlying mechanisms with focus on biotranslocation  and  transformation  of chromium  by  AM  symbiosis.  The  key  findings  are  as follows:
    (1) AM symbiosis can enhance plant Cr tolerance
    In a greenhouse  pot experiment, we  investigated the effects of  arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis on  the growth of dandelion (Taraxacum platypecidum Diels.) and bermudagrass [Cynodon dactylon (Linn.) Pers.] in Cr(VI)-amended soils [0  mg kg-1, 5 mg kg-1, 10 mg kg-1,  and 20 mg  kg-1]. The results showed  that the dry  weights of the  two plant species were dramatically increased by AM symbiosis. AMF inoculation increased plant P concentrations, while decreased Cr concentrations and  Cr translocation from roots to shoots  for dandelion. However, as for  bermudagrass, AM  symbiosis decreased  plant  Cr concentrations  without  improvement of  P uptake. Besides, the study also revealed that AM symbiosis changed Cr species and bioavailability in the rhizosphere. The study confirmed the protective effects of AMF on host plants under Cr(VI)contaminations.
    (2) Extraradical mycelium contributes to Cr stabilization in mycorrhizal roots
     We explored  if  applying exogenous  P to  the Cr  contaminated soil  could  match the  positive effects  of AM  symbiosis  on plant  growth  under Cr  contaminations  for  the highly  mycorrhizal dependent   plant-dandelion  (Taraxacum   platypecidum   Diels.).   The  results   indicated   that   P application  could   not   increase  plant   dry  weights   as  well   as  AM   symbiosis   did.  Besides enhancement of  P acquisition, AM symbiosis  can also stabilize  Cr in mycorrhizal  roots and thus restrain  Cr  transport  to  shoots.  Synchrotron  radiation   micro-focused  X-ray  fluorescence  (SR μ-XRF) analysis of metal distribution in principal roots also confirmed the immobilization of Cr in mycorrrihzal   roots.   Furthermore,    by   using   a   three-compartment   cultivation    system,   we demonstrated that extraradical mycelium (ERM) can take up and transport  Cr to mycorrhizal roots from distance, but may  not translocate these Cr  from roots to shoots, and  therefore contributed to Cr stabilization in mycorrhizal roots and relieved Cr phytotoxicity.
    (3) Cr translocation and transformation in AM symbiotic interface
    In a two-compartment root-organ culture system,  the uptake, translocation, and transformation of Cr(VI)  by ERM  of AM  association was  investigated  using inductively coupled  plasma  mass spectrometry   (ICP-MS),   scanning    electron   microscope   equipped   with    energy   dispersive spectroscopy  (SEM-EDS),  transmission  electron  microscope  equipped  with  energy  dispersive spectroscopy (TEM-EDS)  and X-ray  absorption fine  structure (XAFS)  technologies. The results showed that ERM can actively take up  and transport Cr [either in the form of Cr(VI) or Cr(III)]  to roots but  retains  above 70%  of  the total  Cr in  the  fungal biomass.  The XAFS  analysis  further showed that Cr(VI)  in the ERM  was reduced completely to  Cr(III) and subsequently precipitated mainly  by  phosphate  analogues  likely on  fungal  surface.  Besides,  through  comparison  of  Cr speciation between  living hyphae and  inactivated hyphae, we  also proposed that Cr(III)-histidine analogues may exist in the living hyphae.
    In  order  to  investigate  the  detailed   biochemical  processes  of  Cr  immobilization  by  AM symbiosis, we further  investigated the cellular distribution  and speciation of Cr in  both ERM and mycorrhizal roots  treated with  Cr(VI) by using  FE-SEM-EDS, scanning  transmission soft X-ray microscopy  (STXM)   and  XAFS   techniques.  The   results  indicated   that  AMF   can  produce numerous extracellular polymeric substances  (EPS) on the fungal  surface upon Cr(VI) stress, and Cr mainly  existed in these  EPS, which  indicated that those  EPS potentially  contribute to Cr(VI) reduction and  immobilization on fungal surface.  The significant positive  relationship between Cr and P concentration (analyzed by EDS)  on hyphal surface further indicated that  phosphate groups may act as counter ions  of Cr, which was also revealed by the XAFS  analysis showing that the Cr in fungal biomass was in the form of mainly Cr(III)-phosphate analogues. Take together the results of  both STXM  and XAFS  analysis,  we concluded  that  AMF can  adsorb and  reduce  Cr(VI) to Cr(III),  and  then  complex  Cr(III)  mainly  by  phosphate  groups  on  fungal  surface  due  to  the functions  of  EPS  produced by  AMF  upon  Cr(VI)  stress.  In  addition,  the  STXM analysis  on sections of  mycorrhizal roots (grown  both in  vitro and in  vivo) exposed to  Cr(VI) also indicated that AMF structures  (arbuscules, intraradical mycelium, etc) in  mycorrhizal roots can also  reduce Cr(VI)  to  Cr(III)  and  precipitate  Cr(III)  possibly  by  phosphate  groups,  and  thus  restrain  Cr translocation to  plant  cells across  the symbiotic  interface. All  in all,  the present  work provided strong evidences of  Cr immobilization by ERM  and Cr compartmentation by  fungal structures in mycorrhizal  roots  at  cellular   level,  and  therefore  unraveled   the  mechanisms  by  which  AM symbiosis immobilize Cr and enhance plant Cr tolerance.
    The  study has  demonstrated the  important  role of  AMF in  Cr  biogeochemical processes  in plant-soil continuum. Besides, the present work has  also provided essential evidences of the direct way AM symbiosis enhances plant Cr tolerance, which indicates that AM symbiosis can be used in ecological restoration of Cr contaminated soils.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/37026
Appears in Collections:城市与区域生态国家重点实验室_学位论文

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Recommended Citation:
伍松林. 丛枝菌根增强植物铬耐性机理研究[D]. 北京. 中国科学院研究生院. 2015.
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