【作者】 金崇伟；

【导师】 郑绍建；

【作者基本信息】 浙江大学 ， 农业资源利用， 2008， 博士

【摘要】 缺铁是农业生产上限制作物生产的主要因素之一。对属于双子叶和非禾本科单子叶的机理Ⅰ植物而言，缺铁条件下，根系会作出多种响应来增加吸收利用铁的能力，这些响应包括：诱导高铁还原酶和铁转运体，分泌质子，增加亚根尖根毛发育和侧根发育以及分泌酚类物质等还原性小分子有机物。然而，后两种缺铁响应在改善物铁营养中的贡献仍尚未明确。本文首先以红三叶草（Trifolium pretense L．）为材料，较系统地研究了缺铁诱导的侧根发育和根系分泌的酚类物质在提高根系铁吸收，增加土壤中铁有效性及促进根系质外体铁再利用中的作用及其机制，以及体内酚类物质对生长素代谢的影响及其与缺铁响应之间的关系；另一方面，由于人类活动所导致的大气CO₂浓度增加会促进植物的生长，进而可能影响植物的铁营养，本研究以番茄（Solanum lycopersicom cv．）为材料研究了这一环境因素的变化对植物铁营养吸收的影响；最后，本研究借助病毒诱导的基因沉默技术（VIGS），初步探讨了体内14-3-3蛋白在植物缺铁响应中的调控作用。主要研究结果分为铁吸收机制，体内铁再利用机制，环境因素的影响及铁吸收调控机制等这四个方面，概述如下：1、侧根发育与根际微生物在植物吸收铁中的作用（铁吸收机制）缺铁条件下，红三叶草的侧根数量显著增加，并且侧根数量与根系高铁还原酶活性之间显著正相关。此外，对Dasgan等（2002，Plant and Soil，241：97-104）报道的结果作相关性分析也发现，两种不同基因型番茄line 227／1（P1）和Roza（P2），以及它们的两个杂交后代（‘P1×P2’和‘P2×P1’）在两个不同水平(10^-6和10^-7M FeEDDHA)的低铁条件下生长，它们的根系高铁还原酶活性与侧根数量也呈显著正相关。进一步分析表明，这4种番茄的侧根密度大小趋势又与它们的耐缺铁失绿能力的强弱相一致。这些结果证明了缺铁诱导的侧根数量增加可以提高根系高铁还原酶活性，并由此在增强植物耐缺铁能力上发挥重要作用。此外，有研究表明土壤微生物能促进植物铁的吸收。本研究中，我们也发现碱性土壤灭菌后，红三叶草的生长和铁吸收受到了明显抑制，但叶面喷施Fe-EDTA溶液和土壤接种根瘤菌使红三叶草的生长和铁吸收均得到恢复。后者现象与根瘤菌结瘤后根系的高铁还原酶活性显著增强有关。溶液培养条件下，红三叶草根系在缺铁条件下会分泌大量酚类化合物，在碱性土壤培养条件下，也发现根际土壤中酚类物质积累的现象。鉴于酚类物质具有抑菌性，我们测定了缺铁土壤培养的红三叶草根际微生物种群的变化。微生物16s rDNA PCR-DGGE分析和培养试验表明，缺铁培养的红三叶草根际土壤和人为添加根系分泌的酚类物质培养的土壤的微生物群落结构均发生了明显的变化，且都表现为分泌高铁载体（Siderophore）能力强的微生物数量的增加，说明缺铁诱导的根际微生物群落结构变化很可能是根系分泌的酚类物质引起的。在可培养的微生物中，我们还发现，与根系分泌的酚类物质共培养后，增加了土壤中产生长素类物质的微生物的比例。水培试验证明微生物分泌的生长素类物质能有效地增强红三叶草根系还原铁的能力。结合我们以往的研究，提出了机理Ⅰ植物根系分泌物与微生物互作及其对改善植物铁营养的作用的机理模型，即：缺铁诱导根系分泌的酚类物质首先改变了根际微生物群落结构，提高了根际土壤中能分泌高铁载体和生长素类物质的微生物的比例，新的微生物群落又通过增强高铁载体和生长素类物质的分泌促进植物的铁吸收。2、酚类根系分泌物在红三叶草根系质外体铁再利用上的作用（铁再利用机制）缺铁根系细胞向根际分泌酚类物质时，须先经过根系的质外体空间。在溶液培养试验中我们发现，用吸着型树脂去除培养液中的酚类根系分泌物后，红三叶草新叶失绿症状更为严重，且后期没有恢复迹象，这一现象暗示了缺铁诱导分泌的酚类物质可能与植物体内的铁再利用有关。进一步分析表明，酚类物质去除后，几乎完全抑制了缺铁根系的质外体铁随缺铁培养时间延长而下降趋势，与之相对应的是，酚类物质去除后，地上部的铁含量明显下降，而根系铁含量却显著提高，表明酚类物质去除后可能减少了根系中的质外体铁向地上部转运，从而加剧了地上部的缺铁症状。然而，去除酚类分泌物同时也增强了缺铁植物根系的高铁还原酶活性和质子分泌，说明这种质外体铁再利用过程并不是由质子分泌利高铁还原酶介导的。根系分泌的酚类物质对根系细胞壁中的铁的解吸动力学试验表明，酚类根系分泌物确能有效释放固定于细胞壁上的铁。上述结果充分证明了缺铁诱导的根系酚类分泌物直接介导了根系质外体铁的再利用，并在改善植物地上部分铁营养中起着十分重要的作用。3、大气CO₂浓度增加对植物铁营养的影响（环境因素的影响）目前，地球大气中的二氧化碳浓度（CO₂）正在逐年增加。水培试验结果表明空气中CO₂浓度升高后(800μL L^-1)，不仅可以显著增加低铁（以难溶性氧化铁铁矿粉为铁源）培养的番茄的生物量，而且还可以显著促进铁吸收，改善植物的铁营养。此外，尽管高CO₂浓度处理改善了植物的铁营养，但与正常大气下生长(CO₂浓度350μL L^-1)的植物相比，根系的铁还原酶活、质子分泌量和亚根尖根毛发育反而更为强烈，并且FER、FRO1和IRT3个基因的表达也显著增强。通过比较，我们还发现高CO₂浓度处理显著增加了番茄的根冠比。因此，我们认为，缺铁响应增强和根冠比增加可能是高CO₂浓度处理改善植物铁营养的原因，其中前者可能是主要原因。此外，高CO₂浓度处理还明显增加了根系的NO含量，这可能增强缺铁响应的原因。4、酚类化合物、生长素和14-3-3蛋白在铁吸收上的调控作用（铁吸收调控机制）缺铁处理显著增加了红三叶草根系的IAA含量。生长素极性运输抑制剂TIBA或NPA处理植物茎部后，显著抑制了缺铁诱导的根系高铁还原酶活性，质子分泌和亚根尖根毛发育等缺铁响应，但并不影响缺铁诱导的根系酚类物质的积累和分泌，表明IAA没有参与后两种缺铁响应的调控，但参与了前几种缺铁响应的调控过程。此外，缺铁处理明显降低了根系的IAA氧化酶活性。有意思的是，缺铁根系的酚类物质对IAA氧化酶活性具有很强的抑制作用，并且这种抑制作用要显著强于铁营养正常根系的酚类物质，表明缺铁导致IAA氧化酶活性下降很有可能是酚类物质在缺铁根系中积累引起的。综上所述，我们认为：缺铁机理Ⅰ植物根系通过增加酚类物质积累来抑制IAA氧化酶的活性，减少IAA在根系中的降解，提高IAA的含量，进而参与调控植物缺铁诱导的高铁还原酶活性、质子分泌和根毛发育。利用VIGS（病毒诱导的基因沉默）技术，沉默了TFT7 14-3-3基因在番茄中的表达，缺铁处理后，我们发现TFT7 14-3-3基因沉默，明显抑制了根系FER基因的表达，同时也抑制了FRO1基因以及IRT基因的表达，根系的高铁还原酶活性也明显降低。基于转录因子FER在植物缺铁响应中的调控作用，我们认为14-3-3蛋白可能是通过调控FER基因的表达来调控下游缺铁响应的诱导。更多还原

【Abstract】 Iron deficiency is one of the most deleterious factors limiting crop production in the world.Innongraminaceous monocots and dicots,the so called StrategyⅠplants,roots respond to Fedeficiency stress by inducing FCR,IRT,proton secretion,subapical root hair development,lateral root development and releasing of reductants such as phenolic compounds.However,relative little is known about the fuctions of the latter two responses in plant Fe nutrition.In thisresearch,an iron efficient plant,red clover （Trifolium pretense L.）,was employed to investigatethe roles of Fe-defciency induced lateral root development and phenolics exudation in enhancingplant Fe uptake,increasing soil Fe bioavailability and facilitating root apoplastic Fe reutilization.The relationship between phenolics accumulation and auxin metabolism in root in the regulationof iron-deficiency induced responses was also investigated.On another hand,the elevatedatmospheric CO₂ level could significantly stimulate the plant grwoth,which would eventuallyaffect the plant Fe nutrition,in this research,the tomato （Solanum lycopersicom cv.）were alsoemployed to investigate the influence of this envrironmental factor on the plant Fe uptake.Finally,the role of 14-3-3 protein in regulation of Fe-deficient induced response in tomato wasalso primarily studied by using VIGS technique.The main results can be devided into foursections:Fe uptake mechanisms,Fe reutilization mechanism influences of environmental factors,and regulation mechanisms of Fe uptake.These results are summarized as following.1.Contribution of lateral root development and rhizosphere microbes to the plant ironuptake （Iron uptake mechanisms）We found that the lateral root development of red clover was significantly enhanced by Fedeficiency,and the total lateral root number correlated well with the Fe-deficiency-induced FCRactivity.By analyzing the results from Dasgan et al.（2002,Plant and Soil,241:97-104）,we alsofound that although the two tomato genotypes line227/1 （P1）and Roza （P2）and their reciprocalF1 hybrid lines （‘P1×P2’and‘P2×P1’）were cultured under two different lower Fe conditions(10^-6and 10^-7M FeEDDHA),their FCR activities are significantly correlated with the lateralroot numbers.More interestingly,the-Fe chlorosis tolerant ability of these four tomato linesdisplays similar trends with the lateral root density.Taking together,we proposed that theFe-deficiency-induced increases of the lateral root should play an important role in resistance toFe deficiency.The soil microbes have been demonstrated to have benefical effects on plant iron uptake insome researches.We also found that the red colver grown in sterilized soil had significantly lessgrowth and Fe uptake than the plants grown in non-sterilized soil.Growth and Fe content ofthese plants were improved by foliar application of Fe-EDTA or rhizobium inoculation which was related to the enhancement of FCR activity after nodule formation.In solution cultivation,the red clover roots released a large amount of phenolics when theplants were subject to the Fe deficienct treatment.In addition,the phenolics accumulation in thecalcareous rhizosphere soil of Fe-stressed red clover was also significantly increased.Generally,phenolic compounds have both antimicrobial and growth beneficial effects to mirobes.Therefore,we analyzed the rhizosphere microbial community structure of the red clover plants withdifferent Fe status.The microbial 16S rDNA PCR-DGGE analysis showed that the rhizospheremicrobial community structure was varied with the plant’s Fe nutritional status.Interestingly,thesiderophore producing ability of the microbes in the rhizosphere was stronger in Fe-stressedplants than in Fe-sufficient ones.Moreover,the compostion of the siderophore-producingmicrobes of the rhizosphere soil from Fe-stressed red clover plants could be mimicked byincubating the soil with phenolic root exudates.In addition,the percentage of microbes that canproduce auxin-like compounds was also increased by incubating soil microbial suspension onthe agar plates containing phenolic root exudates.The solution cultivation experimentdemonstrated that the ferric reduction capacity of red clover roots was greatly enhanced bymicrobial auxins.Based on these observations and our previous research,we propose as a modelthat root exudates from Fe-deficient plants selectively influence the rhizosphere microbialcommunity,and the microbes in turn favor plant Fe acquisition by producing siderophores andauxins.2.Contribution of phenolic root exudates to the iron reutilization in root apoplast of redclover （Iron reutilization mechanism）Phenolic compounds secreted by Fe-deficient root cells should pass the root apoplast first.Wefound that removal of secreted phenolics from the root-bathing-Fe solution by sorbing resinresulted in severer chlorosis of new leaves,implying Fe deficiency-induced phenolics secretionmay be involved in the of reutilization root apoplastic Fe.Further analysis shows that phenolicsremoval almost completely inhibited the reutilization of apoplastic Fe in red clover roots,and asa conquence,shoot Fe was significantly decreased but the root Fe was increased,indicating thatthis approach may reduce t root Fe transporting to shoot,and hence stimulated Fe deficiency.Inaddition,phenolic removal also significantly enhanced root ferric chelate reductase activity andproton extrusion,suggesting that the reutilization of root apoplastic Fe is not mediated by protonextrusion or the root ferric chelate reductase.In vitro studies with extracted root cell wallsfurther demonstrate that excreted phenolics efficiently desorbed a significant amount of Fe fromcell walls,indicating that a direct involvement of phenolics in Fe remobilization.Taking alltogether,these results stongly demonstrate that Fe deficiency-induced phenolics secretion is involved in the reutilization of root apoplastic Fe,and should play an important role inimproving shoot Fe nutrition.3.Eeffect of the elevated atmospheric CO₂ level on plant Fe nutrition （Influences ofenvironmental factor）The CO₂ concentration in atmosphere has been being increased annually.Here,we foundthat the elevated atmospheric CO₂ level (800μL L^-1)not only significantly increased thebiomass of tomato cultured in low available Fe （the insoluble ferric oxide as the Fe source）nutrient solution,but also improved the plant Fe nutrient status.Interestingly,although thetomatoes cultured in ambient atmosphere (350μL L^-1CO₂)was more Fe-deficient,their rootFCR activty,proton secretion,sub-apical root hair development and expressions of FER,FRO1and IRT were weaker than those of the plants treated with elevated atmospheric CO₂.Moreover,the root/shoot ratio of tomatoes was also significantly increased by the elevated atmosphericCO₂ treatment as compared with ambient atmosphere treatment.Based on these observations,we suggested that the enhanced Fe-deficiency-induced responses and the increased root/shootratio may be the reasons that the elevated atmospheric CO₂ treatment improved the plant Fenutrient status,and the former may be the major reseason.In addition,the NO levels in rootswere also increased by the elevated atmospheric CO₂ treatment,which may be the reasonleading to the enhancement of Fe-deficiency-induced responses4.Roles of phenolic compounds,auxin and 14-3-3.protein in regulation iron nutritionuptake （Regulation mechanisms of iron uptake）The IAA accumulation in red clover roots were also significantly increased by the Fe-deficienttreatment.Application of TIBA or NPA to the red clover stem,which could decrease IAAaccumulation in root,significantly inhibited the Fe-deficiency-induced FCR activity,protonsecretion,and subapical root hair development,but did not inhibit the root phenolicsaccumulation and secretion,suggesting that IAA should be involve in the regulation of theformer four Fe-deficient responses but not the phenolics accumulation and secretion.In contrast.the Fe-deficient treatment significantly decreased the root IAA-oxidase activity.Interestingly,the phenolics from roots of Fe-deficient red clover inhibited IAA-oxidase activity in vitro,andthis inhibition was greater than with phenolics from roots of Fe-sufficient plants,indicating thatthe Fe-deficiency-induced IAA-oxidase inhibition probably is caused by the phenolicsaccumulation.Based on these observations,we propose a model where under Fe-deficient stressin dicots,an increase in root phenolics concentrations plays a role in regulating root IAA levelsthrough an inhibition of root IAA oxidase activity.This response,leads to.or at least partiallyleads to an increase in root IAA levels,which in turn regulates Fe-deficiency-induced FCR activity,proton secretion,and subapical root hair development.We silenced the TFT7 14-3-3 gene in tomatoes by using VIGS technique As a consequence,the mRNA levels of FER,FRO1 and IRT were significantly reduced.The FCR activity ofFe-deficient treatment was also obviously lower than than that in non-slienced plants.Takingaccout of the FER functions in regulating Fe-deficiency-induced responses,we suggest that theTFT7 14-3-3 protein may be involved in regulating the downsteam Fe-deficiency-inducedresponses by controlling the FER gene expression first.更多还原