The Fe-deficiency Induced Responses And Its Regulatory Pathway In Strategy Ⅰ Plants

Iron deficiency is one of the most deleterious factors limiting crop production in the world.Innongraminaceous monocots and dicots,the so called Strategyâ… plants,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.