《大量元素氮》PPT课件.ppt

Plant Nitrogen Assimilation and Use Efficiency,What did you learn from the review?,Signaling Network in Sensing Phosphate Availability in Plants,第四讲 大量元素（氮）,Section 1 General,In general,0.3-5.0%of dry weight.Varying among different species,organs,growth stage.,Increasing with Application of N Fertilizer,especially leaves!,Species:Legumes Cereals,Maize Wheat Rice,Organs:Grains Leaves Stems,Growth Stages:Tilling Seedling(Rice),Vegetative organ Reproductive Organ,Forms of nitrogen in plant:NH4+、NO3-，Low molecular organic compounds;High molecular organic compounds.,Vital Element!,Increasing crop yield and improving quality,NH4-N and NO3-N are the major sources of N.Plant can also absorb NO2 N,N2,aa and urea.,NO3-N uptake is an active process,Assimilation pathway of NO3-in root,Many affecting factors:Light,Temperature,pH,and so on.,Which are tightly related to nitrate reductase activity.,Nitrate Assimilation,Two separate enzymes:Nitrate reductase（硝酸还原酶,NR）reducing nitrate to nitrite;Nitrite reductase（亚硝酸还原酶,NiR）reducing nitrite to ammonia（氨）.,Nitrate reductase is a complex enzyme,NR contains several prosthetic groups:FAD,cytochrome,and molybdenum.,NR is localized in the cytoplasm and requires either NADH or NADHP as an electron donor.,NR has a half-life of only a few hours.,NR is present only in low levels in plant not receiving nitrate,but can be reduced within a few hours by the addition of nitrate.,NiR has a low molecular weight and is associated in leaves with chloroplast and in roots most likely with proplastide(前质体).,As a rule,nitrite rarely accumulated in intact plants under normal conditions,because NiR is present at much higher levels than NR.,In most plant species both roots and shoots are capable of nitrate reduction.The proportion of reduction carried out in each location depends on various factors.,Assimilation of ammonium,Absorption of ammonium,Absorption form:NH4+or NH3,After NH4+-N was absorbed by roots,it will be rapidly assimilated into amino acid and amides,then be transported to shoots.,High NH4+concentration is toxic to plants,which is related to the rapid permeation of ammonia across biomembranes.,Amides is the form storing nitrogen.,Urea can be taken up directly by the roots or aerial parts,Two opinions about the assimilation of urea:,Urea is rapidly hydrolyzed into ammonium and dioxide carbon by enzyme urease either in roots or after translocation to the shoots;,Urea is directly taken up and assimilated,Simplified sketch showing urea generation,transport and degradation in plant cells.,Urea is taken up from the environment through HAT and LAT.Urea generating enzymes are(1)arginase(精氨酸酶),(2)agmatinase（胍丁胺酶）,(3)canavanine hydrolase（刀豆氨酸水解酶）,and(4)amidinohydrolases（脒基水解酶）.Urea could be compartmentalized between cytoplasm and vacuoles by tonoplast-targeted transporters such as TIPs and active urea transporters.The pathway for urea movement out of mitochondria is unknown.Cytosolic urea can be hydrolyzed by urease to ammonia,which can be assimilated by glutamine synthetase.,Rhizosphere:pH changes,NH4+assimilation:pH(acid)-often whole root surfaceNO3-assimilation:pH often(alkaline)-sometimes in patches;may be also be pH patches elsewhere on same rootMaize(in soil&agar+indicator)NO3-NH4+low NO3-,Marschner&Rmheld(1983),NH4+amino acids,etc+H+(acidifying)-H+effluxed(rhizosphere pH:e.g.to pH 4)NO3-+8H amino acids,etc+OH-(alkalizing)-OH-neutralized by organic acid synthesis;leading to accumulation of RCOO-biochemical strategy,and/or:neutralized by net H+influx biophysical strategy:rhizosphere pH:e.g.to 6.5-balance between the 2 strategies for NO3-depends on external NO3-concentration,plant type,etc,Nitrogen assimilation,Nitrogen Deficiency symptom,Nitrogen deficiency is charactered by a poor growth rate：A：The plants remain small；B：The stem have a spindly appearance；C：The leaves are small and the older ones often fall premature；D：The roots growth is affected，in particular branching is restricted。,Plants suffering N deficiency mature earlier，and the vegetative growth stage is often shorted。,N deficiency in cereals is charactered by poor tiller；the number of ears per unit area and also the number of grains per ear reduced；The grains are small；yield decreased。But often relatively high protein content。,大麦：下部叶片淡黄，中部叶片叶尖发黄并逐渐向叶基部扩展，新叶保持绿色而挺直。,玉米：下部叶尖发黄，逐渐沿中脉扩展成倒V字形，中脉发红，中部叶片颜色淡绿。,菠菜：老叶几乎全部黄化，新叶相对留有一些绿色,菜豆：叶色变黄，干枯脱落,番茄,过量 正常 缺氮,过量时植株徒长，株型呈倒三角形，生长点所形成的花芽多发展为乱形果,缺氮的株型呈正三角形，结果差，产量低,Section 2Molecular and physiological aspects of nitrate uptake in plants,Most plant species are able to absorb and assimilate NO3,NH4+,urea and amino acids as nitrogen sources,but varies from species to species.In general,most crop plants prefer a mixture of ammonium and nitrate.Optimal growth of tomato roots occurs in soils with a ratio of nitrate to ammonium of 3:1 and is inhibited if the concentration of ammonium is too high.In a typical aerobic agricultural soil,both nitrate and ammonium are present,but nitrate is the major form.,1.Availability of nitrate,When provided with unlimited supplies of NO3,root and shoot NO3 concentrations can reach up to 100mM,most of which is stored within the vacuole.Cytoplasmic nitrate concentrations are somehow held constant,perhaps by the competing processes of influx,efflux,xylem and vacuolar loading and nitrate reduction.,(1)Reduced to amino acids;(2)Undergo efflux out of the cell;(3)Stored in the vacuole;(4)Loaded into the xylem and transport to the shoot.,2.Transport mechanisms,In root,The energy for NO3 uptake is provided by the proton gradient or proton motive force(pmf).,NO3 uptake is mediated by a 2H+/1NO3 symport mechanism for both the high and low affinity NO3 transporters,Roots possess at least three NO3 transport systems:Constitutive high affinity transport systems(cHATS),are characterized by low values of both Km and Vmax(typically 620 M and 0.30.82mol g.h1,respectively).High affinity transporters(iHATS)with higher Km and Vmax values(typically 20100M and 38 mol g.h1,respectively)are induced within hours to days of exposure to NO3-.Constitutive low affinity transporters(LATS),which can significantly contribute to nitrate uptake at concentrations above 250 M,fail to saturate at NO3 concentrations as high as 50mM.Thermodynamic evaluations demonstrate that NO3 uptake by LATS is also active.The cHATS and iHATS activities correspond to mechanism I and LATS to mechanism II for the uptake of nitrate.,A 高亲和力系统的硝酸盐吸收表现为米氏动力学特征B 低亲和力系统的硝酸盐吸收表现为非饱和的动力学特征,3.Physiological regulation,Both high affinity uptake systems(cHATS and iHATS)are upregulated in response to nitrate.,iHATS is known to be induced by NO3 or NO2.In Klondike barley,the fully induced iHATS flux was approximately 30 times higher than that resulting from the cHATS;while in CM72 barley,it is 10 times.This regulation is important for coordinating root uptake with shoot demand for nitrogen during the growth cycle of the plant.,The iHATS activity typically overshoots plant demand for NO3 uptake when first induced,and is rapidly downregulated after the initial exposure to NO3.Several nitrogen forms,including NO3,NH4+and amino acids,may participate in this downregulation,which can occur at the mRNA level.This regulation is important for coordinating root uptake with shoot demand for nitrogen during the growth cycle of the plant.,Two families of nitrate transporter genes have been identified to date:the NRT1 and NRT2 families.These encode cotransporters can be induced by nitrate,but play distinct roles in nitrate uptake and have no sequence similarity.,4.Molecular genetics and physiology,Table 1.NRT1 family of nitrate transporters,Table 2.NRT2 family of nitrate transporters,野生型吸收氯酸盐，并将之还原为有毒的亚氯酸盐离子，从而使植株退绿，而硝酸盐吸收缺陷型不能吸收氯酸盐，因而保持绿色。,The role of the CRNA NRT2 family was first uncovered in the chlorate-resistant mutant crnA of Aspergillus nidulans(曲霉素).Nitrate uptake in Aspergillus is nitrate-inducible and has an apparent Km of 200M.,CRNA is located in a gene cluster that includes the NR and NiR genes，which encodes a hydrophobic protein with 12 putative,membrane spanning regions typical of cotransporters.,The mRNA transcripts of these genes are found primarily in roots,are induced by nitrate,and are downregulated by reduced forms of nitrogen such as NH4+and glutamine.Levels of NRT2 mRNA correlate with nitrate uptake rates in both Nicotiana species and Arabidopsis.,The NRT2 family of genes encode nitrate-inducible,high affinity nitrate transporters.,Three NRT2 genes were found that encode high affinity nitrate transporters with differing specificities.Nrt2;1 encodes a nitrate/nitrite-bispecific transporter.Nrt2;2 encodes a nitrate-specific transporters.Nrt2;3 has been found that is not part of the cluster.All three genes are nitrate-regulated,but Nrt2;1 and Nrt2;3 are more substantially induced by nitrite.,When plants are shifted from media containing ammonium with no nitrate to media containing 25mM nitrate,CHL1 mRNA levels increase dramatically 30min after treatment,peak at 12h and level off to a high steady state level after 8h.This response is very similar to NR observed in roots of nitrate-induced plants.,The first member of the NRT1 family was identified in a chlorate-resistant mutant of Arabidopsis called chl1.CHL1 gene expression is nitrate-inducible and occurs primarily in roots.,CHL1 transporter is a component of the low affinity nitrate uptake system.Two NRT1 genes have been isolated from tomato,one is constitutively expressed and the other nitrate-inducible.The latter could correspond to CHL1 in Arabidopsis.CHL1 participates in both low and high affinity uptake.,Table 1.NRT1 family of nitrate transporters,Interactions between AtNRT2 and AtNAR2.1(also known as AtNRT3.1)form a 150-kDa plasma membrane complex,thought to constitute the high-affinity nitrate transporter of Arabidopsis roots.,New Phytologist,2012,194:724731,Predicted functions for the rice NAR2/NRT2 nitrate transporters,1)NO3-induction2)Freedback repression Glutamine,NH4+,3)Diurnal regulation Uptake rate peaks during the light period and reaches a minimum in the dark4)Spatial and developmental regulation NRT2 genes are expressed more strongly in roots than in aerial.,5 Regulation,In plants,the expression of NR,NiR,and HANT gene expression is co-regulated with respect to the nitrogen source,the intracellular amounts of reduced-nitrogen compounds,light,hormones,and the carbon status,6 Nitrate assimilation,A:NR单体有3个结构域,分别与钼辅助因子，血红素和FAD结合。B:NR的带壮结构图，黑色为钼辅助因子，紫色为血红素，蓝色为FAD,NR基因表达的调控A:NR mRNA在大豆根和叶中的表达受现有的硝酸盐影响（15 mM 硝酸盐处理硝酸盐匮乏条件下7d的大麦苗）B:有硝酸盐供给条件下NR mRNA表现为昼夜节律变化,NRT1.1 and NIA1 Are Up-Regulated in Various NR-Deficient Mutants,A large proportion of nitrate(NO3-)acquired by plants from soil is actively transported by NRT families of NO3-transporters.There are three families of NO3-transporters NRT1,NRT2(or NAR2/NRT2):Most NRT1 members are low-affinity besides NRT1.1(CHL1);NRT2 members are high-affinity,some require a partner protein(NAR2);CLC members amediates nitrate accumulation in the vacuole.In Arabidopsis,the NRT1 family has 8 members,NRT2 family has 7 members All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues.There is significant separation between NRT2 genes fromdicots and grass species.,Mini-summary,Section 3Molecular and physiological aspects of ammonium uptake in plants,1 Ammonium uptake,Many plants do not grow well with NH4+as its sole source of N,probably reflecting its adaptation for growth in aerobic soils,where NO3 is the main form of inorganic N,and NH4+concentrations rarely exceed 50 M.,Significant leakage of NH4+occurs from root cells,even when NO3 is the N source,so that an uptake system with a high affinity for NH4+is important for conserving valuable N supplies.NH4+transport is also important for a variety of processes in the shoot,which include unloading of NH4+from the xylem,absorption of atmospheric NH3 and retrieval of photorespiratory NH3 released into the leaf apoplast.,The kinetics of NH4+uptake in plants are biphasic,being separable into a high-affinity system(HATS)and a low-affinity system(LATS).The LATS only becomes evident at external NH4+concentrations above 1 mM.,2 Kinetics of NH4+Uptake,The high-affinity system displays typical Michaelis-Menten kinetics,while the LATS has linear(non-saturable)kinetics,consistent with it being mediated by a passive transport system such as a channel.The affinity of the HATS estimated Km values for NH4+in the range 20360 M for plant species.It is now generally accepted that NH4+,rather than the unprotonated species NH3,is the transported form.,The importance of the H+gradient in driving NH4+influx by the HATS(H+/NH4+co-transporter)in rice roots has been demonstrated using ATPase inhibitors.The net uptake of any ion is the result of the opposing processes of influx and efflux,and under some conditions it is known that NH4+efflux can have a significant influence on net NH4+uptake.Rates of NH4+efflux are positively correlated with the cytosolic NH4+concentration.,When Arabidopsis seedlings were grown on NH4+-rich medium(1 mM NH4+),the rate of NH4+efflux was estimated to be as much as 35%of the influx rate.,Possible mechanisms for high-and low-affinity NH4+uptake by Arabidopsis roots.,a,Monomer with adjacent CCT(substrate channel amino acids,red;permissive amino acids,green;CCT-stabilizing amino acids,yellow;D198,orange;adjacent CCT,purple).b,Proposed interactions of the CCT,within and between monomers.Monomer A,yellow;Monomer B,blue;hydrogen bonds,dashed lines.,Nature 446,195-198,2007,HATS for NH4+(but not the LATS)is highly regulated according to the N nutrition of the plant.For example,transferring Arabidopsis plants from high NH4NO3 to low led to a 12-fold increase in HATS activity,and when N-depleted plants were resupplied with NH4NO3,the NH4+influx rate declined rapidly.In the same experiments,the LATS activity was independent of the external NH4+supply and the plants N status.,3 Regulation of NH4+Uptake,Perhaps the very high-affinity HATS is synthesised or activated only when the external NH4+concentration is very low or zero.,NH4+influx in Arabidopsis roots is diurnally regulated,reaching a maximum at the end of the light period and declining markedly with the onset of darkness.This suggests that the C status of the plant may have a regulatory effect on HATS activity that is somehow transmitted from shoot to root.,The Arabidopsis AtAMT1.1 gene was the first NH4+transporter gene to be cloned from higher plants.Subsequently,two closely related AtAMT1.2 and AtAMT1.3 were isolated.The completed Arabidopsis genome sequence reveals that there are two further members of the AMT1 family(AtAMT1.4 and AtAMT1.5)A sixth gene(AtAMT2),which is more distantly related to the other AMT genes,has been identified.The AMT genes encode hydrophobic proteins of 475514 amino acids.,4 Structure of the AMT Family of NH4+Transporters,Phylogenetic tree for AMT transporters and selected membe