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Influence of Inorganic Nitrogen and pH on the Elongation of Maize Seminal Roots

In?uence of Inorganic Nitrogen and pH on the Elongation of

Maize Seminal Roots

ARNOLD J.BLOOM 1,*,JU

¨RGEN FRENSCH 2and ALISON R.TAYLOR 31

Department of Plant Sciences,University of California,Davis,CA 95616,USA,

Cyberonics Europe S.A./N.V.,Belgicastraat 9,1930Zaventem,Belgium and 3Marine Biological

Association,The Laboratory,Citadel Hill,Plymouth PL12PB,UK

Received:8August 2005Returned for revision:28September 2005Accepted:15November 2005Published electronically:22December 2005

Background and Aims Root absorption and assimilation of inorganic nitrogen usually alters rhizosphere pH,but the immediate in?uence of such pH changes on root elongation as well as that of exogenous inorganic nitrogen itself has been uncertain.

Methods A differential extensiometer that monitored on a real-time,continuous basis root elongation in an intact 3-d-old maize plant was developed.Treatments included root media at pH 6á5or 5á6that lacked nitrogen

and ones at pH 6á5that contained 100mmol m –3NH 4+or NO 3à

Key Results Acidifying the root medium from pH 6á5to 5á6nearly doubled the elasticity of the seminal root,but slightly decreased its elongation.Plasticity of the root apex was not detectable in all treatments.The presence of ammonium or nitrate in the medium stimulated elongation by 29%or 14%,respectively.Addition of an osmoticum to the medium had no effect on root elongation in the absence of inorganic nitrogen,but diminished the stimulation of elongation in the presence of ammonium and nitrate.This indicates that these ions or their by-products serve partially as osmolytes.

Conclusions In nutrient solution,root elongation of a maize seedling—even one with ample nitrogen reserves—depended most strongly on exogenous inorganic nitrogen,and less so,if at all,on either the pH of the bulk nutrient solution or the mechanical properties of cell walls.

Key words:Nitrate,ammonium,rhizosphere pH,Zea mays ,acid growth,cell wall elasticity,root growth,nutrient solution,turgor pressure,osmotic potential.

INTRODUCTION

The below-ground environment from which plants extract nutrients and water is highly heterogeneous,both spatially and temporally.For example,inorganic nitrogen concentra-tions in a soil may range a 1000-fold over a distance of centimetres or over the course of hours (Bloom,1997b ).Given such heterogeneity,plants depend on various trop-isms (e.g.gravitropism,thigmotropism,hydrotropism and,perhaps even,chemotropism)to guide root growth toward soil resources (Epstein and Bloom,2005).The physio-logical mechanisms through which these tropisms alter growth remain uncertain,although the ‘acid growth hypo-thesis’is often raised as a possibility (e.g.Chen et al .,2002;Peters,2004).

According to the ‘acid growth hypothesis’,plants regu-late cell expansion through modifying the pH around the cell wall and thereby its extensibility,which increases at low pH (Cosgrove,1999).A large body of evidence supports this hypothesis in shoot coleoptiles or expanding leaves (Rayle and Cleland,1992;Peters et al .,1998;Van Volkenburgh,1999;Kotake et al .,2000;Schopfer,2001;Friml,2003),but results on roots have been less conclusive.Lowering the pH of the medium may either promote root elongation (Edwards and Scott,1974;Evans,1976;Winch

and Pritchard,1999)or have little effect (Bu

¨ntemeyer et al .,1998;Peters and Felle,1999;Walter et al .,2000).The standard approach for examining the in?uence of pH on

cell extension has been to examine tissue segments subjec-ted to severe treatments:for example,in Wu et al .(1996),frozen root segments were abraded with carborundum,thawed and squeezed between two glass slides to remove cell sap;in Tanimoto et al .(2000),lateral roots were killed in boiling methanol;and in Schopfer (2001),the segments were frozen,thawed,and abraded.Such treatments have been deemed necessary because the mechanical properties of cell walls in fresh,turgid tissue could be complex (D.J.Cosgrove,https://www.wendangku.net/doc/3616613752.html,m.).

Rhizosphere pH changes as roots absorb and assimilate inorganic nitrogen;the assimilation of NH 4+strongly acidi?es,whereas absorption of NO 3à

slightly alkalizes the media near the root apex (Smart and Bloom,1998;Taylor and Bloom,1998).These rhizosphere pH changes may be responsible for the differential patterns of root

growth observed under NH 4+vs .NO 3à

nutrition (Bloom,1997a ;Bloom et al .,2002).Alternatively,NH 4+and NO 3àthemselves may be responsible for the root developmental responses (Forde,2002).

To test the acid growth hypothesis in roots and the short-term in?uence of exogenous inorganic nitrogen on root elongation,a new approach was developed that pro-vides measurements of the mechanical properties and elongation of the root apex on a real-time,continuous and non-destructive basis.Here it is reported that,in this device,exposure of a maize seminal root to a more acid medium dramatically enhanced its elasticity,but factors such as the availability of inorganic nitrogen in the medium

*For correspondence.E-mail ajbloom@https://www.wendangku.net/doc/3616613752.html, Annals of Botany 97:867–873,2006

doi:10.1093/aob/mcj605,available online at https://www.wendangku.net/doc/3616613752.html,

For Permissions,please email:journals.permissions@https://www.wendangku.net/doc/3616613752.html,

had a greater in?uence on root elongation than cell wall mechanical properties.

MATERIALS AND METHODS

Maize(Zea mays L.cv.WF9·Mo17)seeds were placed on germination paper(thick,?ne weave,paper towelling) soaked in1á0mol mà3CaSO4for2d and transferred to a0á004mà3light-impervious polyethylene container ?lled with an aerated nutrient solution containing 0á15mol mà3NH4NO3,1mol mà3CaSO4,0á5mol mà3 K2HPO4,0á5mol mà3KH2PO4,2mol mà3MgSO4, 0á2kg mà3Fe-NaEDTA,and micronutrients according to Epstein and Bloom(Epstein and Bloom,2005).The con-tainers were placed in a controlled environment chamber that provided a photosynthetic photon?ux density of 400m mol mà2sà1at plant height for a14h light period at25 C and a10h dark period at15 C.

The next day,a plant whose seminal root was120–180mm in length was placed into an extensiometer[for a black and white illustration of this,see?g.3in Bloom et al. (2002)or,for one in colour,?g.9.5in Epstein and Bloom (2005)].The seedling was supported in the extensiometer by its caryopsis.The seminal root lay against a surface of the extensiometer that was tilted4 from vertical.The side walls of the extensiometer extended outward from the sur-face to form a trough.A nutrient solution?owed down this trough,bathing the root.The solution contained1mol mà3 CaSO4,200mmol mà3KH2PO4and either100mmol mà3 NH4H2PO4,100mmol mà3KNO3,or no nitrogen,and was

adjusted to pH6á5or5á6with KOH.The osmotic potential of these solutions wasà0á082MPa.Their pH was continu-ously monitored throughout an experiment and did not vary>0á2pH units.At the midpoint of an experiment, 68mOsm KCl(y s=à0á14MPa)was added to assess the response of the root elongation to a shift in osmotic poten-tial.The end of a small plastic pipette tip was cut off,a large knot tied in one end of a nylon thread,the free end of the thread passed through the narrow opening of the pipette tip, the tip attached to the root cap with surgical-grade cyano-acrylic glue,and the other end of the thread tied to an arm connected to the shaft of a rotary variable inductance trans-ducer(RVIT;Schaevitz15–60,Pennsauken,NJ,USA). Weights of1á2,2á4,3á6and5á2g were placed on this arm to stretch the root and assess its elasticity plus plasticity and then were removed to assess elasticity alone.Applying a weight of5á2g was approximately equivalent to subjecting the root to an osmotic potential ofà0á14MPa[based on F=P·A,where F=force,P=pressure and A=surface area,and given that the roots had a radius of about0á5mm and an effective cross-section of5%as estimated from measurements of root hydraulic conductance(Frensch and Steudle,1989);and from micrographs of the apex (Bloom et al.,2002)].Five minutes or longer were allotted after addition or removal of weights to permit the elongation of root apex to resume a steady rate(Fig.1).A small piece of a wooden toothpick was glued to the root initially about 14mm from the apex,a part of the root that is no longer elongating(Taylor and Bloom,1998).A nylon thread connected this toothpick to a linear variable differential transducer(LVDT,Schaevitz050DC-D).A two-channel chart recorder logged the output from the RVIT and LVDT. A?at-bed scanner and an image analysis program (Digitize-Pro,Dr Yaron Danon)digitized the chart recorder tracings.Apical root length was taken as the difference in the positions of RVIT and LVDT.After smoothing the data using a Gaussian kernel to compute local weighted averages,the elongation rate was calculated from the changes in length over time through numerical differenti-ation(Mathcad12,Mathsoft).An FIR(?nite impulse response)high-pass?lter(Mathcad12,Mathsoft)was also used to assess the sudden shifts in length when weights were added or removed.An ANOVA(General Linear Model;CoStat,CoHort Software)was used to test for signi?cant differences among means(P<0á05). Neumann used a similar approach to examine the in?uence of NaCl(Neumann,1993),polyethylene glycol (Chazen and Neumann,1994)and nutrient supply(Snir and Neumann,1997)on leaf extension,but employed a single transducer.Consequently,his extensiometer mon-itored the leaf as a whole and did not isolate the changes in a speci?c region.In the present study,to monitor the elongation of just the root apex and to eliminate any signal generated from movement of the whole plant when weights were added,the difference between two transducers was monitored.

To determine segment mass,NH4+and NO3àconcentra-tions and osmotic potential along the maize root,individual seedling roots were exposed to the various nitrogen treat-ments for18–24h,and gently blotted dry before they were 14

Time (h)

(

)

F I G.1.Examples of changes in the length of a root segment versus time for3-d-old maize seedlings.This segment,which was between the apex and a point initially14mm from the apex,contained the apical meristem and elongation zone.The root was exposed to1mol mà3CaSO4,200mmol mà3 KH2PO4and either100mmol mà3NH4H2PO4(NH4+)or100mmol mà3 KNO3(NO3à),or no nitrogen(No N).The solutions were maintained (mean6range)at pH5á660á2(No N,pH5á6)or pH6á560á2(NH4+, NO3àand No N,pH6á5).The labelled arrows indicate the times for the NH4+treatment at which various weights(designated in grams)were added near the tip.The same series of weights were applied to the NO3àand nitrogen-free treatments at the times indicated by the shorter arrows.

868Bloom et al.—Root Elongation and Rhizosphere NH4+,NO3àand pH

rapidly(<2s)frozen on a thermoelectric cold-plate mounted under a dissecting microscope.Axial sections of1mm length were made with a?ne razor blade at1-mm incre-ments from1to10mm from the apex along each of ten roots.Root sections from each location were oven-dried and weighed to determine dry mass per unit length.Other root sections from each location were pooled and collected in Eppendorf tubes containing1á5ml of1mol mà3CaSO4, which was adjusted to pH3with H2SO4.These sections were sonicated for30min and then centrifuged.The super-natant was withdrawn and analysed for NH4+and NO3àas described below.There were at least three replicates for each N-treatment.Root NH4+and NO3àcontents were expressed per segment water volume based on root radius measurements at each location.Two other frozen root sections from each location were immediately placed after excision into the sample chamber of a Wescor5100 thermocouple psychrometer(Logan,UT,USA)to assess osmotic potential.

To analyse NH4+concentrations in the samples,a?uori-metric method based on the reaction of NH4+with o-phthalaldehyde(OPA)was modi?ed(Goyal et al., 1988).An autosampler(Shimadzu Sil9-A,Japan)injected a50mm3sample into the?ow of a buffer solution consisting of126mol mà3K2HPO4,74mol mà3KH2PO4, 5mol mà3OPA and0á39dm3mà3b-mercaptoethanol.The stream circulated for several minutes through a cabinet controlled at64 C to optimize the NH4+-OPA reaction. The NH4+-OPA in each sample was quanti?ed using a?uor-escence detector(Shimadzu RF-551,Japan)set at410nm excitation and470nm emission.The time from sample

injection to peak detection was3á4min with a pump?ow rate of2cm3minà1.

Analysis of NO3àwas conducted via HPLC(Thayer and Huffaker,1980).Samples of50mm3were injected into a stream of35mol mà3KH2PO4(adjusted to pH3á0with H3PO4)before passing into a100mm·4á6mm column packed with anion exchange resin(Partisil Sax 10mol mà3;Whatman Laboratory,USA).The absorbance of column eluent was monitored at210nm.The time from sample injection to peak detection was1á8min.

RESULTS

Elongation rates of the seminal root after placing a3-d-old maize seedling into an extensiometer equipped with two high-resolution position transducers became uniform in about1h(data not shown)and remained so for several hours(Fig.1).These rates ranged from1á5to2á2mm hà1 (Figs1and2),values similar to those reported for seminal roots of well-watered maize growing in vermiculite(Sharp et al.,1990),in solution culture(Frensch and Hsiao,1994; Taylor and Bloom,1998;Winch and Pritchard,1999)or along germination paper(Bloom et al.,2002).Recovery to steady rates required<5min after addition or removal of weights and<15min after the addition of68mOsm KCl (Fig.1).These?ndings indicate that the procedures used were benign and did not signi?cantly damage the root. Elongation of the seminal root in the nitrogen-free nutrient solution was slightly faster at pH6á5than at pH5á6(Fig.2).Providing100mmol mà3NH4+or 100mmol mà3NO3àin the nutrient solution at pH6á5 stimulated elongation by29%or14%,respectively,in comparison with the nitrogen-free solution at the same pH(Fig.2).The addition of68mOsm KCl had little effect on elongation in the nitrogen-free solutions,but decreased the rates under NH4+or NO3àby7%and18%,respectively (Fig.2).

The stretching of the seminal root was proportional to the weight applied(Fig.1)and the root fully recovered when the weights were removed(Fig.3).This shows that the stretching response of the root apex was predominantly elastic,not plastic.Root elasticity in the more acidic solution(pH5á6)was double that in the more neutral solu-tion(pH6á5).In the neutral solution,root elasticity was insensitive to nitrogen treatment(NH4+,NO3àor nitrogen-free)and the presence or absence of68mOsm KCl(Fig.3). The mass of root segments were slightly higher in the more mature root zones of plants receiving100mmol mà3 NH4+than in those receiving100mmol mà3NO3à(Fig.4). Concentrations of NH4+near the root apex were slightly higher in the plants receiving100mmol mà3NH4+than in those receiving a nitrogen-free medium(Fig.4A). By contrast,NO3àconcentrations were negligible in the plants receiving nitrogen-free medium,but increased to over14mol mà3in the more basal parts of the root in the plants receiving100mmol mà3NO3à(Fig.4B).The overall osmotic potential of the apex(mean6s.e.)did not vary signi?cantly among the nitrogen treatments 0

Salt treatment (mOsm)

(

–

)

F I G. 2.Root extension rates for3-d-old maize seedlings.The root was exposed to1mol mà3CaSO4,200mmol mà3KH2PO4and either 100mmol mà3NH4H2PO4(NH4+)or100mmol mà3KNO3(NO3à),or no nitrogen(No N).The solutions were maintained(mean6range)at pH5á660á2or pH6á560á2and at the two different osmotic levels (0or68mOsm KCl).Shown are the mean6s.e.(n=6different plants for each of the four N treatments;each plant was exposed to both osmotic levels).Bars with the same letters did not differ signi?cantly(P>0á05).

Bloom et al.—Root Elongation and Rhizosphere NH4+,NO3àand pH869

(–1á1360á06MPa for the NH 4+,à1á0560á05MPa for the NO 3à

,and à1á1760á07MPa for the nitrogen-free),and the values observed were similar to those previously reported for maize root apices (Sharp et al .,1990).

DISCUSSION

The acid growth hypothesis of roots,although well-known,has been subject to only limited experimental testing with only seven studies that have focused on this topic (Edwards

and Scott,1974;Evans,1976;Bu

¨ntemeyer et al .,1998;Peters and Felle,1999;Winch and Pritchard,1999;Tanimoto et al .,2000;Walter et al .,2000).Of these studies,several only examined elongation of root segments

(Edwards and Scott,1974;Bu

¨ntemeyer et al .,1998;Tanimoto et al .,2000)or subjected the roots to relatively large pH changes [pH 6á5–4á0(Evans,1976);pH 7á0–3á4(Winch and Pritchard,1999)].The present results for root apices of intact plants demonstrate that even moderate changes in rhizosphere pH (between pH 6á5and 5á6)can dramatically in?uence the elasticity of the apex (Fig.3),but that this may not in?uence the elongation of this region (Fig.2).By contrast,the presence of exogenous inorganic nitrogen signi?cantly enhanced elongation (Fig.2),even though the seedlings had ample nitrogen reserves in the caryopsis to support growth for several more days (Bloom et al .,2002).

The meristem and transition zones (Baluska et al .,1996)near the root apex differ from more mature root zones in that they lack fully differentiated phloem tissue.Import of carbohydrates or some nutrients (e.g.K +)from more mature tissues into the root apex may depend on diffusion through the symplast (Bret-Harte and Silk,1994b )or may be enhanced by pressure driven ?ow from sieve tube ele-ments (Boyer and Silk,2004;Gould et al .,2004).Never-theless,little of the nitrogen absorbed in the maturation zone moves toward the apex (Siebrecht et al .,1995;Walter et al .,2003);therefore,the nitrogen required for cell division and isotropic cell expansion may derive prim-arily from nitrogen that the apical zones themselves absorb and assimilate.

The stimulation of root elongation in the presence of

exogenous NH 4+or NO 3à

is an appropriate response with ecological implications (Bloom,1997a ).Plants predo-minantly obtain nitrogen through root absorption of NH 4+and NO 3

àfrom the soil solution (Bloom,1997b).Spatial and temporal availability of soil inorganic nitrogen is highly heterogeneous,as mentioned above.To survive under such heterogeneity and under competition from soil microorganisms,plant roots must be in the right place at the right time.Appropriately,roots proliferate in soil regions that are nitrogen-rich (Hackett,1972;Drew,1975;Grime et al .,1986;Sattelmacher and Thoms,1989;Bingham et al .,1997;Robinson et al .,1999;Zhang et al .,1999).Speci?cally,proliferation of lateral roots seems critical for exploiting nitrogen-rich regions (Bloom et al .,2002;Forde,2002).The phenomenon observed here—acceleration of seminal root elongation by exogenous inorganic nitrogen—would rapidly position the mature zones of seminal roots,from where lateral roots emerge,adjacent to nitrogen-rich soil regions.The presence of NH 4+stimulated root elongation (Figs 2and 3)and accumulation of root biomass (Fig.4)to a greater extent than that of NO 3à.This is consistent with other studies (Bloom et al .,1993)and may re?ect that assimilation of NH 4+to glutamine consumes the equivalent of about 2ATPs per NH 4+,whereas assimilation of NO 3àto glutamine consumes the equivalent of about 12ATPs per NO 3à(Bloom et al .,1992).In the carbohydrate-limited apical meristem (Bret-Harte and Silk,1994a ),the lower energy requirement for NH 4+assimilation may permit cells to maintain higher elongation rates and to accumulate more biomass.

Diminishing the osmotic potential of the nutrient solution from à0á08to à0á22MPa by the addition of 68mOsm KCl had no effect in the nitrogen-free treatments,but depressed

root elongation under NH 4+and NO 3à

nutrition (Fig.2).In maize,apical zones of the root rapidly absorbed NH 4+and NO 3à(Taylor and Bloom,1998).Most of the NH 4+

absorbed promptly disappeared from the tissues (Fig.4A),presum-ably,as it was assimilated into amino acids (Bloom et al .,2002).Some of these amino acids may serve as meta-bolically benign osmolytes to support cell expansion in the elongation zone (Rhodes et al .,2002).By contrast,a

portion of the NO 3àabsorbed remained as free NO 3à

within the apical zones (Fig.4B),providing another metabolic-ally benign osmolyte (up to 29mOsm or à0á063MPa)to

0·3

0·2

0·1

0·0

Salt treatment (mOsm)

D i s t o r t i o n (m m )

F I

G .3.Immediate changes in length between the root apex and a point initially 14mm from the apex for 3-d-old maize seedlings when weights (5á2g)were added to stretch the roots and when they were removed to permit recovery.Five minutes or longer were allotted after addition or removal of weights to permit the elongation of the root apex to resume a steady rate.The roots were exposed to 1mol m à3CaSO 4,200mmol m à3K

H 2PO 4,and

either 100mmol m à3NH 4H 2PO 4(NH 4+)or 100mmol m à3

KNO 3(NO 3à),or

no nitrogen (No N),and either 0or 68mOsm KCl.The solutions were maintained at pH 5á660á2or pH 6á560á2(mean 6range).Shown are the mean 6standard error (n =6different plants for each of the four treatments).Bars with the same letters did not differ signi?cantly (P >0á05).

870

Bloom et al.—Root Elongation and Rhizosphere NH 4+,NO 3à

and pH

support expansion (Bloom,1996;Bloom,1997a ;McIntyre,2001).The addition of 68mOsm KCl to the nutrient solution depressed root elongation possibly because it counteracted the osmotic effects of the stored amino acids and NO 3à.Although the osmotic potential resulting from the amino acids,NO 3àor KCl was small in comparison to the total osmotic potential of the apex (1á1MPa),these metabolic-ally benign osmolytes were probably not distributed evenly throughout the root,but concentrated in the more metabolically active compartments (Aspinall and Paleg,1981).

It is unlikely that the effects of 68mOsm KCl on the

plants receiving NH 4+or NO 3à

were speci?c to the ions K +and Cl à.The high-af?nity transport systems for NH 4

+and NO 3

à,which predominate at 100mmol m à3

,are insens-itive to the presence of K +(Scherer et al .,1984;Bloom and Finazzo,1986;Smart and Bloom,1988;Bloom and Sukrapanna,1990)or Cl à(Glass et al .,1985;Bloom and Finazzo,1986;Deane-Drummond,1986).In previous experiments on maize roots,mannitol and KCl were indis-tinguishable in their effects on cell turgor and root elongation (Frensch and Hsiao,1995).Neither elasticity nor plasticity of the root apex changed with the addition of 68mOsm KCl (Fig.3),and thus cell wall properties seemed unresponsive to additions of these ions.

In Lockhart’s model (Lockhart,1965),the rate of cell expansion depends upon its extensibility times its turgor pressure in excess of a certain threshold [RGR =m ·(P c –Y ),where RGR is cell relative growth rate,m is volumetric extensibility,P c is cell turgor pressure and Y is yield threshold turgor pressure].Cell wall elasticity and plasticity were assessed by rapid shifts in root length produced by the addition or removal of weights.Cell wall elasticity at pH 5á6was nearly doubled from the value at pH 6á5;plasticity was negligible under all treatments (Fig.3).Such shifts might derive from either stretching cell walls or altering cell turgor.For example,if cells growing at pH 5á6had less turgor than those at pH 6á5,then stretching could occur more easily,independent of cell wall extensibility.

Winch and Pritchard (1999)examined maize at the same age as the plants in the present study and grown under similar conditions.They found that cell turgor pressures were similar in roots exposed to pH 7á0and 3á4.Presum-ably,cell turgor pressures remained unchanged over the narrower pH range of 6á5–5á6used in the present experi-ments.Therefore,the responses to mechanical perturbation that were observed were derived primarily from changes in cell wall elasticity of the root apex.That the root apex responds in primarily an elastic fashion seems reasonable.Cell walls are reinforced in the maturation zone,a region basal to the one examined in the present study;thus,the maturation zone presumably would demonstrate more of a plastic response with irreversible increases in length.Elongation of the root apex was compared at pH 6á5and 5á6,a pH range found in the soils around Davis,California (DeClerck and Singer,2003).Maize yields are relatively insensitive to soil pH in this range (McLean and Brown,1984).A change from pH 6á5to 5á6doubled the elasticity of maize roots (Fig.3),but slightly slowed root growth (Fig.2).These results indicate that cell wall properties alone do not regulate root elongation.

Some researchers have questioned whether cell wall mechanical properties can be measured on live,turgid tis-sue because walls suffering both strain in all directions due to turgor and strain in a unidirectional vector due to the addition of weights might behave in a complex manner.Nonetheless,it was found that roots of intact plants exhib-ited simple mechanical properties;elongation was linear with the strain applied (Fig.1)and recovery from the strain was complete (Fig.3).These results permit a simple inter-pretation.

Proton pumps in the apical zones of roots generate what are known as ‘growth currents’that have an important role in the determination and regulation of root polarity (Weisenseel et al .,1979;Miller and Gow,1989).A previous

812160

406080

Distance from apex (mm)

Mass (mg m –1)C o n c e n t r a t i o n (m o 1 m –3)

F I

G .4.(A)Tissue concentrations of N

H 4+or NO 3à([NH 4+]or [NO 3à]in mol m

à3

based on the per segment water volume)and (B)mass of root segments (mg dry mass m à1

)at various distances from the apex of a maize seminal root for plants receiving nutrient solutions that contained either 100mmol m à3NH 4

+(NH 4+),100mmol m à3NO 3à(NO 3à

),or were nitrogen-free (No N)for 18–24h.Given are the means 6s.e.(n =3–6different plants of each treatment)

with small error bars incorporated into the symbols.

Bloom et al.—Root Elongation and Rhizosphere NH 4+,NO 3à

and pH

871

study(Taylor and Bloom,1998)found that maize roots exposed to either100mmol mà3NH4+,100mmol mà3NO3à, or nitrogen-free media pumped suf?cient protons to main-tain the root surface in the elongation zone at least0á4pH units more acidic than the bulk solution;the greatest pH differentials,however,were in the nitrogen-free treatment. These results indicate that the differences in root elongation observed in the current study were not simply that the vari-ous nitrogen treatments produced different pHs in the elongation zone.

Walter et al.(2003)reported that elongation of maize roots was faster in pure water than in a nutrient solution containing both NH4+and NO3à,a?nding contradictory to those presented here.This solution,however,provided NH4+at3á85mol mà3,a concentration several times higher than the highest levels measured in agricultural?elds in Davis,California(Jackson and Bloom,1990),and the maize roots grown in this solution accumulated high levels of free NH4+(40m mol gà1)in the meristems.For compar-ison,in the present study,the nutrient solution used contained0á1mol mà3NH4+,and free NH4+concentrations in the root meristems were<6á7m mol gà1(Fig.4A,calcu-lated on the basis that root segments were87%water).High accumulations of free NH4+in tissues are toxic because they dissipate pH gradients in mitochondria and plastids (Epstein and Bloom,2005).Thus,NH4+toxicity might explain the differences between the results of the two studies.

In summary,the results of the present study indicate that in well-watered maize plants,exogenous inorganic nitrogen more than pH or cell wall elasticity or plasticity in?uences the elongation of the root apex.

ACKNOWLEDGEMENTS

We thank T. C.Hsiao for the use of equipment,and T.C.Hsiao and Nigel Crawford for their comments on the manuscript.This work was supported in part by the USDA NRICPG Grant2000-00647and National Science Founda-tion Grants IBN-99-74927and IBN-03-43127to A.J.B.

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