Plant Physiol. ( 1966) 41, 173-180

Transport of Amino Acids to the Maize Root'Ann Oaks2

Department of Biological Sciences, Purdue University

Received May 21, 1965.

Situnz,ary. Wh-lienl 5-mmi111 miiaize root tips were excised and placed in ani inorganiic saltssoltution for 6 lhours, there \\as a loss of alcohol-iinsoluble nitrogeni. The levels of threon-ine, proline, valine, isoleucine, letucine, tyrosine, phenvlalaniine, and lysine in the alcoholsoluble fraction were severely redutced, whereas those of gltutamlate. aspartate, ornithinie.anld alanine were scircelv affec.led. There was a 4-foid inicrease in the level of y-amino-butyrate. Those amilino acids \\hose \v nthesis appeared to be (leficienit in excised root tipsalso showe(d poor incorporationi of acetate carbon. In addition, the results show thatasparagine and the aminlo acid.s of the nieutral alnd( basic fractioni w\ere preferentiallytransported *to the root tip reggion. T'he results therefore sugges,it that the synthesis ofcertaini amino acids in the root til) regioni is restrictedl. ani(l that thi. re(quireimienit foram'11ino acids in the growing regioni coultd( regulate the flow of amino acids to the root tip.

The movemient of materials in plants is a well-docunmented pheniomenoil which has recently been re-viewed I)v Kutrsaniov (13). The general theoryevolved in(licates that certain tissues, i.e. a leaf par-ticipating in photosynlthesis or the endosperii of acereal seed, serve as a source for mlaterials requiredby a growinig region. It has been shown in systemiisuitilizinig the mlovemcnt of the products of photosyn-thesis (1, 10, 13) that suigars are the principal con-ponents traslsported out of the leaf. It has been as-

sumed that, after their arrival, these transportedsuigars are extelnsively metabolized to viekl all thecell conistitueents. Further support for- this idea hasbeen derive(d fromii observations conicerninig amiinioacid interconiversionis. principally the extensive me-tabolism of glutamiiic and aspartic acids in growingtissues (6, 29), anid the ubiguitv of transanminase re-actionis (28, 29). However 3 types of experimentsare inconsistent with the notion of an extensive miie-tabolism of glucose or interconversioni of amiinlo aci(lsin the growing region.

A) When maize embryos are detached fronm theirendospermis anid grown tunder sterile conditionis, thereis, initially, a dlecline in the soluible aimiino aci(d coii-tent aln(l anl insignificant increase in embryo l)rotein.This initial deficiencv is overconme bv the a(lditionof anl ap)propriate n)ixture of amino acids tro thleCu1ltuire mediuim. (21 ). If glutcose were the major pre-cursor for the amino acids and enmbrvo protein, thisiniitial lag in protein synthesis Nvoul(d niot be expecte(l.

J ) It is usually folnd that C''-laheled aminoacids are recovered fromii plant tissues mailnly in theforni in wvhich tlhey were administered (9, I.5). Glui-

Research sponsored by contract AT-11-1-330 withthe United States Atomic Energy Commission.

2 Present address: Reseaivh Unit in Biochemistry,Biophysics, and Molecular Biology, McMaster University,Hamilton, Cana(la.

173

tamic and aspartic aci(ds, therefore. represent the ex-ception rather than the rulle for the i(lea of an ex-tensive interconiversion of anmino acids.

C) Both Kursaniov (13.) and Ziegler (31 ) havefound that the miinor componienits translported inl)lants vary considerably, wvhen the environnmental coln-ditionis were altered. Their results are suggestive ofa selective transport of certaini conlstituenits. Thevariations in the aminio acid contenit in their experi-imienits cotild. for example, reflect a restrictioni in theuiptake of specific comiiponienits into the transport sys-tem unider certaini enivironmiiienital conditionis or theycould reflect a chaniged requirement for comiponentsin the receiver region. This latter possibility is pre-ferred since, in at least onle instaince, the germinatingimlaize seedling (19). the miovenment of amiiino acidsfromii the souirce (the endospermn) was inhibited by theadditioni of the required amino acid.s to the sink (theembryo). If the amino acids were niormally syFn-thesized from glucose in the receiver regioni a selec-tive transport of aminio acids related to the require-ment for amino acids would not he expected.

'rhus, it appears that glucose miavlnot be exten-.sivelv miietabolized in a growing region, that the ma-jority of the amiiiino acids miay be conserved alnd usedas stuch and finally that a transport of aiiiino acids toa receiver region could he limited by the amiiino acidrequirement of that regioni. To test these possibilitiesit was necessary first to kiowo the amiinlo acid comii-position of the protein in a regioni where a niet synthe-sis of l)rotein was occirring. T'he 5-mm root tip ofmaize represents suchl a regioni (17). As the cellsin this regioni mature there miay he miinor losses oftotal protein (4.12) and a redistribution of existinigproteins (3, 25 ). As new cells are formed, how-ever, the proteins which are required for cell divisiolnain(I elongationi muilst be maintained. T'he amino acidprecursors for these particular proteins imlay he de-rived from synthesis and protein turnover within the

PLANT PHYSIOLOGY

til), anid by traiisport fromii other regiois of the seed-ling. The relative re()uiiremiieint for the aimino acid,neceslarv for growth xx as established by anialy7zillngthe amino aci(d composition of the proteini in thlenm root tip. This, the relative rate of supply of the

am111ino acids from all sotnrces (synthesis. l)rotein tnirn-over, and transport) couild be establislhe(l.

WVhen acetate-2-C Ias fed to intact root tips. itxvas fotind that the spread of acetate carlbon ilito tIleamlinlo acids of the r-oot til) lproteii) dli(l n1ot l)l)roachthe spread that wvould be reqtiire(l if all the aminoacids xvere miia(le exclutsixelv in the root til). TI ui,for example. the synthesis of glntainic aiidl asparticacids and glutamine appeared to be relatively easyfor the root tip cells: the synthlesis o)f snich aminoacidis as theoninie, proline and( letncinie restricted.WNThen the root tips wvere excisecl ail(l groxvn in ctul-tlire for 6 houirs, the soluble piools of this lattergrotul) of amino acids (referred to (sa the neuitral and(Ibasic aminlo acid fraction) xvas drasticallx rediuce(l.Tn another set of experiments, acetate-2-C'4 xxas fedto somiie distant part of the seedling, the scnttellum orat region of the root 3 to 4 cmll from the til). and the dis-tribution of C14 in the, growing region analx-ze(l. Inthis case the contribtution of acetate carbon to amideschiefly asp)aragine. aiul( the nenitral and basic aminoacids wvas considlerable. 'The resnlts, tberefore stug-grest that many of the amino acids are not made inthe roOt tip in amiotints sufficienit to sul)l)ort growrth.andl that this requireniienit may iii fact. he influentialin oletermining wvhich amino acids are traansporte(d tothe root tip.

Materials and Methods

Al aize seeds (hiyb. var. Wfo) X 38-11) xvere sterilize(I briefly wvith Chlorox (a commercial bleach conl-taiming %s5oditnm hvpuchlorite rinsed, and allowedto grerminate onl a thini layer of agar- (1.5 %) in the(lark at 30(). Seedlings with roots 4 to 5 cm longwere tsed in all experiments.

Idetnltificatioln of thle Amino .- cid Pools. OInehlunldred 5 mm root tips were either extracted im-uediately with 80 % (v/v) ethyl alcohol or incubateclin a salts solution (18. 21) for o) hours before ex-tractioni. The alcohol soluble fraction and the in-.;olutble residtle were eaclh hydrolyzed wv'ith 6 NT HCI at100° for 12 hours. Each fractioni was thein taken todrvness in va-ctua at 40'. A 'I'echniconi amino acidaluto-analvzer wx as used to determinie the indivi(dalamino acid pools.

Applicationi of Radioactive 4cetate. Acetate-2-C14 (7.3 mc/urmole) Nvas adde(l to a solution of 1.5 %agar (450) containing the uisuial salts (26). 'rhelabeled agar was then hardenied in molds 2 mm X 50nmm. One mold xvas polaced oni a imicroscope .slide,and the critical regions of the intact root (5 permold) were anclhoredI on1 the labeled agar with stripsof unlabeled agar. Alternatively acetate-2-C14 wasplaced in a small watchglass containing a salts solu-tion and gluicose. The expose(d scuitella of excised

emnbrvos were p)laced directly in the liquidl. In theptilse experiments, the scutella xxere washed xvith de-ioi)ize(l \\ater and p)lace(l in fresh salts solution xvith-ouit tracer. Nfter the experiment, the critical regionsof the root, were excise(l anid treated as (descriledporev-ioisly ( 17. 18. 21).

Results

.-Iuuino 4cid( Coutcnt oJ the 5-min Root 'I'ip. Thetotal amiiounts of the individual amino acids in thealcohol-soluble anid insoluble fr-actions are shoxvni intable I. In the 5-nim root tips, glutamic and( asparticacids mnade up half the alcohol-soluble amino aci(dpool but only 20 % of the alcohol-insoluble amiinio aci(dcontent. Tt is clear from this data that as the roottil) grows and as those lproteins characteristic of theroot til) are miiade, the requiremenit for the aminio() acildof the neutral an(l basic fractioni as a group is conl-siderably greater than the requiremlenit for gltinmmicand aspartic acids.

Changes in the lev-els of the amiillno acids after ex-cisiQn shoxv more genierally which amiiino acids aremade in adequate amounts within the root til) andwhich may be sul)p)lied preferentially by the transportsystenm (table I, columnis 3 and 4). 'he soluble poolsof gluttamic and aspartic acids and of alanine anldlornithine were scarcely affected when the 5-mlm tipswere excised auicplaced iii a salts soluitioni forlhours. The level of y-anlino) butvric acicl showe(d at4-fold increase, wxhereas the levels of all other amiuitloadids declined duriimg this treatnmelut. 'I'lme leveleach of the neutral andl )basic amiiiio aci(ls in the ale((-lhol-insoluble fraction wvas slightly lower- in the cx-cised roots. The reduction of proline in the alcohol-soluble and insoluble fractions was iiiore pronounce(dthan with any other anmino acidl. Thus, as with d(-tached embryos (21). there was, ti(ler culture coindi-tions, a preferential loss of certaiui aminio acids in theI-oot tip. During this timiie there wo-uld ha\ve heel) amiieasurable increase in length in the 5-imm tips hadtheyr not been excised aln(d an increase in alcohol-ini-soluble nitrogen (17). Tlhere was, hoxxever, a 26 %loss inl alcohol-ilnsoltible niitrogeni during the 6-hourincubation. The reduced r, te of l)roteii synthesiV,relative to protein degradation. coulld be directly re-lated to the reduced suppl)l) of amnilno acids. HIox-ever, it should be stressed that other reasonable ex-lanations exist.

Kinietics of Incor-porationi of .lct(ate-2-C14 by thRoot T'ip. The kinetics of acetate incorporationl itothe amino acids of the intact r-oot tip are suiimnmarize(din figure 1. Typically, there was a liiiea- increase indutamic and aspartic acids and their amiidles from theearliest times (10 mmin), while a slight lag precededla linear increase in glutainic and alspartic acids ofthe insoluble fraction (fig I A ). \Vhen the individualcolniponents were isolated on Dowex-I (acetate) as(lescribed previously (14, 18, 21), a lag in the incor-poration of acetate carboni into glutamiiic anldl asparticaicids and into gluitamine wxas riot obserTed. C14

*74

OAKS--TRANSPORT OF AMINO ACIDS TO THE 'MAIZE ROOT

labeled asparagine was usually not detected in intactroot tips. There was a slight lag before a linear in-crease in C'4-labeled glutamic acid was observed inthe residuie but no apparent lag for the incorporationof C14 into aspartic acid of the residue (fig 1E). Anal)lparenit steady state was reached in about 20 min-

utes for the soluble conmponenits (glutaniiic anid aspar-tic acids and glutamine). The reduction in the rateof incorporation of C14 into the residue aftel- 2 hotursprobably reflects the exhaustion of acetate-C'4 in theatgar strip)s.

Th'lie incorporation of acetate carbon inito the ainiilo

Table I. Alcohol-Soluble and Inisoluble Anino Acids in .5-nim Root TipsThe 5-imm root tips were excised, washed with water, and either placed in a salts solution for 6 lhours or extracted

(lirectly with 80 s (v/v) ethyl alcohol. The alcohol-soluble and insoluble fractions were hydrolyzed in 6 N HCl at1000 for 12 hours. The amino acids were isolated with Dowex-50 H- resin and then separated witlh a Techniconanino acid analyzer.

GlutamateA spartate'I'hreoniiceSerine(GlycineAlanineProlineValineIsoleucineLeucineTyrosinePhenylalaniineLysineHistidineArgininea-Amino butyrateOrnithineTotal nitrogenTotal GA*Total NB* GA represents the

acids.

Intact tipsSoluble

amoles/20 tips

2.541.450.250.390.300.870.690.320.120.190.080.120.070.170.050.040.10

3.993.86

Insoluble,umoles/20 tips

21.711.320.720.961.561.640.981.350.861.590.420.651.150.340.83

.

5023.03

12.05

Excised tipsSoluble Insoluble

(%lo Intact)

31248758767893396668575742638080

415108

493

11198888889688181788377878481

74

,umoles of glutamic and aspartic acids and( their amides; NB, the ,umoles of all the other amini

GA

- PROTEIN

I

. *_PROTEIN

/ SCLUBLE

20 60 12 ISOTIME (MIN)i

NB

c

-* *Gi

Gku NH,

- PN-GIu

3TIME (HR)

FIG. 1. Kinetics of incorporation of acetate-2-C14 into intact maize root tips. A, glutamic andl aspartic acidsand their amides. B, the neutral plus basic amino acid fraction. C, glu is the soluble glutamic acid; glu NH, glu-tamine; Pn-glu, the glutamic acid of the residue. D, ala is the soluble alanine; Pn-ala, alaninie of the residue. E,asp is the soluble aspartic acid; Pn-asp, aspartic acid of the residue.

The initial activity of the acetate-2-C14 in 6 ml of agar was 979,500 cpm. Approximately 0.2 ml of agar was pipettedinto each mold. Five root tips were placed on each agar strip and 20 root tips were extracted as I sample.

II

0

D

TIME (HR)

175

1PLANT PHYSIOLOGY

acids of the neutral anid basic amino acid fractiongave a somiiewhat differenit picture (fig 11B). Therewas a relatively fast iniitial increase of C14 into thesolulble fraction (luring the first 30 minutes, followedby a linear increase after this time. Furthermore.after the first 30 miniutes the C' recoveredl in the resi-dlual nieuitral anld basic amino acids exceele(de that re-coveredl in the soluble fractioni, a situation lever ob-serve(l xwith glultamic ald aspl)artic aci(ls. The pat-terns of inicr-ease of the ind(livi(duial nleuitral and basicamino, acids could be divided rou,hl\v inito 2 cate-gories: .\ ) Those in which a. stea(ly state in the solu-lIe amino acidl was achiexved at ablolut the .s111ame timeas the linlteaI inicrease of C'4 illto the resi(lue w\as ob-serve(l. 'T'his is represenlted in figure 11) for alanine.Th'lie synthesis of serine and glycine ha(l similar kine-tics. B6) Those aminio acids in which verv little C14Nvas recovere(l in the soluble fraction. This type ofilncorl)(ration, characteristic of leucine, l)roline, thre-onine, valine, arginine, anid lysine, h1ats been discussedextensively with respect to the incorporation of leu-cine into root til) proteill (17). Similar kinetic4udttldies vieldiing essentially simiilar results have beenlperforn ed with regionis of the root 3 to 4 cmii fromthe tip.

Rel(/tivi Rates of S'yntlhesis. Calculations of therates of svynthesis of the individuial amliino acids ar-efraulght with comiiplications (2, 10, 17). Most seriousis the 1)ool phenomena wlhich has already been dis-ctusse(l in detail (17). It is critical to note, h1ow-ever, that when a linear increase in C14 in a proteinaimino acid is observed, a saturation(of the protein-precuirsor 1)ool is assulmed. Thuls, for example, theincorporation data indicate a small protein precursor1ool for glutamic acid since there w\as a lag of sev-eral minutes before a linear rate is achieved; a large)rotein-precursor pool for alanine since there was alag of 30 minutes. By this dlefinition the protein-precursor pool for aspartic aci(l should be very smallsince no lag was observed. These 3 amino acidsslhouild be synthesized efficiently in the root tip sincetheir levels are not appreciably affected bly excisioni(table I). Using either the cpm achieve(d after a 2-houir incubation with acetate-2-C"4, the rate of in-

crease of C14 in the insoluble fraction, or the pl)ecificactivitv of the protein precursor amino acid as crite-riia for the rate of synthesis, it is apparenit that therelative rates for aspartic acid and alanine wfere lessthan wvould be predicted from their relative contribu-tioni to root til) protein (table II). The reduced rateof inicorporationi of C14 into aspartic aci(l may he at-tributed to pool pheniomiiena or to the contribution ofassparagine, wlhich is not miiade in significant amou)nt,sin the root tip. The latter alterniative is lpreferred.since the kinietic data suggest that the l)rotein precur-soI pools for aspartic acid are small. .\ consideral)lepool diluition of alaninie miCacle froml acetate-2-C'- is.however, iui(licated from the kinetic studies (fig 1F ),and, accor(lingly, may be a mlajor factol iii the ap-parent redtuce(d rate of synthesis.

Despite this discrepancy between the incorlporatioilrates and the l)redlicte(l rates, conmparisons of therates of incorporationi of acetate carbon into glutamic,-acidl andl leucinie of the alcohol-insolUble residute areinformlative because A.) the relative amiountis of gIntamic acid anid leuicine in the root tip are similar,B) they are rel)resentatives of those aiuiio) aici(ldwzhose synthesis in excised root tips is either adequateor (leficient, and C ) the formation of acetyl Co.\followved by 4 enzymnatic reactions is requiired in thesynthesis of eaclh (30). Comparisonis show thatwhen the incorporationi of C14 into the amino aci(l ofthe resi(lute was linear, the rate of incorporation intoglutamic acid w-,as; 428 cpm p)er hour for 20 roottips, and inlto leucinie, 284 cpnm per houir (table T[).It would appear, tlhen, that glutaniiciacid is mliad1emluch more rapi(lly fromii acetate in the intact root til).A simiilar argumienit couild be presenite(l for niost ofthe nleuitral an(d basic aminiiio aci(ls (e.g., threoninie aiviproline in table 11). However, lack of iliforiniatiolnabotut the a-ctual hiosynthetic sequenice of these aminoacids in plant tissues makes the argume"i t lCss cdTtain.

Tranisport of Anmino Acids to the Root T1ip.\Wiebe and(i Kramiier (27) have demloinstrated, by selec-tive adnminlistration of P32 anld S3' to barley roots, ageneral moveement of these iniorganic ionIs to the

Table II. RclafaiVe CoWntibutios ,,f 'fOtal .tlinno .Icids and .Alntin(o Acids .Sv)ithtl'5iCe(ifromn Acctatc-2-C'4 in. Root Tip Protein

The results are calculated fromil (lata obtained from the experiments described in tables I and(I IV ani(l in figure 1.I'he calculationi for the specific activity of the newr amino acid inicorl)rated inito the root tilp protein lhas b)een describecd1rev,.ouslv (21 ).

10035.427.47.8

16.652.5

Rath10052.413.512.627.868.5

Specific activityo f newx amiiino aci(l

in p)roteinl

10046.48.(

12.917.627.4

The cpm in glutamate at 2 hours was 985; the rate after a linear incorporation of C''wa4 s aichieved was 428 cpm in 1

bour for glutamate. and the specific activity of the new protein glutamate xvas 5175. These values for -luttamate\wert set ait 100 0/.

\niullo acid

(ilutamate\ spartateA\lanine'I'lmreoniineProlinecucine

,ulil( tcs

0.770.960.420.570.93

1 76

OAKS-TRANSPORT OF AMINO ACIDS TO THlE1 AIZE ROOT

growing regioni. Furtherimiore, when they fed thesetracers to the root tip little radioactivity moved outOf this region. A similar picture pertains wheneither acetate-2-C14 or leucine-1-C'4 is given to dis-crete regions of the maize root (table III). In eachcase, much of the C'4 was recovered in the region ofapplication, showing that a large part of the tracerwas not available to the transport system. WVhenthe tracers were given to the root tip, considerableC14 was recovered in the 1 cm adjacent to the tip,but very little in other regions of the root or shoot.\Vith more basal applications the C14 was more gener-ally distributed throughout the seedling; however, amovement to the growing tip was apparent.

C'4-labele(d acetate represenited an ideal tracer forthe experiments illustrated in table IX'. because neg-ligible amounts of acetate carbon are converted tosugars in the maize root (8). Acetate-2-C14 was feddirectly to designated regions of the intact root, an(d.after the required time, the critical regions of the rootwere removed and extracted as described previously(17. 18). The results in table I\N show that, as witlhisolated pieces (14). the greater part of the acetatecarbon was recovered in the organic acids and in glu-tamic and aspartic acids when acetate was fed directlIto the root tip) or to a region of the root 3 to 4 cnmfrom the tip. In contrast to this, most of the acetatecarbon transported to the root tip from a region 3 to 4

Table Ill. Effect of Region of Application oni the Redistributtioni of Carbon Derivedfrom Leucine-1-C04 and Acetate-2-(C'4

The tracer was given to the designated region of the root for 2 hours. After- tlis time the critical regions wereremoved and extracted as described in Materials and Methods.

Tracer a)pplie(d ;at

C14 Recovered in the alcohol-soluble fraction(cptn/20 )iece,.)

Region* of the root

Acetate-2-C' 4

I eucine-l -Cl4

Tip2 cm from tip4 cm from tip

9600**22752160

11

15601497830

Tll

1104699**330

Tip 13z,24()*- 7785 872 cmii from tilp 3160 6364 63,(640*14 cm from tip 3018 2124 3064

* Region I is the 5-mm tip; II, 0.5 to 1.5 cm from the tip: III, 1.5 to 2.5 cmn from the tip;the tip.

* The radioactivity recovered inl the region of application.

IN Shloot

2034906566**

n.d.6851022

38 101422 162

52,4801 * 512

IV. 2.; to 5.0 cm from

Table IV. Distribution of Carbont Derived frojmt Acetate-2-(C 4

Acetate-2-C'4 (7.3 mc/,umole) was applied to the root in agar strips that were 2 mm wide. Twenty 5-mm tips orsections 3 to 4 cm from the tip were excised at the times indicated, washed with water, and extracted with 80 % (v/v)ethyl alcohol.

Time(hr)

Tracer givenl(lirectly to tip 2(tip sectionsanialyze(l)Tracer giveni3 cm from tip(3-cIml sectionsanalyzed)Transport to

Total C'4in thesolublefractioni

( cpm in 20)pieces)

5816

4487

% Distribution within the soluble fraction

Fther-Soluble

()

OAS-S:

42

559

.Amino aci(is

GA AM11XI

21

18 ()

tip (tracer- 540 17 20 l l 15 37applied 3 c;ii 4 1130 14 21 8 21 36from tip) 8 2087 7 25 14 21 33

* OAS represents the organic acid and sugar fractions; GA, glutamic and aspartic acids; AM, glutamine and asparag-ine; NB, the other amino acids.

** When acetate was fed directly to the tip, all the detectable C14 in the amide fractioni was in glutamine. Inthe sections 3 to 4 cm from the tip, the C14 was almost equally distributed between glutamine and asparagine. Inthe transport experiments, approximately 80 % of the C14 in the amide fraction was in asparagine.

BPI

3

177

f

PLANT PHYSIOLOGY

cim from the tip) was reci\veredl in the amide fractioni,chiefly asparagine, ai(Il in the iletitral anid basic aminoacid fractioii. Thtis, thier-e does appear to be a selec-tive transp)ort of nieutral anid basic aminio aci(is anidanmides, to the root tip region.

Tranisport of Al,ui,,o( Acids froiii thle Scittelliuii totile Root. Wh11eni acetate-C'- w-as fed to a regioni ofthe root 3 to 4 Ciem froiii thle tip, it proved difficult toget stifficienit C1 4 to the tip to deteriniiie accuratelyw-hich amino acids w-ere being transported. Ani alter-native approach was to feed acetate-2-C'1 to the scu-telltum, alnd theni to deternijie which amilino acids w-eretransported to the root. The results fronii suich aniexperimenit are presenited iii table V (coltimniiis 5-8).A\cetate-2-C''4-as also fe(d directly to the initact roottip (colulmni I anid 2) anid to a recgionl 3 to 4 cim fromilthe tip (columniii 3 and 4). 'I'he (listribution of C'4 inthese exl)eriments rep)resents thlat wxhichi might be ex-pecte(l if glucose, imade froii acetate-2-C"' in thescutellum ani(l tranisportc(l to the root, selredl as themiiajor soturce of aiiiiio aci(Ils i-ecor(le(l ini coluiminiis 7anid 8.

In the root tip regioni (colulmnls 1 and 2 ). by farthe greatest proportion of the acetate carbon was re-covered in the soluble glultamic antd aspartic acids andglutaminie. Very little C'4 xvas recovere(d in asparag-ine. Abotut half the C' in the insoltuble fractioni wasin glutanic anid aspartic acids. Likexwise. most ofthe acetate carbon recovered fronii a regioln of theroot 3 to 4 cmii from the til) (columnliis 3 ali 4 ) wvas iiiglutamlic aiid aspartic acids. Inl the older regions ofthe root con.siderablv imiore asparagine was nia(le, and(a, greater lprol)ortion of the acetate carboni wvas re-co\ ered as y-amino-butvric aci(l. Again. iTost of the

C' ' in the othelri- amino acids was in the alcohol-ini-soltible resi(Itue. .\lanine wvas ani excepitioii. In thesctitelluimii nmost of the C'4 in the amiinio acid fractioniwas recovere(I in glutamic acid and glutiamine (col-tiniiis 5 and 6). TI'he major differenice in this tissuewas that a greater plroportionl of the C'4 in the othel-amilno acids x-as founlid in the soluble fractioni. Pro-linie was the most strikinlg example. \When the sctI-tella w-ere washe(d and place(I in freslh salts solutionaii(l glucose for ani additional 10 houirs (pulse experi

enits), over 80 % of the C14 was lost fronti the solubleaminio aci(ls (22). The excel)tion waas asparagine.w-hlos_e C14 conltent tloubled during this tiliie.

At 2 hours most of the C' I in the amnino acidI frac-tioIl of the root \ as in glutamic and( aspartic acids.This distributioii p)rol)ably reflects the remarkaldleability of the scutelluimii to imiake aiid( export sugars(22) anid the ready equilibrium lbetween these 2

aiminio acids and the acids of the trica-rboxvlic acidcvcle (14). \Vith time,. a greater proportion of theCl'' was recovere(d in the nieuitral andcl hasic aimiino aci(lfractioni. No special significance is l)laced onl thistren(l, sinlce the kinietic stucdies (fig 1, A an(l 18)sholowed a simiiilar trenid wi,th time wvheni acetate \vasstipplied directly to the root. In contrast to localal)lplicationls to the root, hoxv-ever, considerably miioreC"' xwas recovered as asparagine, and a greater pro-portion of other amino acids, notably glvcine, serine.alaniiie, proline. and leucine, was recovered in thesoltuble fractioni. In addition, the distribtution of C'4in the amino aci(ds of the root did not reflect the (lis-triblttion in the scutellum, indicating that all theaimino acitls mlialde in the sctitelltim \vere llot eclivllvacce 5sible to thce transport system.

Tal)le V. IuieMrPi')'ti''uMi of I((('tate-)-C' 4 into the -1')ibio . cids of thlei Jla[aLe .Scittellnui (1a1(1 I t)()tThe values rel)resent the recovere(I after a 2-hour local fee(ling at the scuitelltumii the dlesigniatedl region of the

root. Values for the transport of aminio acids to the root (the whole root wxasause(l in this case) are 10 lhours aftera local feeding at the scuitellum. Twentl pieces wvere usedl per sample.

Root(direct application

of tracer )51mm111 til) 3-4 cmii froimi tipSo'* Res` Sol

Scutelluii(dlirect ap)plicatiol

of tracer)IRes Sol Res

GlutamateA s1parta te

nltaiiniie

\ sparagineSer-inie- glytvciiie

AlaiiineProliiie-y-Atinino butvrate

alineLeucine-i soleucinic

Cl' inl GA (tcpill),C '4 inl B'cl)'

2080

54673420

1 1011A.(133

S.n.d.10460s

337 )

468

2

985

227

3_ 2

77270163

1435;1437013371764

31682338

960

ii.d.

ii.t.

191

n.d.ii.d.nl.d.

3526387

4744360

81110155277

12728(23711041219

5

101,00039,800

61,0003210084()in.d.

21,95023802935tl.d.841

n.d.205,01034,946

6

16,85012,140)

211 .10.63402380

nid.

10,0510

28,99021,670

7

4940720

1 90()4440530n.d.860

891380ii.dl.470n.d.

12,0003131

814821050

512178727515

376760303

25323371

* Sol is the C14 iin the alcolhol-soluble fraction; Res, that in the insoluble fraction.** The total C-14 is the acetate-2-Cl4 incorporated into all the cell components; C'4 in GA is the C14 recoveredl in

glutamic and aspartic acids and the amides; C14 in NTB is the C14 recovered in the other amino acids.* nd.(].: C14 too loxx' for dletection.

Transport toroot (tracerapplied atscutellumn)

Sol Res

OAIKS-TRIANSPORT OF AM\I[NO ACID)S ro THE MIAIZE ROOlI1

Discussion

Folkes and Yemni have shown that the amino acidcomposition of reserve proteins in barley endosperm isverv different from that of the cytoplasmic proteinsof the embryo (5, 30). From their extensive nitro-gen balance sheets, they concluded that the majorlosses in glutamic acid, asparagine, and proline couldhe accounted for miiainly in the significant gains in'other bases' (more recently defined as nucleic acidsand their precursors; see ref. 11). in chlorophyll, andin the amino acids aspartate, lysine, arginine, andalanine (6). The other amino acids showed onlvminor increases or decreases during the germinationprocess. This represented the experimental founda-tion for the notion of an extensive interconversion of-amino acids in the embryo. Further evidence forrapid synthesis or extensive conversion of amino acidsin the embryo was derived from the ubiquity of trans-aminase reactions. The rates of the glutamate-aspar-tate or glutamate-alanine transaminations were, how-ever, considerably higher than the rates of trans-amination between glutamate and the other aminoacids tested (28, 29). From these observations itmight be preferable to place glutamic and asparticacids, and perhaps proline, in a special category ofamino acids un(lergoing rapid metabolism duringgerminatio;i. T'he other amino acids stored in theendosperm appear to be conserved and used directlyais protein precursors in the embryo. The supplv oforeforn'ed1 anino acids to the embryo is apparentlycl prerequisite for the increases in embryo protein,-,ince emibryos grown in culture do not augment theirproteiin content unless an appropriate mixture ofamino acids is added to the medium (21). Further-more, this requirement for amino acids appears to becritical in regulating the degradation of storage pro-tein, since addition of this same mixture of aminoaicids to the medium delays the disappearance of ni-trogen from the endosperm of normal seedlings (19).

Reactions leading to growth in the embryo are con-centrated in the linmited meristematic regions; that is.iM those regions where a net synthesis of macro-iiiolectiles is occurring. Using the root tip as agutlideline is obviously an oversimplification of theIrrowth processes in the embryo, since new proteinsare made in other regions of the embryo (3. 25). Tnfact, the enhaiiced synthesis of asparagine and y-ainohbutyrate in older regions of the root lendssupport to this contentioni. However, using the roottip does permit comparisons which are not possiblewith the miore heterogeneotus tissues of the wholeembryo. Thus, knowing the amino acid compositionof the root tip protein, it is possible to say that foreach micromnole increase in glutamic acid, an increaseof 0.42 Mmniole of threonine. 0.96 ,umole of alanine,0.57 pumole of proline and 0.93 ,umole of leucine wouldhe required. Ideally, it should be possible to deter-mine the rates of synthesis of each of these compo-nents with radioactive tracers; and if various sourcesare .suspected. it should he possihle to determine the

r-ates of supply fromii each of these sources. Suclhcalculations have proved to be useful paranmeters illbacterial system1s (23). However. at present, 2 com-l)lications prohibit similar calculations in tissues fromhiigher plants- A) the infinitelv more comiplicatedP0ool Structure (2, 17. 24), and B) the iniadequatesteadv state conditionis, which are in part the natureof the diversitv of the cells involved and in part theless carefullv controlled methods of administeringboth ntutrient and tracer. Nevertheless, useful coin-parisons are l)ossible. The results of the present in-vestigation show clearlvy for instance, that acetatccarboni is preferentiallv incorporated into glutamicand aspartic acids in 2 regions of the root, and alsoin the sctutelltumii. Futlhermore, less acetate carbon isincorporated into leucine, an amino acid whose syn-thesis involves a series of reactions verv simiiilar tothose required for the synthesis of glutaniic acid(30). In cultured roots the level of such aminoacids as proline, leucine, and threonine is not main-tained. These amino acids show relatively poor in-corporation of acetate carbon. Taken together. theseobservations suggest that the synthesis of many amiinoacids is deficient in growing tissues. Hence, the en-tensive interconversion of amino acids previousl-assumed to be prevalent in plant tissues (2, 5, 24)mlay be of minor importance. Additional support forthis argument is provided by the fact that amino acidssuch as leucine (17). valine, or lysine (Oaks, unpub-lished) are not extensively mietabolized by root tissue.In contrast to this, those amino acids closely associ-ated with the tricarboxylic acid cycle, glutamic andaspartic acids, do appear to be made in sufficientamotunts in embryonic tissues and are extensivelymetabolized by the root tissue (14).

If the synthesis of miany amino acids is, in fact, (le-ficient in growing tissues, 2 types of questions maybe asked: A) Does the low rate of synthesis rep-resent the genetic capacity of the plant cell; i.e., canthis rate be altered by altering the levels of specificsubstances in the cytoplasm? B) Does the loxv rateof synthesis influence the compositioll of amil ) acidstransported to the growing regioni fromn sc:escingregions? Answers to the first question hay beenattempted with regard to leucine biosynthesis in nmaizeembryos (20). The results show that instead of in-hibiting the developnment of 3 enzvmes in the bio-synthetic sequence. a result that mig-ht have beeln pre(licted fromii bacterial systenms (7). leucinie actuallycaused a slight increase in th!c recovered activitv(20).

The present investigation represents an approachto the secon(l questioni. TBoth the altered pool levelsin cultured root tips aln(l the incorporation of acetate-2-C14 into root tip amino aci(ls suggest that the syn-thesis of many of the neutral and basic amino acidsis deficient. Significantly. this same group of aminoacids is preferencially transported to the root tip.Although identification of specific amino acids in thetransport system was difficult because the recovery ofC14 in the root tip was low, it was possible to identify

l/'?

PPLAN r PHIYSIOLOGY

asp)aragillne as a nIj(ajor c( ' nenlt f the transport sys,terni. Furthernmre, the synthesis of asparagine fromiacetate-2-C' in the inltact root tip was alinost- corni-pletely absent. Tlhus in this one instance a specificaminiIio aci(l, requiired in the synthesis of root tip) pro-tein buit Inot ilia(le in the root til), was stupplied 1y thetranlsport systemn. In this olne case, then, it is reasoni-able to say that the requiremlienit for a particular amlinoaci(l in the -;rosilig re-ion could be (lirecting themovemient of that amiino aci(l in the transplort svstenm.

The experiments concerned with the transnortfrom the scntellulli are not as precise. since sugarsrepresenlt a imiajor componenit of this systemi (22).However, thle higlh recovery of mianv of the neuitralaicl l)asic am-nino acids in the solll)le fractionl of theroot is a (listinctive featuire. It has been stiggestedthat the svnthesis of amino aci(ds in the growing re-gions couil(d be controlled by the supply of amIinio acidsfrom the transport systemii (16. 17 ). This coutld beinvoke(d lv the repression of enzymie formiiationi or byallosteric inhibition of the action of one of the en-zynmes in the biosynthetic sequence. I )enonstrationof the first pihenomenon has, to date. been eluisive.The latter phellnomenon, however, appears to be ofmajor inlip)ortance in the biosvynthlesis of i-nanv amiiino,acids in the maize root til) (16 18 . The involve-mlent of either phenomenon wonmld require the buildupof a l)art of the )ool aminio aci(l. It is ther-efore sig-nificanit that the amnino acids transported to the root1o cortNrillilte extenisively to the soluble pool.

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l <0