Citation
Sugar transport in the maize scutellum

Material Information

Title:
Sugar transport in the maize scutellum
Creator:
Whitesell, Joseph Henry, 1936 ( Dissertant )
Humphreys, Thomas E. ( Thesis advisor )
Anthony, David S. ( Reviewer )
Biggs, Robert Hilton ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1971
Language:
English
Physical Description:
91 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Bathing ( jstor )
Fermentation ( jstor )
Flasks ( jstor )
Hexoses ( jstor )
Hydrolysis ( jstor )
Ions ( jstor )
Sugars ( jstor )
Table sugars ( jstor )
Water tables ( jstor )
Water uptake ( jstor )
Botany thesis Ph. D
Corn ( lcsh )
Dissertations, Academic -- Botany -- UF
Plants, Motion of fluids in ( lcsh )
Sugars ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Characteristics of the uptake of sucrose, glucose and fructose by maize scutellum slices are presented. Sugars were taken up at almost a constant rate until the bathing solution was depleted even at concentrations well below those which saturated the uptake mechanisms. The effect of DNP, phloridzin, uranyl ion, and anoxia was to inhibit the uptake of sucrose approximately twice as much as the uptake of hexoses. Maltose was taken up without hydrolysis. Turanose was not taken up but slightly inhibited the uptake of sucrose. The following conclusions are dravjn. (a) Sucrose is taken up actively without inversion, (b) Hexoses are taken up by two processes operating simultaneously, diffusion and active transport. (c) The active uptake mechanisms for sucrose and the hexoses are located at the plasmalenvna. (d) The active uptake mechanisms for both sucrose and the hexoses are driven by glycolysis. (e) Metal binding characteristics of the scutellum are different from those of yeast in that binding is not specific to the uptake sites and bound metal ions are apparently not released during sugar uptake.
Thesis:
Thesis (Ph. D.)--University of Florida, 1971.
Bibliography:
Includes bibliographical references (leaves 87-90).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Joseph Henry Whitesell.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030470530 ( alephbibnum )
37786673 ( oclc )
ACN3671 ( notis )

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P lI. IJOULE IGE n l lTS


The authjr IxrerZSies his since-*** rhaniks to- [rr. T. E. Hu.nIphre.ls

forT hir 41uidncz throughohut thr. graduote Iprogi'ri m and fo~r hi5 red ice,

pa~tiencei and help ;n conducting e...perimecnts and Tpjrept.rin the r-a~nu-

ECr i p.. It is j pleasure to ....*ork in h~iE IdjTlabor Tor bTnd ch;are h

e~quiprrien t. The hEp oF 1.* rs. D. S. IAnthcr,, F., L. 5irat h ;ind r.. H.

b~cggs a roalitta.;r~ i,,chllers ;s jlEC ajppriciated. The. Forjn= D department[















FABLE OF CONTENTS



Page

ACKNOWLEDGEMENTS .. .. . .. .. ... . ii

LIST OF TABLES .. .. .. .. .. .. . iv

LIST OF FIGURES .. .. .. .. .. .. .. v

ABSTRACT ,,. . . .. .. .. . . .. vi

INTRODUCTION

LITERATURE REVIEW . .... .. .. .. .. .. .. ..

METH1ODS AND MATERIALS . .. .. .. . . 5


Preparation of Scutellum Slices .. .... 25
Analysis of Sugars .. ... .. .. ... 25
Manomectry . . . . . . . . . . 26
Metal Analysis ....... ... ... 27

RESULTS . ... .. .. . ... . .... . 2?

Kinetics ... .. .. .. .. .. ... 29
Sucrose Uptake . ...... . . S
Ga: EvChange .. .. ... .. .. . .. 2















LIST OF Trs3LE J


abli page

I, lehod of Cajlculjting Data ,, ,. .. . .. 31

2. Gluicrt UIptijke jS e'ifated b, Ulrjn*,l
loa n thieBatlhing Solution ....,....,, LS

3.Fermrentct onl in Water And Suigar
:olutions .. .. ... ... .. .. .. S

4. Thc E ffec t o f Uran,1 Iri t -ae Pre rea t-
me~nto~n ~uci'c w~ptjCe ............. 56

5. InhibitiOn of lugar Uprake b, Phlcridrin ,. j3

6. The linhibition b, 00lP of Uptake fromi
0.0111 Eugar SoluIon1 .. ... .. .. .. 59

7. ljltose Uptne . . , . . . . .. I61

?. Thez EiFfcts of' Turaneose and flanni rol
ojn Sacro; e U~prLke . .. ... ... .. 03

1. Suc~Tros TiiSue Lee I and CSuirise Uptake~ ,, . O'r

10, Sucrsse Uptake a Affected h, Various
Cations ... .. .. . ..... . .. . 701














LIST OF FIGURES



Figure Page

1. Sucrose Uptake Vs Time .. ... .. .. ... 30

2, Sucrose Uptake Vs Time .. .. .. .. . ... .. 35

3. Sucrose Uptake Vs Time . ,. .. .. . ... 36

4. Sucrose Uptake Vs Concentration . ... .. .. 37

5. Sucrose Uptake at Constant Sucrose
Concentration .. .. . .. .. .. .. ... 40

6. Glucose Uptake Vs Time .. .. ... .... 41

7. Glucose Uptake Vs Tinie .. ... .. .. .. .. 42

8. Rates of Fructose and Glucose Uptake
Vs Concentration ,. ... .. .. .. . .. 43

9. Glucose and Fructose Uptake as Affected
by Uranyl lon Pretreatment ,. .. .. . 44

10. Uranyl lon Pretreatment ard Glucose
Uptakte . ... .. .. .. ... ... . 48

11, Cluroze Uptakle at Constant rCo~ncntraton .. .. 50

12, Fi rre n rjt i on i n r Ij e r and Su; r ose .. .. .. 6b

13. Gas E chjlnge in r'jucr snd 0. 111 Surore ,. . .. 70

IL. Cjf Echangc in riata ,-ndj 0.lrIGlucee .. .. .. 7l

IB. Ecccr.ercatson ir. Eve:lar :Llurion .. .. .. .. 72

Ii. r~crsl tsin.~lr.:i ..............,.... 75

II, Effeci of Urari,I I;n Pretrtr c rncir ConrcLntra-
tiojn or. Supr Uptake, .. .. .. .. . .. :7

15. Fletal Einding Fo~llrow~ig ar. Acd P~ctreateentr . .. 78






Abstrjct of' Dircertation; Presenred to the
Graduate Council of the University of FloridJ in Parr;al iulfillImen~t
of the P~equirements for the Degree oi Doctor of Phillosophi

SUZG'.f. TP. USPCIFT lu, THE 1141:;E SCUTELLUM;

B;

JoscFph Henr, Wn~tosell

June, 1971


Chairman: Dr. Thioma~S E. H~umphre-:s
Major Deparrreeent: Ecn

Characteristics of the uptaki- of sucrose, gluicoe and frucloto b,

maze scutellum slices are pre~sented. CSugarj Iere taken up at alm~oi!

a conrstnt raite until thej bjthing jclullo~n wal depie.edl e.cn at co~ncen-

[rations+ u:11 belou those whlich saturated the uptar.0 rmechanirms. Th

effect of DUP1Y, phinf'idrir, uran, I ion, jnd anor.io twas to ir.h~ibit r e

uptake of ucrorr e appro-.ii satel mic a~iE s bmuch as the UpEra ke ot h;hl5se.i.

Malrics wasr talen up wi; hoiur hydroli sis. TurCAnoSe I-.i1 not taken up

but slighri, inhiibiced the uptal.r of jucirose. The following conclulsions

are c~r awn. (a: Eucrore- is taken~ up -jCrivcl, aithiout in.erjion. (b)

Hesxose; are taken up bi 1'0 pr3 ~ocesce operating Simultadneou5i,, dirffu-

sion and cci'..*e transport. (c) The actise uitake mechianisms for sucrose

and the he.:-.oses arre locate at the plasmalerr~ain (dj The acti.; uptar."

mechasnisrms for bothi sucrose and thL he>;ose5 are driven by glicrlysiC.

(2) hotel binding chiarac reristics of the scutellum art different from

those of ;east in that binding is not specific to the urptaker rtes and

bound ralttal ionis aire apparently not released during :ugaJr uptalke.


















Much research has been conducted on the movement of sugars in the

cells of animals and microorganisms. Stein (1) reviews this work in a






p~ljnE?, jrn.; ,r the Probt le.. is I:jhtrj l II0 [hi L..II r~: L rldin3 ilf plinE

ph,.CIMAt least it.. rcsprcr ;c. h;lhcr :r-3.n;:r~:I;i th.. re ..Trt orr



;) up :.-.1 c.-nos acc.:1.ride 12 Sucrose 'i 1, Fir the ireson .ii:Crver

st...I ib .~i~lr i i, rr- ,.10 -t d .. plan i [" IId IL.









rea.o :I ~ i:: ,*5. 0 .;r uni lil I: IIIln:.n-s of ~Ch.-- [rITI:.s.5 of 00 e.-:







su r: pro 1 .se .3irth iU.. Lhd th Lr I:E.i: ?nh:...~T~ rt ::in ii( i reser


Fr~lia c r ~ ~ ldl, 'Ir *:ced:,; fru l- .-cor and.lP:.ter partE of lj ants-~ J h)L are-


I NTRODUICT ION







neeir;al to supprrt juch concepts ir 50.11 grear.1, inedcquar-_. ILc iljo

5ta!Cs that the verry exists.*nce of~ c`rri rs has not: tie1 pro;.cn.

studir?5 or BiideSki (:) orn the~ acculrsulation of~ sugars b, plhlolm~ L1ssue

jdd support to th( CO[Lncentin that an acrlt., mostment or su~croSE ;s

probabl, a part of phlopm transport.

The rrork reported herein wa~s underrtake:n rrO 3-in a better ulnder-

sranding or thea process ofi sugjr uptake;,, espc~i;Jlly t."r.;t of ucrTe.

Several .h-racterscists makeZ the c..rn ;s~cullum well F.II;.:Jl for Lhe.

sHudy of iugar uptake.: 1,.3) Ths uptak3 of sucars is real oilorwing



in rhe- Form of sucrose rather rhan~ itarch: alucsse :s not -accur~Iulated:

(c) there ij nro aJeinJ phenomenon durirag which 4n adjustnerr~t in thr-

reSpirtory rT3[ ccurs;~j jnd (0) mOrphologicall,, the scutellun i:

leaf rissua which functions ir. cha mosement. Gi sugar r'r001 Ehe end~~iperm

to .he Je.loping seedling.













LITERATURE REVIEW


Several systems have been described in which sucrose is taken up

without prior inversion.

Weatherley (6) measured sucrose uptake by floating leaf disks of

Atropa belladonna on 10% sucrose solutions and taking dry weights be-

fore and after uptake. At times the amount of hydrolysis was very lower

and the amount of hydrolyzed sucrose in the bathing solution varied

independently of uptake. Whereas pH affected the amount of hydrolysis

it had very little effect on uptake. The amount of hydrolysis was

greater whien older leaves were used. Washing disks prior to treatnment

decreased the amount of hydrolysis. He concluded that sucrose was

absorbed as such. He found (7) that the uptake of glucose and sucrose

on a per mole basis was nearly identical.

Iweatherley (8) also found that the uptake of sucrose wras revers-

ibly inhibited about 75% under nitrogen. He followed the loss and up-

take of water as well as dry weight and included a discussion of the

osmotic situation prevailing in the tissues. He concluded that thle

uptake of sucrose was probably an active process.

Experiments by Pennell and Weatherley (9) showed that sucro:- up-

take was in~hibite-d about 5070 by 2,4-dinitrophenal (DNP) and not at all

by phloridzin (both at 1 mM). In this paper the amount of dry , 2.ht

increase was shown to be caused partly by an increase in s ar;se.

glucose and fructose. The amount of increase not due to th:.:- siy r

a 5.1 jssur.. *J t h. .: E .(. st -ar th ferrat ioi:.- s= .t s g F r..






aenou~nr of .insight inre.=.*~d rr:. jlas due ;o starh ;t w93 iArluLed thatL no

"uphalll" mrjl~ncnt. of sucrore need Occur,

Pojrter a4nd liev, I' 1) wo~rrked rwi h to~bsico leaf disks~ jndi measures

uptake, accumulation and Lgaj errhange in SE solurio~ni of juccose~ andl

in.*err sugaJr. Wrhen ;nterE 5uqrr was5 supplied, ?luicse dis3F~appeard a

3 faster rate thiar. Fruirtose. InvrrE sugjr cjiusedj ~a re rapid a.:cumu-

latio~n of starch than did juCrjse. The totajl uptake~ ojF 5ugar vaS [he

samie wrhethe'r indfrt 5Jgar Or SUCTCIse w35s Suppl;ied symual

late led sucrose could be r~co*.red fromn the le~af disks jfrer bei~ng

jpplied in the~ bathing solution ;hroling that juirose could be taken up

without ;ns\erjion. Thla! around ,n PQ? oF 0.74 du~ring ixubatiojn of tijjue

in varer, but this ;Increased to aboute 1.1 when [he Lilsue' was incubated

in ?ucrose' or he.*ose. The speCi fic ac t i it*I Of th;- CO2 c.o=.ed was5

about Ihe jiame aS thjt Of thC applied iugar leading thrm ra belireve

rhac rhe Obser~~td rate of gss l.changz .-jos noi the result of a hiigh

rare of formn.tnacior soverimposed on the 9.3' exrchange" i rraterl. They

ruggelead~ rather a crhif[ in subjrrarste LO~ Suyr win j,...prs wrCe jddedd

to the bathin3 solcrion.

Viccer: and fIerrcer (II) studied the urt'l:.? o' jurrose by bean

lea tee.-.The bjrhir.g Iolution contajined1 e-l; Sucro-e andj a !rjce

of fractateC jf t incubation, in';:aring that ex.trartellulj r .r,drclasi s

..Ja= ;mall or no~neiictnt. Wrhen tijjue sampFles Nere ant.lued .roit of

rhe Sugary woSS Sucrose I*;th small Snounts of reducing sugars present.

Gas e.*.changel was5 measured jnd1 rh-. P.2 increased fromi about nS to I.0)

r.hln sucrosee was Addd to .he barhing solution, The notiiced a liner

phase2 of jucrJSe Iip(? e wirth lime2 despite large~ concentration diffe-r-

ences in the bjfthJr jcj].jo;in. Tht ste.ao l ract ofi jsaro~se uotae Iwaj







about 9 umoles/g fr wt hr from 1% (0.028M) sucrose. An inhibition of

sucrose uptake of 55% was noted wi th 2.4 x 10dMW DNP. Uptake was mnea-

sured over periods of 8 to 26 hr.

Vickery and Mercer (12) reported increases in 02 consumption upon

the addition of surcrose that were of short duration and independent of

concentration. They stated that the rate of C02 production showed no

correlation with the concentration of sucrose in the external solution

(and hence, in the free space) but was strongly correlated with concen-

tration of sucrose in the apparent osmotic volume. This is used as an

argument that the sites of carbohydrate metabolism are included in the

asmotic volume for sucrose and that part, at least, of the cytoplasm

is included within the membranes involved in sucrose transport. Su-

crose accumulated against a gradient and the initial rates of sucrose

uptake followed a concentration vs uptake curve adaptive to Michaelis

and Menten kinetics, After several hr of uptake the rate decreased.

This decrease was attributed to inhibition of further uptake by sucrose

inside the cell. They argued against the possibili ty that sucrose

pumps occur only at the tonoplast.

Hardy and Norton (13) studied the uptake and utilization of 14C-

labeled sucrose, glucose and fructose by slices of potato tubers.

Glucose was taken up faster than fr-uctose. All three sugars were found

when untreated tissue was analyzed for sugars. It was suggest--I o,n tha

basis of the labelina of various intermediates that sucrose wa- asre.

unchanged and transported to storage where part of i t was hydro~l,:-c

resulting in storage of all three sugars.

Sacher (14), using bean pod tissue, found an extracellules ;I

o~uter p;ci ..-,.:rr a~i c ..n e- ricd .cas~n jl I n i ts activi ty. Hot-rei .-c







suiroie uptak~e ass njLe depenldent on in~tertsiF act..i C a; Snealrn b, the

obser.ation rhat :Jiose wajs taken up in rth abse~nce of ourc~r :pace

irler tose a;t; vi ttl. ClucO-;e uptate <--.;; thre times as5 Ifat J: Frur. CoSi

uptakte from 0.030l solutionl.. There L4-Sa no aiitct o: 10~ t o 10 'tI

uranyl nitrate on uptral.e ofi 0.0?11 glucosa or sucrjas. The Fact that

Sucrose~ wajS tken as as 5suich.j jlii als~OhST[ deosr trd bySholi~ng thjt the

giveme/frucT~i tose rljdoiour i .It rtjio kzs l iti ct:hajngd b:; Iiptjl.e a~han

us ii n, fruc tose -labr led SuCrose, Uni forlily labeler d sacrose relmaincd

unifo-ml, ljbeled ic .n in the presence of unlabe led fructose or glucose

in the~ ha .hing solution. DlrfP (: n 10- 11) inhtibied uprakei from 0.0003Hl

jucrosc 6b ,and ircm 0.0!n suc~ros e 881, In Freshl; cut !;jssue the

endogenous Sugun5 cnisted4( lajrgell of glucose a-nd :ruicts. dnd only

trjici jrounts of suiTrOe. Upon incubation in sugqar silurionn juc.-esee

Wji storedJ, apparentl i ini ths .acule, and the suloC.C/reducingnl jllljr



wrLd the sucrose .-as hyorollzed and the r~tic rjreiltl decreased.

jacher jrouei on cnhe bjsjisl ofSe.eral linesj of I::;jrdence thb[ L:-a rate

l im' rin1 5 tp in hcrose uprake lilj i n Ehd Formjtioi of ;uCTC;2 ind

that the cit~plicam iS frae space to the ta.(ese.



In the gterminating~ :astor bcan, suirose 15 syntrhsi.zed In the; iidospirsT

jt '.h e apensi of ijt. Thr suicree is ca'-.en uoj b, the- ijt**lldon5, and

It ii transplorted- fromr thean into the de.elc~oing ieadlling ar;s. In the



lui... Isest of thr .ugar~ is in the iorn of ;Lucrose with ..irtu-lll ro free

Iietuies present. h;heds ner.pa-o oreriht b

th~e rotyledoins and' suc:rose .>a accurT;ullated aginer olCentl. tion







gradient. DNP partially inhibited the uptake of sucrose. Sucrose was

shown to be taken up without inversion by several lines of evidence

including the retention of asymmetry of labeled sucrose applied in the

bathing solution. In this tissue sucrose was taken up at a higher rate

than either hexose and, more unusual, the r-ate of fructose uptake was

considerably greater than that of glucose uptake.

Kriedemann (17), on the basis of microautoradiographs of castor

bean cotyledons exposed to labeled sucrose for 20 min, suggests that

the cell walls and intercellular spaces provide a diffusion pathway

by which solutes can gain access to the vascular system from an external

source,

Kursanov (3) refers to work in which it was shown that the fibro-

vascular bundles from sugar beet petioles took up sucrose, glucose and

fructose but exhibited a much higher affinity for sucrose.

Grant (18) studied the JptakC of glucose, fructose, and several

other monosaccharides by carrot and corn root tissue. Some of his

results were as follows. Carrot root tissue exhibited a )ag of several

br before uptake began. The uptake of glucose under N2 was less thouI

2E~o tha in ir.He sho.--ad that Jlucor entcred the ice II re jccu-

v~ulated as thc l'ree; sugjr jgjinst .j conecnrtrjrion gradient~. t l-

i'Cas co-~ernicr.11;Cr in the carrOI. ti5Clle oC~iEedd 1!.0!5tt j51C.rir~3 Squjl

d i r ribu! io~ r; aLh.;r [th ire: h us5 i ght o:f the t i .u-. Uptated of glueseI.

rroceededj at j conr rant ratec from~~ 0.0010II sclutionn urst" the batting

solution ~ ~ l ase-as e.H how' e Ehjt th~e ur Eal e OF 3luiiGSGI )nrJ

:e*,e~ril nwhrr csuaors folion- uFptake .: Concen[ trat~C1Tio curse accrdi

t~ liichi eiI ar..i rfierer r, I.iraticl.. 110: did n:t ferii rc. be co~n.; rni d

w~ith rth facr that! a coni cant irat c.1 uputjle t*Jih tiii6, in Fpite OF i






declining sugar' cor..:"'retra on, i r incon sir E::n t .-ai h reactio n~ ratci. ;a

pred~i ted t., rlchaeli5 anid hcntrn k;itt i cs. The mar i nes. ra r-i GI

surgjr uprjl.e b*; carror d-isc; werer frorc 3. to 10 unnolclc*'g b~r and frGD=~

corn roots sure 1ro.0? to 24 umolc.'g br (1 l. I t wa ElIcO:.n th;;t in

carrotl t;Isue the repIredT~ CO., wasr der;.*d preferrentiall*, fror, thi

e nte Pran I sjiuga r L.

Apee..s jnd Eee~ers (203) userng iarro[ and pOtato slices mieasured

0-, consulptionl 3nel C0-. ev~Il[olutio ;i r. o (0.5 umoles m~i) cor~c.-nrati;. ns~

of glui'ose, Thle T.Q did noj :..ar,* Li r.irr fiicanti ,l fromn ..aci: jnd the

addiction of glutose did rootL induci iign~tf cjnt changes~~ ;Ir 02 upruse or

C02 cutpu?.

PE i nihold and i~nhar i 'I dtlror.5:ra~ed ;in jci rie uptak~e me~chan nis

i n car rot root tIssu wC\hic was capal of~j atCI CCrIITmulat in rl -o-meth-,*gl('1]u-

cose. TIhe rate of uptake~ of ,-o-r..ath*,dglu.:ose -.-.ried uith con~ce.-.ra-

Si on approxima tEly a; pirtdicedL b iiichje i njyd fIern itn k~i netfi cs A .

a period of uptal.E the rirnue \ia rinse~d for !0 min ;rnd Lther plactJ in

uater and a concentrationi ratio of ;5: 1 ras mjirtair.=d britsmEn theI Can-

centrations in the ticsue and a-.ele. Chron.oOrlgraphy and jnd lis oif

C02 i ndi cj ed rhat r -r -w thlg iacose rra not ~~ rnetaolIized.

Harley prnd Jeranirage (22) Studied the uptake; of jugari by. becch

mycorrh; :as. They fournd that the~ rate c*1 uptak~e *. concentration

curses for glucsejs and fructo'e formed rectailgular h**pertolat butr the

shapes o~f thi curses wcre different, the m.iir:mum rcat for fruCrGle

bi i ng h;9ghr thar the me..imium rate for gluicesc. In rlhi; rssu ch re-c

is considerable hydrolysis :Ihen sucrose is supplied. iPmrd,

i nh ibi td the abjOrp [i on oi: he.-.oi-.i. When lnoitures of glu~cose2 and

Sr uc toid~ s-tre ;u~p pi ed g lu~c~te w~s prefeiren t;i I ly absorbe-d. The

addition of sugarr raused a re:.pirjrar, siuaie n rmeuooa







concentrations the stimulation caused by glucose was considerably

greater than that caused by sucrose in spite of the fact that a roughly

equal amount of sugar on a weight basis was taken up during the measure-

ments.

Mlorgan and Street: (23), studying the carbohydrate nutrition of

excised tomato roots, found an RQ of about 0.75 in water and about 1.0

in sucrose. The root segments had been starved prior to measurement

and the uptake of 02 was stimulated by the addition of sugars. The RQ

of root tips supplied with sucrose, dextrose, galactose, or reffinose

was within the range 0.90-0.96. The endogenous respiration had an RQ

as low as 0.70 and in mannose as low as 0.60.

Thomas and Weir (24) measured the uptake of sugar by tomato root

segments from solutions of 0.05M glucose and 0.025M sucrose. It was

found thjr mord~ Suqjr Co i rriighl basgi2 Irj Ejiin up arh;r suirOlt \ ii

zupp~lied! a co~mpar~ di to ucO-;t. Sucrose ii mortedI; SUpCTrO [r t

gluci.Zd ini 5urpoT[r tJ ng gro[h :I deCi fed [.:rnl.m l roo:[ .

Whenr radi Ih rec.([ ilicGE firr IFICu~baE.1 ir. Euiroie thereT if i COF.i-

-ider~abl. jmmount of :.
a hoerh~ or nojr 3-, zucrczC is t aler. up a 1 ho~ut ;n.trilon. .ac~rose at

0.029tri and 0.i05rf 1 hroh rirnulated the i-.luciion ofl CO, und i t rw-- rug-

cie_ d F.MEr this vjs duc to~ j ;1Uturade..i ofi respirT~jOTrZ, enz*tn

BIald:Ci (5) areasurtd the upukel; 01i iuiirot b eciterd .cEcular

buindle: Or phI~eile thf~ue: iriii a *jiC.rit* ilf plards.. II. appFl- phlomi

and celtr, .Jc'uljr bundleCS abou~ ;C.~ .0l [r. of L6. th ptk rom~ 1 1 suirose

could bei fourid ir. the- til.ue ii [he form of :u.:rose. ureupa,

irom 0.0031 andj 0.011: tolution, proiedLedd at 6 priogressi-.el, skwarr

race until rhe e-.:er nal sollut;ion contaiind abou[ 10: r of h- original

amoT~unt iof Iucre't.





101

Sucrose waSj takeni up, jgainst icncsntration gr..Jlents of the jrdcr

of 103. Thi ,rates o accumuljtionn t. .aScular LbundlL'; cr phlocle ti;s be

weire muCh higher th~an rates b; pjreracn,ma froml the samie plant. Vsua

cissue jccu-.aul3Led~ Microse at ratej of J to- 16 orales.i'g El- vt br from



P-i.haldJ aid Ellsion (2.; addresled. the qlucstion as to *.*hrether thr:r

wajs j difiusion barrier bet.-seen irternal jubstrjt: ~nd i i~es of resps-

rur.The ,* applied SurnilOw-r hrpojCot,*I segme~nts usi r labeled :3lucose

o~r Iluitamric aci.1 lrn the preihhnce or absence oi 00N ar~d liCO3Ured thC

total jnmoint of i.12 r.ol.id and l'd spec Ii I ac ti ,i t,. The,* .uibj ected

the data tr lrerlce arsalysis and concludedj that !here .*:35 110[ an affe~c-

ti-.c d; ffus~an barr;er beltween thel eaternal subs~rite and! thrIe '.s t

ritich jubjrrates Lre res.pircd. n lt ra a ie I re-a n

JU-'t possiible, ho.ae;er, that sorch j mechanism L(act;i:e rr.miip:.rC) d~f

oi"erate in Use- ;bsen.ce of EdIIP, buc that in its presence !he irrojecules






Euga~r car~e Ic probat.1,~ the ii.ost .tudJied of higher plints ,n

regrdsto uga naecets.Biolass.. (27, p ?i00) state the uptake.

prole a fales:"...IL was~ (cun.1 ihat d.:iI:. i f ju,3di jltr [ I IUC

plj:.d i7 acrarted de cillEd .vate, lus. **er, lircle of their enjogenousi

sug1-r to- the *,ater. Tniis e-ither the~ toniopiast ir retr.:mel.iprm.l

to :ula.* c~.oge..*:r. 01 rhare I Le aC'ri acumuiLatin trach~?ljm in the tell

which a~;...i.el oppose:; the Cui r.Trr' Jlrrusi;nal mn*.ement of iu3dr.

The firot is per~haps r!h. jlapJ.p r Cplanatiucn, but ra~ier the irotica.

o: r-plaJi n i ng not. the rougar c r ignl, be,~ II; came arcumu~ilated behi;nd thle

iniptermeable tOnorljst."






Sugar cane exhibits a large, rapid (1-br duration, 8-min half

time), apparent-free-space uptake followed by a slow uptakte which can?

occur against a gradient and which results in sugar accumulation. The

accumulation uptake will1 proceed over a period of 72 hr. In comparing

rates of uptake of various sugars he found that glucose uptake was mtore

than double that of sucrose uptake on a molar basis. Uptake of fructose

was similar to that of glucose.

Bieleski measured respiration during sugar uptake and found an

increased 02 uptake upon the addition of sugar to the bathing solution.

He does not mention any change in RQ associated with sugar uptake.

Bieleski (28) found that sugar accumulation was completely inhib-

ited by 10-SM DNP, Phloridzin at 2 x 10-3H caused from 10 to 80%/ inhi-

bition of the uptake of glucose. Wlhen tissue was prewashed in 2 x 10-3M

magnesium chloride it caused a 0-20%/ increase in the amount of glucose

accumulated. Double reciprocal plots of sucrose, glucose and fructose

uptake rates vs concentration yielded straight lines (29). The Vmax

reported for sucrose was 0.7 umoles/g hr.

Glaszlou (30, 31) suggested that the outer space consists of the

cell walls and cytoplasm and is in diffusion equilibrium with the ex-

ternal solut ion. "Hence the cytoplasm is part of the outer space

where outer space is defined as the tissue volume which comes to rapid

diffusion equilibrium with sugars in the external solution (the outer

and inner space for this tissue may be quite different for solutes

other than sugars)" (31, p. 178). Tracer studies showed that the

hexoses in the inner space came from hydrolysis of stored sucrose,

Hatch etal. (32) reported on some of the enzynies involved. TI- ,

report characteristics of sucrose synthetase in the direction of su-







rose 5,n the~s;s. / i ~cti titi n th- rie-. rse J Ireci Ion coulId notr beL de-

Eccred becauISi. 3' thE presence or i phosphcetsse which r pidl, hl;Jrol; ic

UDP to UJMP. Stidence For the prrtsence of SUCrG~c--P 5,ntheLr.i 10 wras pre-

3enred, and acdJ jnd jl kaline in.ir Ljces ...reT dcr~CCTbed. The;, couIld not

f indr Sucratre phO ~hor* I as,] En_-jmes for the C,ntheSi-,, ir.te~.rce~n.*ersion

nnd breakdiot-en of hccoce phosphst tes erre identified. The Jamounts 3f

scld and ilk.alineL in'.ls*F.35i S .jry wri h the growth ruC and~ Zhe SUCrose~

scorarge rare (.33) suggesting a 1.0,? role- for insecrt.ise in rejulating rhe~

meo.esnen[ and utiliijtion of uCriioje. Sjcher ct al, (34) p~resen~t a

schemer for' the sugar jccurnuljtion C,cle in immirure 3ugar ceni. Acid

in.ertate occurs both in thce outer CpaCE andj rhlo jtorjgC iC~impa1rtii l'-nt,

Sucrose is h ydrol,-- fd prior to up[JIkE. and, glu~coJ i:. [al.en iip SC..ral.~

times~ us Last as fructose. Sucrose is re-leased tro.T. ;toljgge .;a

ht~drol,sis 0..3 dif'fu'.lon of the booses~t Outt of 3iOrage.

Ilatchi (35) dc--imontra'ted rhe pretence oi iucrose-P syntheta~se in

bjoth lefi andl storage tissui of 'ug-r cane. H!e also showedJ thL; 5,r-

thesis; of Suirose-P by tisslue supplied .4twil luccee. Scoea

r tored nmore ri-pdl, fewii -uirose tha~n froma suerose-P an~d Irere rjpid ,

froml fructose~ th;n r m :r3~ iucrose-P.. Thi5 ic CongiStrTCent~ wih t prop-

ojitionn thjt sugar phaseshares do not penerrat iembra.ws~ as eaiil, as

do non~-phas~osphor~lat suga,-s. Age~nietr, of labeled Jucrcse -a~s lo~st

during storage. Imrile onl/ :Inj.ll qu~anLt S of Sucrose~ --.er Ctored

wrhin iuirosc-P \*.J: supplied t~he s-mmetrr of labell wa: large lly na-in-

acie.Thi-; w-as coil. is tenet w*i h a sc 6.10 or, a-hichi ;ucro ,e-P is

formedc b\ thei actioli Of rucroseC-P c,ithetse ;Ind 5uiroseC i5 stoed

jgainst a sucrose :orcentrraicsl gridiene *.ia the hiJrolesi; of suciase-P

to ;ield Sltored iucrose.




13

in further support of such a scheme Hawker and Hatch (36) demon-

strated the presence of a specific sucrose phosphatase in sugar cane,

carrot roots, etiolated barley, oat, and pea seedlings, parsnip root

and potato tuber. The enzyme was associated with particles which be-

haved l ike mi tochondri a duri ng di fferent ial centri fugat ion. Mend ic ino

(37) had earlier described enzymes in wheat germ and green leaves that

included sucrose synthetase, sucrose-P synthetase, and a nonspecific

sucrose phosphatase.

Hawker and Hlatch (38) present a scheme for the mechani sm of sugar

storage in mature sugar cane tissue. Evidence was presented to show

that the hydrolysis of sucrose is a prerequisite to storage and a rate

limiting step. Mature cane tissue contains an acid, wall-bound inver-

tase and a neutral invertase apparently located in the cytoplasm. The

storage compartment invertase found in immature tissue is absent in

mature tissue. Sucrose storage takes place more rapidly from hexoses

than from sucrose. Uptake of both glucose and sucrose as a function

of the concentration of the bathing solution had the kinetic properties

of an enzyme-catalyzed reaction. In studies on the localization of

ctlr, fir; ii e-r-.. foun j rlh.-lit .mos if n~o[ all of thef Iucr oser s,*nthat-.jse

s.=ar loca ted ir. rhe condulcr ing tiissue, .srd i r i.' poiin ted out th3t i t



Hatchj an ljridou IJ7) prtltented direct evidenic th3e sucrote i.

thet prudJo~nin=.at mornner~lrrs of traniloCatc d pjhow~iiTrthjre ilc. :ugarj care.

71.- jsi.nrncr ofr Ilabelrd Iucrore Ius. .ma~intained through theC siculjr

tis~c v th la. chath and.ter..Pardonizjtion did occ~ur duiring



3iche-r (40)) p~c~rll.cotd -.. .-ar!.-0.nln for e: [TractioPlelrnic I1ucro..




14

Unthesis in the bean endocarp. He supplied UDPG and labteled~ ructose

ind obtlained sucroise ;n wrhcich the labl *r:- predomin~atcly ;n the iruic-

lose moler,. Exper-imennts alrso indicated thE CprcencCe Of UDrPG pyropho;-

ph~or,ldse in the ex.tra3c;[ioplasmic spaci. \lthn latealed fructose nias

sulpplied and suicrol synthsisi occurred i r. he citopls asm~ ;he 1 glu-

case/114C fruitose ratio was apprsroimatell one; thi; sucrose remin~ired

in the tissue e'.Cn aftlcr eve~lnsi-.e w~aching.

B, usinS aceton-extracted chloropljsst from ugjr cjne-, Ha,, and

Ha~Sid (4I) wlere able 10~ ShOu the srnthesi5 OF Sucrose-P fromi UDPC. and

fructose-P and the synthesis of su~crose fromi~ UE=FG jnd fructosr:. The

preparjtion contained phc.5phatajses that h;Jrolyzedd sucrose-F jnd frac-



Schoolar and Edeliia.n (L2) mieasur d stcrete~d iLugar, CO., fi;xicn,



ficated oni \ar~ious solutions. The amount of sucrose rccreted intLo the

bathing solution wasr ineirsease by 10 ft s~odium lodoactate (10-'.

About one-third of the E0E81 Eucrose synthlsizcd during .j 4-d.i, period

ras sre re ted. The ;nh~ibi ter caused no change in rhe- amiount of soluble

sugar \,ithin th-e dist.s .nd i t caused an inrcrsi in the amount of total

soluble: phorns,~n:hate produlcrd. Faspi ration leasurcd in the J.ir).

shoved an FQ of considerable less than I.0 snd this wajs reduced even

furherby 0A.Otherr respiratory inhibitors did not ilicir similar

responses.

Mlany investigations hJ.te been made ol various cn;~mes in~ol:ed in

sugar translorm tionsr~. Onl,; : few will e ment~ioned h~ere. In h-is

re,ica arricle on jugar transfori..ations in plants, Hajssid (Iy,) jis-

cussed the chjracteristics of sucrose SlnthPLjSE anid -ucrose-P c/n-







thetase, the two enzymes most likely to be involved in the synthesis

of sucrose fromn glucose and fructose or from either hexase alone.

Putman and Hassid (44) studied the transformation of sugars in

vacuumn-infiltrated diskts of Canna leaves. When labeled fructose or

glucose was provided, labeled sucrose was recovered which was labeled

in both hexoses; however, no free la'oeled glucose could be found when

labeled fructose was provided and vice versa, an indication that sucrose

was formed via phosphorylated hexose intermediates. When sucrose was

provided in the bathing solution there was rapid inversion of the su-

crose with the appearance of hexoses in the bathing solution followed

by a resynthesis of sucrose within the tissue.

Cardini et al. (45) point out that the equilibrium constant of

sucrose phosphorylase lies in the direction of sucrose hydrolysis and

that sucrose phosphorylase has not been found in higher plants, A

study of the characteristics of sucrose synth~etase from a variety of

plant tissues is reported. The equilibrium constant, K = (sucrose x

UDP)/(UDPG x fructose), varied from 2 to 8 at 370 and pH 7.14 in dif-

ferent experiments.

Leloir and Cardini (46) studied the properties of sucrose-P syn-

thetase but point out the difficulties caused by the presence of in-





IG

ber.tween sucrose conrent jnd jcid ins~ertise actritic,. cdinets

ac Cini; L: .,j high .~iur In3 cr~nas of hi h sugar usage snd 1-.r. dur ingl times

o~f hiigh -ucrosez sto;rage. The, sulggest that~ high i~ver~tueS aCti*ity

pl~re.n~i suirojSe -.Lorage and1 that during periods of 10=;1 hc ost: dema-nd

hydroljjts is due toj ~)alkalin ;ra=ertaic br-;hch ;s not, assoC Dscd **;ith

the ..acuille but locrated in rlhe c*;top~Fleam. ;he; jcuggenr rhat rhe -acidj

Inerrrl se ;j located ir. [hS rrll and~ ;1; the tienopl jgt.

YKurianl. ct al. (4j) ;omosied the locali4liat~innd p~r...erl.ies of'

herok~injje .rithl upatak chjrscterisre c~i ofco~~~cndutn rirsus ircAln sugar

bee-t. TIhis lissues takes; up glucos~' e rust!sr th:lrl irructjSF andc ihe: boIXi-

kinese- jssociated vi ch the; sru~ctural 2lements~ of the rells h:1s a highe-r

afillnit, for gluCcje thjn iiDir frlctose. On rhis basis rhei ;lugge~st

that. he.,so.irase on1 [h-- ilmemrin mre m be part of the uprej'.e process.

The uprjke o; sugars :nd~ the Inrsetl binding choairacteristcs r

;ee,[ I-avre been Srud;'ed ;ntensi.ely.

P.0thst-;n (5Sr' ~resents~ s.eral l~ines of~ e.idenTce LO sh;eu that[

uranl icr. aifecits the up[;Ike ojf glo-ICose I0, east due to: ;ts bihinfrg

to-. rhe zurface and nor io an uotake~ into the iatoplarSm of the Ce 11.

Rathsrtin and Mcier 151) describe the com:pedE~c on fr urBan,I ;or, b;-

rrran thre :east cor~plrrming Ir;:; .arrd rar'ous~ c;.-le;raq agents 3dde*Ul ro

the baithiig solution. On1 rne basis of rhis ..,rk [he*; cond.uded th::E

the" banding sires on the suresee 01the east cag[ Cllj rere pal~pr ophaaces,

UJ~;.-aii.. blrck.l jbour 40~.; jf the uotie of glucose in ,part I50).

Se-.eral other iorions, iincluding io2 ng Ci *,nd win ,trdt

rhe s1 'aice of y/fdit ells butr ujrarl ;or. fOrms ? much l.cori stable-

romp le- 152). Data ..ere presenr-. to Shiou r'lat .hreas~l thC other

cations rere bound to the ina ;rne sic hjt b~nrd ur n~il lon, rhG,- did

nMr Inhibit the upr!tak of 31ucose.;






Data showing the amount of various ions bound to the surface of

yeast cells as a function of the ion concentration are also presented

by Van~teveninck and Booij (53). They showed that in the case of

N2+ or Co2+ when glucose was added to the cells the metal was dis-

placed from the surface of the cells and appeared free in solution.

When the glucose had been taken up by the cells the metals were again

bound. If cells were first poisoned with 10A and then supplied with

glucose a small amount of glucose uptake occurred but was complete in

15 min. The amount of glucose uptake by the poisoned cells was the

same (on a umole basis) as the amount of uranyl ion bound (on a uequiv-

alent basis) by nonpoisoned cells. It was possible by adjusting the

growth medium to vary the amount of phosphorus per yeast cell without

causing irreversible damage to the cells, The amount of uranyl ion

bound and the amount of glucose taken up after poisoning were both

reduced in phosphorus deficient yeast. 'There was a good correlation

between the amount of uranyl ion bound and th~e amount of glucose taken

up by poisoned cells. When yeast was poisoned and then provided with

glucose the uranyl binding capacity disappeared. The addition of 10A

alone caused a 50%0 inhibition of cation binding which could be reversed

by washing the cells in water.

VanSteveninck and Rothstein (54) present an argument to show that

in yeast, sugar uptake can proceed by facilitated diffusion or by an

active uptake mechanism. The faci 1itated diffusion systerc-c r, bei

demonstrated wi th gilj; rosec upr jke~ by uni nduced cel ls and e i h g luii:e



thnl Cneryj re please b, !l,CC I,si. i r. sire of~ [he f at that gluc65i






,s~stms are diff~erent rrlth rellpect to I[Jln bind~na, iclFcr o fi. on;:

upE.-rke, concentration-, of uran,l ion requirid to ir~hitbit uprjake, k~inetic

psramerers, and pjtterns of pecificiti.

Fothui r n Andl Vanirtevennind : () sulnrnarized wrork. donc on, uIprake.

jnd mental b~ilding~ t, ,east cells. It ~as p~ointed out tha. r .e ;nisii~i-

Ctr, CfifcI' of ur?... I on jnd NliL jrl: n1ot du to diTFpClicefler of: i

requi red car for.. 1he corac lusion is r TCjh- d that II.5 phoCphOTr, I i tes

to which uran, I o,i binds, are used continuoulsl, il. gluicoi- tra..sp~ort

and are; rc'ciinerTa td cont i nuouslyI b; j, glCol ,''C. In the ,east s,rtem

a close corlciaticn is pictulred b~r~etwen gl~ccolsis and uptak~e ;rnd

gl~oi*, tic ATP ;E arsumetd to be the: encrg, source for dr;.ingJ uptake.i

Cjrrier jnd gl,Col,tic reactions are thought to be in close geegreph~ic

prox~imi t,. In their Chscre~ to Explain the trsr.5portt of sugars the

cjrriers for fac;l;tated d;i."usion and3 for icti.e Ltrnjport jre ion-

silered to be the s.-c. WIhen act;.e tranlsport occuri the jmouunt of

carr ier jmai l jble for fac il1itra ed difuior I' s rO Edu1ced.

Wheeler and Hancher, (561 p~laced oat roots into n. I and 1.0 mM~

uranil acerate for ..arling periods of time and then made- ilcctron

reiierogrbphs in rwh ch cristjls, apparenli, comlposed oi a ur;anium~ complex,

could e abl, be iecn. Afer A 30-mir. treaciment fo~llorwed b, a :0-min

desorpt~ion the u~ran.71 complex wasi sharply; local;zed its Ceii rrillk

irsteircllular spaci-t jnd secretor, products ir. dirctr contact 4.ith c~il

w~alls. Wlith lonij.r L~-tretment times, up to 50 br anid the iowetr concenl-

tration, thec uran*,i comTple. Crystlalb could be: found in .e~;cls in rlhe

cICt6rl~ater3a in the .j-uole, Othcrs-sise the ccleli \ere nomasl w~i t' no

uran,l ion free ;n Lth c~toplsmi or in ccall orgajnillei, Lrn o

applrent;, caused a deiinite dilatic~oreo the~ mmbranes~ from a normal






width of 90 A to a width of from 150 to 200 A. This effect could be

seen on the plasmalemma of treated cells and in vesicles which con-

tained uranium. They concluded that few, if any, free uranyl ions

passed through the protoplast and that uranyl ion in addition to being

bound to the plasmalemma is bound to cell walls and to secretary prod-

ucts along its surface.

Roseman (57) has reviewed the literature on a bacterial phospho-

transferase system that is thought to be responsible for the uptake of

sugars. The system as it operates in Escherichia coli consists of

three protein fractions: Enizyme 1, Enzyme II, and a low molecular

weight protein designated HPr. Phosphoenolpyruvate (PEP) is the phos-

phate donor, and a variety of sugars including some disaccharides can

serve as acceptors.

Enzyme I and HPr are found in the cytoplasm and Enzyme 11 is

associated with the membrane. Enzyme I and HPr are constitutive where-

as Enzyme 11 is constitutive with respect to glucose. Most Enzymes 11

are inducible. The specific sugar requirements of the system are due

to Enzyme II. Enzyme I and HPr are common to all sugars phosphorylated

by the system. Enzyme I catalyzes the transfer of phosphate from PEPP

to HPr which serves as a phosphate carrier. Thi specific Enzyme 11

then~ cu tl onesr~ 1~1-. Errinster ced" pl-.cr Fphate f'ri.T ph~or phate-li-frr to th








exagernO.Ou iuqri -nteir thli cell ias Fugar Fho~Sphates ar.J this i-. d-



proceiC ; ij.Cll-3 jcti'.e traiport.






Ctlp loocu Jureu'- jcTcullated SUCrose1-P when ;ncuba~ted in

sucrose anJ it i: thought ;Ihat the phi'SphotranSferaS' Sjiem is opera-

Si ve 'T thv uptuike aInd FhospihoriIh[ion (5.0).

Edeslmjn et al. !.ES) wo~rl.e~l rwith scutlle,; roots and shots of CoLs,

rpie, whea~r, jand borln:,, Thiey ShiJwed that the scutellum Contrained a

higher rjtior of salcr350 to heicose thAn did the~ rOOt or 5hoort. HomGe

absorcplion wasd ;r.hibltedd by about h~alf wheln e.-.perimieints Ivre run under

reitrogen. :ubstantial sucro~se form~ation rcok place ii, the scuttiliuni

under rlitroge~~r., whreos incorporation~ into jmino-acidr, Dr..Ides, mralii

jcid, jnd sugar Iph:-sphaeS waS co~nsiderabl, reduicd. In t1.850 li.Lu?5

frucrosP is ib~srbedl ;t aIbo~t half the rare of glucoir. ur o-

phates, sucrose, gluci;e, rruc tase glutarnic and asparrtic acids and

their ;miic~e,, mnalii acid, i02, jnd pol,*sacchairide wreT found to contain

label after appl,ing trrccr bmounit5 of labc led f'ructose or glucoje.

The scurlclumT C.js :hour.ll to contin I;Ichl lowei(r lE.elS of~ h~Cdroi;tc

enz,.ne= than th~e r~orl or Shoots. All of the en-,*nles necessal,; for

the fouraition of so.:rosi fror, herose wercr found ;n [be Siutellum and.

ir jchenP is presen~ted to Ehowr (hC path of SUCr.oSe 5,nthe-5iS wh;Ch in-

.oldes5 thi entimi sac3rose-P S,nthe tase.

Hurophre;s and Carrard hia~e publiihcd a series of paperj dealing

w~i h 110i uptake productLion, s toraqe and lealkage of Sugari b,. the

corn scute lIlure Thci dlemronstiritd Ehat glvcose Iiprsae pro~ceeded jt a

ionstant rare e.ea through glucose in [be bat~hing SolutiGO was Iargeli

deple red of gl~ucose ai ar risult of uptjlke (60). The racir of givCose

urptake ,ras shown to war,- depending7 on the conditions anid length of the

prior incubation of the L;lsue. Changes in the tissue content of

various csugarS and -.urjr phosphatss jfrer ,erving periods of tilie in





21

water were presented, and it was shown that mannose inhibited the up-

take of glucose. Data were presented to show that the corn scutellum

accumulates carbohydrate mostly in the form of sucrose, the content of

starch and hexose being low,

Experiments concerning the glucose-free space of the scutellum,

which involved measuring the amount of glucose in the tissue after in-

cubation in various concentrations of glucose in the presence and

absence of DNP and mannose and the measurement of glucose exi t fol lowd-

ing transfer into water, indicated that the space was intracellular

and that a carrier was not involved. Fructose and mannose occupied

a space of similar size (61).

When incubated in high concentrations of fructose (0.1-0.9M),

scutellum slices synthesized sucrose, some of which was stored and

some of which leaked into the bathing solution (62). The leakage of

sucrose was reduced in th-e presence of Mg2+, r,2+ or Ca2+ and EDTA

increased the leakage from the synthesis compartment (63).

When sucrose storage was measured after incubation in fructose


im. lurLITe .hit in =0und~i EMEi the O lufj IjCe 2n ut ation .T.@J urn un. .. re~




abo t a lo, dcre se in cor d ucrole air.-c Iruics c r jr .5 l;-. e-o-enousi







Irlltj ; el :1he-n Iu.:rilj e .ij -dIrJd rio the barh-,n.) ;;lu~ti j.. .:onr:.ni n..)

Ops.I.Tau' .. il''unts .JI' he-,it. ThIs *:.tul dj not look. beer, Ilhe iase if jn.cros

.i.-ru twi~n.; ~...Jr Cl ..=3 ,r:sr- r : I p tal~e .:.r .b rin3j [l process-i r upt-si'-e.




22

The loiSs from~ st.ragi e was mecasured b, loading thie Stora'ge CompF~rtmen[

uith~ o Euirose andj then ;ncubating the tissues at different pH \jlves

and ri th- ,nd wi thout "cold" su.:rose. Ilore sucrose wras lost jt the

higher pH Lalues and th~e loss was greater in the presence of "cold"

sucrose than in wraEtr ind;cating an erch .n7gi betueecn errornal iind

c tored sucroe.

The addition of Fructose or Jlucose to s~~caeilum sl~cice (L)

resulttd ;n a strong, aErObic Fermenration and the conccmmicant pro-

duction oF ethanol. Increase jucrose i,nthesis~ upo~n ;rcubation ;n

fruCtoseE accompaniid an inlcreasee In gl.iol.sis i cholrt an increase ;rl

0., uptake~. This sujpiorted- the ;de3 that tli,col,tic C.TP 11.ugh1t be re-

sponiible for sucrose- synthesir. ihen ;ncubated in rnrir thef Fi: ior

intact scutElla wras about ), while that for slices wras near Lnit,*

It rwrs cone lud-.d, on th~E bisis of the lete It of ad;Ous phosphO-

fructokinese regulators during d~iffrent rates of gl,col;sis, th~at

control ofi glcolsis ;n rhs sctiure 11.1 a c -crtetd through thea ;avil-

abi lit, of SUbstrteC a-ld th~e distritu;uton o~f aden:n- nuleoti~es and

inojrganic phosphate.

Prcticatmncit s.'ith tr; S!hldro> .nethil I)aminlOnethane (trisi pjretenltd

the StorGTe~ of exog~nous su!crose bult the inhibition could be reserred

b. h1,drogen ion or L,- fit Mn2+, and to d lesser Ea tent tig andl (O

Sucrose storage from fructose was5 little affected b, the pretresatiment

rl th tris (66).

Pretreatruent wri h uran*,I ni trate (67) <--eS Simi lar in i rs effects

to pre[treament with triS in that storage of: eogenous sacirC-a was

inhibi ted and the inihhibitin could be rev-ersed by. H, HI+ and~ lo Soile

er tent Mrl21- Uran,l ion pretre~arment only slightl,- inhibi ted sucrose





23

synthesis from hexase and the inhibition was not thought to act through

the uptake of hexose.

It is possible, by incubating slices in high concentrations of

fructose, to build up considerable concentrations of sucrose in the

synthesis compartment. That this is sucrose and not sucrose-P has

been demons tra ted. Upon reducing thle external sugar concentration and

inhibiting leakage, the free sucrose in the synthesis compartment will

be transferred to the storage compartment. Experiments with mannose

(68) indicated that whereas mannose inhibited the storage of exogenous

sucrose, it did not affect the storage of sucrose which had accumulated

in the synthesis compartment. The suggestion was offered that the

storage of synthesis compartment sucrose involved a non-nucleotide

phosphate doner such as occurs in the bacterial phosphotransferase

system.

Pretreatment of scutellum slices with HCI (0.01M) did not inhibit

the storage of exogenous sucrose or the synthesis and storage of sucrose

from fructose (69),

Recently Humphreys and Garrard (70) suggested that leakage is

from the sieve tubes and is the end result of a series of events which

include intercellular sucrose transport, vein leading and phloemn

transport, Several compounds, all of which can either displace or

form complexes with Ca2+ and Mg2+, protect the leakage process which

is labile at 300 in water.

Since some confusion exists in the us;. of v~ri;,ii-..o!, cjrcerning

uptake studies, definitions of several ter...s ui-j n the,? -r. -e:ntrn

of data and the discussion are given -.ere.

Ilpr~lak--Th 5 tcr-in i uJ d ijr the disapp..:ranc- e of a iet -.ace~







from the b3thing solution and ;r not nearnt to ;mpl, a parricular neech-

anirm.. 50oe duthors should uie abrorprionr.

Diffiujion--TheP net nso?,ement of molecules as a res~ui t 01' rhC i f

thermal nl.tior. Fromi a region of higher LO One of 10,.r0( concentrration,

Wrhzre a miembtrane 15 r ro ssd rE:istance mayI be due to the l im; ted number

and rize of PoreS in thE miembrane~ or tc the rolubilIi ,* C harac teri stics

of the jolutE in the Intmbrane.

Faciliti ated di fFusion--Thi s is a procerr in which a co~nitr ntratio

grajdient is the dr;.ing forrce as in diffusion and the proics s.lads to

a disapipearan~ce of thec gradien~t. The process is thought LC in.ol~e j

membrane conntitucrnt (carrier) locj;ed o~n or in the Is..embranrr Hhich

"fiac i li tatLs diffu srLi on. Faclitated diffusion of a soluito jiroir J

membrane; requircs ne input of' energ, other than that nee~de to na;ntain

rtructure. Itr me, showr a high1 degree of spC~Cifcicit and Ikinaticf are

likely,* to :hhou rjcurjtion thus n~ot conForl..ung to Fick'=. law. of d~iff-

si100.

Actrive- rransport--This process in*~.ol.S the csz of metabolic cncrg,*

3as dr;.;ng force. Ir. ls capatie of bringing about the accumulat~ion

of a SubStance. agjir=1 its concentration gradient. It ij ?enerally

characterize b; a hi:7h degree of specific~t, and sj~rationo kin-tics.













METHODS AND MATERIALS


Preparation of Scutellum; Slices

Corn grains (Zea mays L., cy. Funks G-76) were soaked in runninJ

tap water for 24 hr and then placed on moist filter paper in the dari

at 24-250 for 72 br. The scutella were excised and cut transverse 1,

with a razor blade into slices 0.5 mm or less in thickness. The 1i~ceJ

were washed in distilled water until the wash water remained clear snd'

then were blotted on filter paper and weighed into groups of from :*,1

to 1.0 g depending on the type of experiment.

During IrFpr jarain,, th- Slier vetre [hroutlhl nli)."*I C0 thjt gjch

groups o f Ille r .*6j: a r indon~ r I, le i onl fromn S :- 10 0 Icu rt Ill Thli

resulted i nr. i cI IeI 3cr.Earemnt rwhcn m~easurem~: nS Oi uptakei .:r &c.;u-

mu~~lationl wrITI .m..,dei~ o. upl~ijca rgrups~ 01 Eslices fr~~ro.. OeJa,'s prcp..-

ration. e uLts werET notl as. cons i i-tnt w.ht n dup lct .e are compaFredl



Urnlu:r cirherrwise nored, iniiubsticon r.Evre carried Out vi th 1,CI r



EralnsuicL icienrif it (r..npar,, rNew truns .ick, II. J.i rotating jt jpp.rorr

imael 10 e*amnThe :LOiu.e oi Soluti~on was~ Uujulli ]Td *il








iC m leS tare i nc la e ri(chi"J '' .r hr .;. i re w rIa inte tas p' "oI ~I I ! lr LO)E






rrucrose jrnd reducinlg disacchairides Here jnalyzed jccording to

thle rielson-So,~c?~ogCi cpper reduction michod I:71, 7-?) as reported h,-

5pio 13).The alternate copper Ieagent suggested b:, C~omog3 I ra

sEid.

L'hin samipling the: barhi~ng solutio;lnS for 511gers, amorunts[ beCs**eer

0.1 ain 2.0 mi wrer tak~en depending on rl~e ioncenrr. ti~on, and appro-

prjiat d~lutions wrerc madr so thar betueer. O jnd 140 ug of gluio:e or

fructose erecr used fo~r an1al,sis. T.Jrice this jmount wasr usd for disac

ch~arider other tha~n rucrose Ab~srban~c e wa rejd Or, a F~.letLL~t-Summeror,

flaoel 5.005 photoelctrirrc colrirrreter.

Ti5Elue 5ucro"e r:.-s etr[a[ted b; pouring 20 ml of boiling 20,:

ethajnal over thr cliCes and cOntInuin~g the boiling fo~r ;0 sec. iThC

slice: nrre usrepdo in thc jilcohol folr .0 nain, the allohol 1-35 dectntifd

anid thes pracedure repeated.j Thi 5 li cs r -*6re ther. rinsed three I ithn

rr; h 5 mi portlan of Citahol. The com~binecd C.*.LraC~ing solut ons wrecr

evjporated almnort to drincrs oil a steam bath. \.'ater vra-: addcd to a

voilumc of 50) mrl jnd 10 darops of 0.1.* Ii Ia0 added ro adjust thes yn. Th~e

Treulting ago~us solu=:ion~ was~ frozen, Afrtr rthandiig rh 50 uri~l n U.9

ccntrifuged for 10 min ir. j clinical icntrifuge. for the de-tereda;ritio-

of' CucrsE~, 0.1 fr1 olf this jolutio~n wa~ u~sed.





Experlments welre carried out r. I warbur? DEspiremlCetr ati j30

Thle direct method~ for CO, uWE used 174). The amount of rissue added

to the flasks~ <.as either 100 or 500 iig. when the sli~cs werrp prcpared

the,; sriE placed <-.i thout weighing~ ;nto 25 mi Eriennme,er fla;ks in 10 mi

of rater and incubiated for 1 br at j00. The wrater incubatiorn remo.en

leakatalr sucrose (64). Foliclw:i ng the i ncubtjlor, he slIi ces re re blot ted






and weighed into Warburg flasks. The sugar solutions were either added

at the time the slices were placed in the flasks or added from the side

arm during the course of gas exchange measurements.


Metal Analysis

Uranyl ion was determi ned by the method described by Rulfs et al.

(75). Absorbance was read at 400 nm as suggested by SilIverman et al.

(76). Aluminum was determined by the method of Gentry and Sherrington

(77) as reported under procedure A by Sandell (78). The extraction

was made at pH 5. The purpurate method of Wi lli ams and Moser (73) as

described by Sandell under procedure A (78) was used for the determina-

tion of calcium. Magnesium was determined by the Eriochrome Black T

Method (80). The permanganate method of Nydahl (81) reported by Sandell

(78) was used for the determination of manganese. Cobalt was determined

by a modification of the Nitrosc-R salt method of Marston and Dewey

(82) as reported under procedure B by Sandell (78).

The methods used for uranyl ion and aluminum are not specific;

however, since samples from untreated controls showed zero values

interfering ions were not present. The methods used for manganese

and cobalt are specific. Magnesium, in amounts that would be present

in the solutions analyzed in these experiments, is reported not to

interfer-e wi th the purpur-ate method for calcium. Copper, iron, and

manganese do interfere to some extent. The value obtained for calcium

in the control samples was low but not zero; however, some calcium

would be expected to leak from the control slices. The method for

magnesium is not specific, but the amounts of interfering metals in

these experiments were too low to cause significant error. The aRouTilsl

of calcium and miagnesiumi flounj b, u:ir.3 these methods agree very cloel,-







\rith tho~e found b, ato~mic absorption FpecCtreacep, in tearler wlork b,

ilumchr-r s r~d C..rrjrd 1.70).













RESULTS


In the first section kinetic data will be presented to show the

rates of uptake of sucrose, glucose, and fructose with time and the

variation of uptake rate with concentration of the bathing solution,

it being assumed thiat sucrose uptake occurs without inversion. In the

second section the assumption that sucrose is taken up as such wrill be

justified and the effects of several inhibi tors on7 sugar uptake will be

presented, In the third section gas exchange data will be presented

and it wi ll be shown that fermentation accompanies sugar uptake. The

fourth section presents the results of a study of metal binding charac-

teristics of the scutelluml slices and the effects of several cations

on the uptake of sugars.


Kinetics

Figure I shows the cumulative uptake of sucrose from two concentra-

tions of sucrose over a 2 br period. In these experiment lth fiirs

sample was taken I min after adding the sugar solution =.0 rte iliice.

When the bathing solutions were analyzed for sucrjic .j s..all

amount of glucose was usually found except in the sample. I; Latr, atI

min after adding the solution. This glucose could hav; cvnli friom the

extracellular inversion of sucrose cr from glucose difiuilrng cut of

the tissue following Intracellular inversion of sucrose. TbeIso-

the data from which the upper two curves in Figure 1 were calcull us.li

The rol;d lirr in FiguLre 1 repf858Dts datj cjlcu~latedd (froni- clui~nr.~,
















/ O'

r, O




o b~
:Ti rise red n

Figur L urs pceV i\e. Oego lcsntue
inf eah la n T lice ee icb td frIb

in a-arergiven I rin e, an then 10 mi f m g r



th as 0 min ha er eue o 80n















~I_~ __ __
~I~


Klett units


Table

Method of Calculating Data"
(0.00750M sucrose)


1 2


Decrease
in sucrose
due to
uptake


Due to
suc rose
(3-2)


Decrease
in column
3


Concentration
of sucrose
M


Time Without With
mi n invertase invertase


0 0

30 1

60 3

90 4

120 4


88 88

84 83

76 73

66 62

55 St


0.00660

0.00623

0.00547

0.00463

0.00383


Values shown in Figure I were derived by multiplying the figures in
column 5 (dottedj line) and column 6 (solid line) by the appropriate
conversion factor.







Tab le II 'a; i gnor ing, th.e non- iniert.1-.e -It reted complei hic.

i t i s asumrd rhjt glucose i s comin.) frm~ exrale li11lar ire.ir:ion Jnd

thel ~Lu:rose` inverUtFP but notI taken.T up i i notL counteI as i5urtiak~. The~

dotrzed line repres*:.-nt. the actual acreasej in :ucrose of rhe =olurio~n

(ca~~ljculJe froml column ',, Tjble 1.. Th~e two Is thods J0 no: result in

large J;iffrences in calculated uprake. Table I is presented~ to deI--

unstrzte t~cime method of m.eaiuring jucrose uptae. he..1.3n-r

'uLroSe uptak.e data p~risented~a ..a alculated t., thi .nethoJ r:Cr~:esened

bi rhe solid lines,

'Ihen thie rinet- ursrr unsc rrichd~rjaun bi' ucrior, i.Tun~edi-sal b.3fore

.adding rlhe sucrose, an uinme-surra s~nou.e o~f .rater adheircd to :he slice:

andl EG :he sides o~f the F lass and caused a dilation ofi trl; Ijdded jugar

solution.. There Is probabit also j r'ree npjce .coluT.. in t~e slice

which causes di Hlrion Co rhjt jnal,*Sis of the r'irst sanples i ndiclted

3 sucrose concenirtratin cornsirc~rjbl lo--aer than that vbi~ch .-,a in:Elalls

a3ddd. Thiis decrcr.s-- ;in con~centlratin can be Jccounted for by dilucinn.

In Ihe i-rfpejriment Of Figure 1, rle iconc~tientrno I mirs after adding rhr:

sucrjse <.asr 0.00fa.0,I whe-n 0.00j':O00 iucroce was jddedj s.]n 0.0132--#.1 Hhen

0.lr)150M1 S..acTros was5 adae.. These c<..rn~rentration canr be accesi~ired sor



case of 0.002500; :..**ro~se. The E p~ro fojr 0.0050%SJC sucrose :would be Imr

ai~l, f l eif frt wis in~de ro ~e~ter~rnine thet amount: o~F vlatr ,nd Eree

space. Thet problems was gnored byl r..e~asuring upr.--'tl *"rr~m melc time [he

fisrs ssample rwas r.ake. Thie ij.[3 Fare prasanted to Ihr.r that !ther is

Iict a large, rapid: pooJr Jf uptal-e -men :uCro.e i. FirSI added t, rth

EIS Sue.

The~ rat1e oiir u tal..e ;ocrease= iifter C:Ie firTC 3 niiri. 6.t hellr Lhe




33

rate gradually increases over the first 30 min or there is a delay be-

fore uptake begins. In subsequent experiments the First sample was

taken 15 min after adding the solution.

Notice that the rate of uptake was constant with time in spite of

the fact that the concentration of the external solution was continually

reduced. In the upper curve the concentration at the beginning of the

last 30-min period was 70%/ of that at the beginning of the first 30-min

period. In this same series of experiments a constant rate of uptake

was obtained with 0.001M sucrose (data not presented) in spite of the

fact that the concentration of the external solution was reduced to

40% of that added.

More experiments were run to determine the rate of sucrose uptake

with time and the erffct of sugar concentration on uptakte rate. The

rate of sucrose uptak~e with time is shown in Figur~es 2 and 3. The

curves are more unifcrm at the lower concentrations and as the concen-

trations increase the curves become more erratic owing to the difficult,

of detecting small changes in concentrated sugar solutions. A water

control showed that the leakage of sucrose amounted to no more than

0.2 umoles per g fr wt over the period of time during which uptake was



Thi. betl app~rOii.ne~tic-r. C\f .ll GIj [hL cur~i; 3 ;-earn to: bet j trlraighr



F; 'ur 4 sho**C (ht ffe;,[ .:.t iucri*(.F i~cC~=ntrjtion Or,n iarote UpI-



perijd j rr rrich uptake~ <..az re:asur ed. Ais seen t, the twior ctical

CUrse,~ the djja~.: cTI r, <...II ll;h the tyIp;Cjl rliihis*-li and~ herntcin

h*pertsolic subotrjte concentrats.-n cur.<- foir which h thF iOnItants werTe

deri.ed r'rcurs j L~ncij..co r .an Burk, pl.:t of the data,
































F i ure 2. Sacrros? Uprtake Vs Tarne. In thtse emptiiirianen I -4 of r I ce-,
vwjs incusjted ;n water for hr. This r1as I:licIEed b;
2-13 c.! rinsr; and then the~ upralke rDlution v*LL add~d to rh-
lis.Tcn mi or' uptak~e C0'uian~ wasj idded sild th: arriot~nt
of sucrose remo3 0.0 in tii 5olvpling was taksn intou jaccoune
<*hen r:. 1 cu la t ;nr the~ du e Th i e e r.p : >= '.'.n
Ii~n jF ter jdd; tion oF th-e upta's solurion I.ir..s _. rr- on r1.<
?I jph).Sml;0"05n wr e !r S.i. Each
L.u..E rieprscnts .jr.a f roJ.1 1 d:*,' cx.


































0.005M


T i mie mi n








r---'--

P


lii3 0. IIt




60 r.3




20. ~r

L. ..*

5 0 351





45 mO 1n .


0)











I~-ll-l---'UII1----------111-1-111


CO


OE
L.

a- G)
X (
Q 0 9



E- m
me t



ERc
OO C

L e-

G)






m -- a







oom~





*0



oa




L- 3C



CCC

1 GI



Tc


\ m
d 1
c


L'S





Jud jon.1 .].]d. O...r._.-







The erp-rim~ent or' F;igur 5 ShlorS Sucrose5 uptaker; wh~an the btjhing

solution is rmain:oined at a constant sucrose concentration. In thi:

ezper ir..ant th .olumae of the t~athing Folution was reduced to II ml fo.~

greajter accuracy. At the end of i.ach 15-m~in upt:4l.; periodj th- solution

\as rzinoved snd fresh 0.0018I sucrose wras a~dded to, the slices. Th~e rate

of uptake cai be seen to increase v,;th time c.er a period of hr.

Figures 6 and i shot** the rate of rjlucose uptake \th ti ime. As

w~ith suirose, the glccosr uptak~e ca~r.es roughly, represent straight l~ine

rwith possibi, a little miore tenden, for the rjtes to iecre~asL a~ r;t

tIme. In thet case of 0.005ti glucose thc concentrratin of the sugar

solution <*as reduced b, about half during th-? course of th~E erocriienen,

,ct the A~te olf uptae c.rr the lajst three: periods Was about thie rjise~

Vler; simil~ ar data uere collutedd usin3 fructose but~ rher-.. are nlor pre-

senterd.

Figure 8 -hou~s the iptalke .5 concentration datj for glucoji and

fr uc tose. n trne iase of gluiose and fructose thle data do not fi t jt

all urhen an attempts ;s ma-de '.0 find the constants Vr..a, and 1:rui bi clort-

ting the data according to a Linewcaserr an~ Burk plort, fleither di the

data agree Ilith whatr would be expirCte if difiusiOn wepre the drivings

force for glu~cose uptt.lli-

Uranyl ion has been jhown to inhibi;t sugar uptalke 1,67) jnd to~ act

at the cell surface (5.5). Uranyl ion caused a partial inl-ibition of

glucose anld fructoSe uptak~e. This was true both e-henn uran,I ion ass

added 10 the uptake solution and wvhen the .lices werre rr-alted r:;th

uran;1l ion prior ri. the uptak~e period. Th. dotted lIn;:s inl Figure 4

show tht uptakLe vi concentrrtior data for glucose and fructose a-.hen

uptak~e -;at Ireasured fo~l loving a pre-treate~ren rri th urar,l ion. Tat..e 2

































Figure 5. Sucrose Uptake at Constant Sucrose Concentration. One g of
slices was incubated in water for I hr followed by 2-10 ml
rinses, 4,2 ml of 0.001M sucrose was then added and 1 min
later a 0.2 ml sample was taken. A second sample was taken
15 min after the first. Then the entire solution was removed
and 4.2 nl of a fresh 0.001M sucrose solution added. This
procedure wars continued throughout the experiment,






16 -





12











30 6090 F2


T remi






28 1


15 30656
Time, min

Fiue6 lcs ptk sTm. Tedaafo hc hs

cuvswr rpae oefo tesm yeo
exprimntsas hon i Fiure 2and: v thth
exepio thtguoewsth ua o r

















200

/ 0. In n

I F.


120o .05n









1 -1









Ti mir .1-



Figure 7. Glucose Uptake Vs Time. The data from -thich these
cur:cs werie p~reparedJ comrie f'rom the senser r.*pe of
periments"[ ac sh~or-an in Figures: 2 and 3 \-si th the
BKCeptican th5t glucrOSC \*JBS thi sugar Lak~en up.





43













Om
LO
VCI


C-LC
(OV

ro




fGL
OX


C -,


OE


ro



oa
OC





OU
o














r.



IJI


a,
ul
O
U


B


, I ~.. I..L. ... .c _


.s t,': 0 n d :o H






















I~ I


I a d





o v


II V
|~ s

t_ O








































It ,


ItI




o~ 3

IT~~ j


lit C
G rar

sc c.





.- a .

r OOC 00
cau a. . .-

C LI o ~
a .-- ai




L-C
L U i O L 5
E P u- >C
n A- ~ .- C




Cr a-c .,

10 -


.I T








-t L. C -
O~ - 130



I-C II
c j .-

o r le OE-r.




4.L3- .C
4.- i D *
I -' C~ G. L E
<[ *** d d






rO JC

(1~ c-- -- O


. 00 O

La O 1 T I
d~ 1. 3~

-- C D

C ~ ii .0 -
















Glucose
concentration Uptake period Inhibition
M hr %


.001 1 58

.005 1 54

.005 2 53

.01 2 44

.01 3 45

.1 3 34


Table 2

Glucose Uptake as Affected by Uranyl
lon in the Bathing Solution






46

jhumsj the effect or' urjin,l ;ion on gluccst uptake'r when urinl;, ion ,.aj

adde~l to ther ulptal e solution, Ilocuce that in tooth pretrreatmorst anid

crearrentr during upcjlke, th-e inhib;ition ras greater at low-.
[;ons3 of Ilucase. The in~hititioni vas5 nreSte when the slices <-.ere pre-

created it urAnI,I ioni. Th is ii perhip5 .I result or' j long~ trm cr'fect

or LranI ion iinic in rhe prelrtreament apesrimentsit uran.l ;oni waj first

applied to~ the jii.:es 105 iil.. prior to the uptjlke per;od.

The glucose uptakle rs concentrjtion cur=.e sitir uranyl ion prc-

trejtmenti rcjsembled, at least at lo- concensrurioni,, a diffusion curse

(Figur~e i). Since glucose is niot dccumulatred b; th.e ..issuc bL ;s

rapidl, used for sucrose J rrlhesiis or ;s (frmanted (60. 65) thi question

arises as towehrtekntc rsne re tose of uptake i.c.

th-ar .glu~osa is. limliTed 'u, diiFUsionl or thojd 01 [h2 hb.-okinese: res~:CiOn.

Jone.. 1,83 studied r1.0 propertiesj of he:
and .rports che Kr. for glucose as 6.5 .s 10-61'.At. o hsmantd

indlcarcs rhat difr'usion and not rhe hiok~inaser reactionr i. linic~ng,

I~f dFfuionl ij rhs. dr;.*ng rorce for uptake and ;f :t ;r s Mr~uwl thje

[Ihe inte~rnal FlucileC cr.cencratio.-, remdins constant anI .er,* 10.? Ehecn

the~ u ata.e should be j srra;ight lIne function ofi thr 3lucate con;'rn~raj-

tion, ** straight line rai, ;1 fact, obtained jt cojn:cntralioior: of 0.OiMi

Und belour. The de.*i~atin frcal lIn:.;rit, at nigi- e* Oncentrat~on mTayi

be due1 to hiviedr intri'ral conclent."3Eijan s 1 i-i jIos- adl: EO- a Si~Uration

of the ?lucose ucilize don procejj .at thet '-iiger concentrations.

To cli=:R this hlpo~lchS;S furhthr CRe rjte of u,.ca~l.e ;nto urran,l

treated ard untreated -lices' *;os follou~ed ith [inic (F;~luit 10),. ).1

the care at glucost alont the uptake Proiced~ J a alm~asr ;1 .:.~rstent

rate u~nt I the 9;oc31e in t",u ''Ctake- 'olutio~i had ec~ 1 depil-!c.! co a





























Figure 10. Uranyl lon Pretreatment and Glucose Uptake. The pretreatment
consisted of I hr in either water or 0.003M uranyl nitrate.
The slices were then given 2 rinses, 30 min in water, .ure
rinses, and then the 0.01M glucose solution was added.Th
first sample was taken 8 min after the solution was ;dded
and samples were taken every 30 min thereafter for a Ftri;d
of 210 min. The curves are an average of 2 determinations.
The volume of the uptake solution was 6 ml so that lbarne
differences in Klett readings were noted over short thi-e
periods.
















/L tr pre tr a r -a t















12 r J0



P ~~rPi U~rlln ior re lr t.nin









60 120 I80
T i c..e mi;n






level below that of the detection system used for glucose analysis.

This curve cannot be explained on the basis of diffusion alone. It

might be argued that the rate of utilization of glucose by the tissue

is constant and that the curve simply represents the glucose utiliza-

tion rate. HowEever, as shown elsewhere by both uptakce and accumulation

data (Figure 7, Table 9), the uptake mechanisms of the tissue are not

saturated even at mnuch higher hexose concentrations. In contrast the

curve after uranyl nitrate pretreatment is typical of a diffusion curve.

If an arbitrary constant is multiplied by the concentration at the

beginning of each period of uptake, the curve represented by the dotted

line is obtained (Figure 10).

It is postulated that glucose uptake is the total of two processes,

one consisting of simple diffusion soon after uranyl ion treatment and

one an active process which is subject to inhibition by uranyl ion

binding.

If' this is true it means that the active component as represented

by the difference in the two curves in Figure 10 is increasing with

time (1.e., since the external concentration ii d creasing, diffus ;or

is decreasing and the active component must b~ inireaijing in ordirr [G;

maintain a steady rate). With this in mind jn cxpiriment -a run ;n

which thle concentration of glucose was kept c.rnety~t b, inreneir~3 the

solution after each sample as was done with fucr;se (Foure 5). The

results of this experiment failed to support the b..othts isFigene II).

Glucose uptake remained constant with time ir. sFit ofc~ the fact t h jt

the concentration was kept Ecentur.. The up tale C.r aj per~iod iof 2 b

was very close to the value obtained with~ delinlr., ?lucose ianicntrh-

tion. When uranyl nitrate was added to the uptale ;;lut~ar. ir. thiE




50?

r~C.




Uran, .o, ded ,-








121







~ ~ ~ ~ ~ ~ s~ ..iad~- rnlin d iezr






Tier ,i ot minl
Fiul I. C uc se U tal a C n ta E(o ce tato is u \/

sub cte to I h nw tr inc ,ad te h
0.0Er upJ:slto s. Te fr tCopew s rle
I i ftr ad ng te sl/o Te.L 'ue ae
a ecndsapl ws akn.. n h ptk outo -/





use~Ilrn,I nitra jte. The uptke o lm a mi







experiment the rate of uptake declined with time. This may be due Ic.

a long term effect of uranyl ion. The amount of inhibition caused by

uranyl ion was the same whether it was added at the beginning of the

uptake sampl ing or added after the tissue had been taking up glucose

for 80 min.

An experiment was run to determine whether or not the inhibition

of glucose uptake by uranyl ion was parallelled by an inhibition in

the amount of sucrose gained by the tissue. The slices were placed in

water or 0.0038 uranyl nitrate for I hr, rinsed, placed in water for




0~r~dr. rin rd, and JLIaCedI ir. .hIl fructose or 9lucse forJ ?O bri star


whi.::1 ~ ~ ~ ~ ~ ~ ~ F~b th sne:vreritd n klldfo .r5 i lj 01 Einu ;nucrose





i [Cd j'. .3~.-.d 4L.: .


tucr~...=c Ugrai

le. Order toj 5Fud sucrc.:e: ult=Le ir should ter urlatbl;; hed !hj

iuicrose :s .uch if thC iulphr b9;nll !Bakeni Up 6.rl ie f .dj

v~ill be preiSenced FO ih~ru thaE suirose ; rla tnkr upF witho:ut in.trsio:n t,



rugar uptake- willI alo be promoted- in thii scioLCTi n.

The Sii.LIutI of ;r..ersio of suerC.:el in [I.6 .-r erma~l zo~luiusl i j

Jiinal jI n.:.32ured t., the spearancer 01 riluco'se ;n the So~lutior~.,

ir.itarli c it .. I.ucIro: e cors.:eat~ji ion l i. 0.0011 [hl r.6,,irlurel .j,.:OUnL 01

gluc;.e noted in the iolut~ion 0.1 0.000i')l. The amount .Bried~ o C-r the

upI3Ce Ieriodl frossI Ehli ..alue LO .30 Samun t [.10 th 1 G~it 01 dtEDrrso








folr [lle daunt oF wlr~plee used, Thi; ..ould mean a endsamliumil I-evote con-

cenrration of (3.U!Jlll Jnd an uiptake scordi, eg o F;gur- .: cl' appro .-



rought~ one~-r~-nth of the rate o~f sucr~ojs uptake o~bsei.rved (njule 41.

This jrigumernt jip3it S :IheL jSSUmptiion,. justifitd by Figure i, thit chrt

t*-so heroses ;Ire t il.-n uIp .t jboutI thJ iameI rtj* hi rutC.l a

beenr piaison ced fore b i Humpnre ys jnd Garr ard (6~4). The possijbili t?

- ..;-.s that In.ersion [jlkis plac: in J posi rion iuch Lhai he:-oses mJe a

prefertnri-all/ toward the point of uptake,.

Ki;ntilc djta on tlF: uproae oi sucrosi glucose and fructose rwere

pr1jnted in che first rition, TheJ on.erjll s~hapes of thr, upl.JI race

. j .ncent ration curies~ is co~ns i drab ig different. \lnesreas herose

uptal..e iicrt as es ..irh co~ne..lrrttrati ove.r the ranger sh.:.L.n. 3uslojse uptake

a~pproaches a ma.ximumi. The conrsiderable dirfferenc in !he ef'iett -of ion-

ccntrationn on up~Ale if an ;ndi-ation that. rv) different pro~cesses ire



B, concering the data in Figures 4 and E. it can; De sen thjt the

total amou~nr. cf carbt~n rjlkn up fr.1,i 0,1 11 solution i 3 highe r when her.-

oser .re suppl ieJ. In e.per;i.Tsent iiearuring c'jssue s*.crnse It <,as

notred tlhar rih amounllt of sucrose ga~ined b,' rhe rissue was thi janme or

highel.r hcn th lices j U.:5I.-eF Inrcubased T.~ :Utrose as iJompare]J to ;rcuFba-

r i on in hthXose. ir. Ordr Eoi shrrk this effec: Onl ?he sarre grculp oF

lI;cesj an I..pel'rine~! r-t wa run in; .hichi uptake wa~s alloued~ to proceed



Dual;cact flasks ..;ere rur, UpF~a!..o waS5 mejSuu c o.er i hr of :hi .Jpcjle

proThe amount ofr caJrbon taken up fr'rn. giis-acese sltir on~~ was 1 O

of rhat taken up from sucr'ose solution although th-e snount of ucrose




53

gained by the slices was the same in both cases, The slices were in-

cubated in water following the uptake period and then rinsed, killed

and analyzed for sucrose content.

When gas exchange studies were carried out using 300 mg of tissue

in the Warburg flasks it was found t'nat the addition of sucrose, glucose,

or fructose to the solution caused high rates of fermentation to occur.

It was assumed that the amount of 02 consumption represented sugars be-

ing completely r-espired wrhereas the amount of C02 evolved in excess

of the amount of 02 consumption represented sugars being fermented,

It has been demonstrated previously that under similar conditions

ethanol is produced in amounts equal to the excess CO2 (65).

The data in Table 3 show the amount of fermentation over a 2 hr

period caused by the various sugar solutions. In the fermentation

experiments the slices were incubated in water for I hr in the water

bath, blotted, and weighed into Warburg flasks which contained the

sugar solutions. The flasks were then attached to the manomneters,

equlil brated for 15 min and t:he readings begun. No effort was made to



the begin.1ir.) of the readil~ing

Th-- r.Ee of resp;rtitoo~~.i :: rc.3:crabi cor..irrntr regar3J1. of the



h o fa combelnricn rri r. Th r i i '-.jz ;n .uicro~.-- n t r so ..o e u l



pe.r bl;C ..:omp.re;~j [o -~ j; rate of 5 u...elesf for tHlc:re.- Up~t-li- of1 3lucos

a.j-..i ;.u TC-se~ fr~oli 0.010 Iiolutionl b, th.C jjir;.06 :i~iices. ga he

followr i lg ri. ults; uro~t 17 uml es/hi~h r (c~r j4I ,,,,c-lc: h; o. -}c, ?lucose

21uale/H.Il' the jToun[ oI' termecnrjtion is a ful'.:rior. I:r the coc.-
















RespFi ratei in Fri Tnen ta ron
clmo les u~nle: I No r. ofi
Eol ut i on li.oe/rher~se,' nr prmn


(Djta j.erjgzd frolll se.evrrl dai'i preparjtio~n;)

W.'aer F.8 (0.4): 4.j (0.E) 5

0.01M suc roe 4.5 (0.41 1 Lt (1 .9) 5

0. 11 fo.to e4. g (0. ) 120 (17

0. 00511 fr uctore
r. 0.000M gllucos 53 0.1 13.8 ( i.2)

0. Irr Jucroje 5, 2.5

0. I1 fructase 5.0 (0. 1) 36.F (LIE.) 2

0.0 Ir lucose 4.6 40.5I



''Nor..ber s in parentheses'r indicate the j.l.erjag draiation. See 10/[, read
materialr and Int~atod;, for er.rprilrental detl I,.


lat~ie 3

Fcrmentraticn ir. Water ar~d Cusjr folul~non





55

centration of hexose in the fermentation compartment and if sucrose is

being inverted, even in the process of uptake, the rate of fermenta-

tion in sucrose should be higher than it is. The sane argument can be

made in the case of 0.1M sugar, since the amount of fermentation in

hexose solution is proportionately greater than the amount of hexose up-

take.

Further evidence that sucrose is taken up wi thout inversion comes

from the effects of uranyl ion on sugar uptake. The effect of uraniyl

nitrate pretreatment on the uptake of hexoses has already been giveni.

Table 4 shows the effect of uranyl ion on sucrose uptake. Uranyl ion

more completely inhibits the uptake of sucrose. UHith hexvose the effect

is to cut the uptake roughly in half. If sucrose were being inverted

prior to uptake, the expected effect of uranyl ion on the basis of

the hexose uptake after uranyl treatment curves would be to cut the

uptake of sucrose in half. It might be argued that uranyl ion is in-

hi biting the inversion of sucrose but the inversion of sucrose as

measured by the amount of glucose found in solution is higher after

uran~yl ion pretreatmer~t than it is after water pretreatment. In mea-

suring sucrose, It was consistently noted that the non-invertase-treated

sample gave slightly higher Klett readings in :he cases where the slices

had been pretreated with uranyl ion.

The effects of anerobic conditions on the uptake of sucrose glo-

cose and f-ructose were determined by incubating slices in waei~r fo.r

Ihr and then transferring the slices to sugar solutions and ir.iutnling

in air or under nitrogen. Nitrogen was continuously bubbled clhrcshh

the solution. ;LucroJ Il ..pacE in Ng2 was only 33% of that in olr r.

wi th gl IcoF 3ndJ frucae roi .r. rates cl u'~ptak~e In 11, were 65% of~ U.l:ez























0.001 4.1 0.i


Table $

The Effecr of U~ron I rritrjte Fretreatmen.. sr. Sucrose Uptak~e


Ulptale uiinnle: hr ~


Colcncetintrtin


Uran,l I ntrate
prc rreatment


\later protrbillaint


C. 005


14.3

11.0



4a1 O)


Slices- (1,0 3 Tr ut) ~ere pla~ced ir. iither us[Er or liran,l
n~itrate for I br. The sli~ice were J;:rn T' 0 ter r;n5Ss p~~lace ;[
rater for 30 Irlin, gi'en r more rwater r~rirnse oind f;nall, .laed Ir. the
batLhi ni 50i utlon. T.*j sampilrs we~re tsken. rlhe firal 15 Pin sc rrr the
bathinglr so;utionT \las addedJ jri thj SecOnd .11 the end of the uIptl.F
pe~riod. Uptakel~ was measured c..er a piriod of ii0 mTn .ri tr. I.00111~ eirtd
0.00)511 scrosc 90! min .-al th 0.001l iucrose .-nd IIO min url h 0.05 ;nd
0.iM CuiTcee. PeteS of Upts~.e in the first thrte are o.:\rajse of the
rcsults 01 two~ e.periments, [hC las~t t1o are Iro~m o~ne rgriment.~n





57

Table 5 shows the inhibition of7 sugar uptake by phlorldzin. At

lx 10-3M this inhibitor caused aboui twice the amount of inhibition

of sucrose uptake as i t did wi th glucose uptake. Witeh nei the~r sugar

was uptake inhibited strongly by the low concentrations used with

animal systems.

Dinitrophenal (DN`P) also inhibited sugar uptake (Table 6). The

inhibition was considerably greater with sucrose than with glucose;

the sucrose inhibition approached 100% whereas the inhibition of glucose

uptake approached 50%/. sucrose uptake in the presence of lx~ 10-4M1

DNP was l inear with time for at least hr.

The uptake of sucrose was measured in the presence of several di-

saccharides to determine if the uptake process was subject to inhibition

by molecules of similar structure. Several of the disaccharides caused

an increase inl thle non-invertase-treated samples. This caused confusion

in interpreting the results since it was not known wh-ether the increased

amount of glucose in solution was coming from hydrolysis of the disac-

charide or from an increased hydrolysis of sucrose caused by the pres-

ence of the disacchalride. Unless otherwise stated these experiments were

run by incubating first for I hr in water and then placing the slices in

0.01M sucrose plus the various disaccharides in concentrations of 0.005,

0.01, and 0.05M. The uptake period' was- Ir.

Lactose was not taken up by the tissue as determined by measuring

the lactose concentration before and after I br incubations by the

reducing sugar method. When solutions were tested for glace-se the

amount was found to be insignificant. When sucrose uptakl.r- ,, menure.1

in thie presence of lactose there wras an inhibition of 33% i. rh c;:e

of 0.0EM lactosee ir i t is assumed that the higher glucose cone~F: ir.?m
















Concntrto Inh it i [ i o

phlo~rid-in
ti Clucose Su. rose







I. 10 1 I 25



2 10 -47







twrice, and placed in a solution of 0.0l1M rugar an~d phlorld.Tin as in-
dicated. F-esulis jre based on a non-inhlbited conrro~l, UpcJake as~
ns:-asured c~ar j L br period.


1shle 4


Inhib; itin of Sug~ar Uptak.e t., Phl;oridzln







Table 6

The Inhibition by DNP of Uptake from 0.01M Sugar Solution



Inhibition
~_~ ~%
Concentration
of DNP Glucose Sucrose


1x 10-51 2 31

3 x 10-58 14 51

1x 10-4MI 35 89

3 x10-4 M 43 94



The schedule was the same as wi th phloridzin. The uptake per iod
was 90 mi n,







the increased h,dial islr of ucrose in the iresence of lac rvte. Ir.

O~thJI woirds. ther rame~ jGrwunt of SucTrofe \iaS iou.10 it thi endC of Lthe

uptaike period vrith or rrithoIut lac toSC. There was mOre qluCOLE in SO u-

tion at thi colo of the vp[ake period w~hen using sucro~Se and lacrose

than ~there unas in the cas6 oi lacrk*Le or sucrose ljnS. If this glucoSC

came from h,drolesis of sucr'ose then u~ptake wars in~hibitedi. This is a

reasonablL assu~,ption s inlce no h,droly iis was i ound i n the cjre ofi

lactose obere asr ar iFLcrease in sacroseS hldrol'.Cis in the pI.E:CnCe of

\ar;ous inhibtlor5 tas commonJrlJ found,

50croji u~ptake Iva; not irnhlblted br m?Iib;ose. rehjrose or cm 1-



When rnaltose wasj suppliied to the ti'sue- there hlas con~siderablt

h~drolysis. As sho.rn in Tible 7 the glucose iconcentrationr incireased

fromi the beginning of the uptake. periodj to the end, w~hilr the co~nccntrs-

ricn of maltorEe declined. The daount of uptake~ is explressed ,-, t,.rn,

of umnOles of 91ucome. The con~centration of oluose i..nsufficient to

account for the uptake (Flgure b) indicating t~hat som~e maltose \rjs taken

up ri t hou t hi droly s ri This experiment jhows firstl*," [h.7 th" discjcha-

ride is being hydrol,red and second, that iGilowr~ina hrrolsis the

roonortc~hande0 cn to detiectej in th~e e.-:rernal so~lut~ion. It miight be

pointed out that maltose would be an expected product of the rreakdo:.-n

of starch in the endojperm.

The app~aranic of gl ease in solution coming from both maltons jnd

sucrose maks i t di fficul t to dcetemine th~e effect of maltosr on jiucrose



:he r. urajnOSe was added to the slices na glucGse appeared in solu-

tiorn. Whin turanose and ucrrose toglether w-erii added to the slices thec
















Concentration Change in Change in Uptake
of added glucose mal1tose umoles
mal1tose umoless/flask umoles/flask qlucose/hr


0.005M 6 to 24 39 to 23 14

0.011 10 to 40 80 to 52 26

0.05M 22 to 78 346 to 274 88



Slices (1.0 g fr wt) were incubated in water for 1 hr, rinsed
twice, and then placed in the various concentrations of maltose, 15
min after adding the maltose the first 2 ml sample was teken. The
second sample was taken Ihr after the first, Glucose was measured by
using glucostat, Reducing sugars were run on the same solutions and
maltose determined by difference. Reducing sugar standard curves were
obtained for bothi sugars. The results are averages of 3 determinations
on 2 days' slices.


Table 7

Maltose Uptake





F. 2

alu..oi 1 a redings ncars littll or nu higher [han uhien 5ruccose ras .dd~dc

alne uran~or = jt j.0511 ijurd jppro ;ijlcatel, a jU01 irhibi t on of;

ucroe ut..,.e(Tale ). he uptale of Eurannse alone was rhecked t.,

using t~E reduicin? sugar rnalr[ed and was found not to~ be :uken up LI., [he

slices.. That the- iel'ect of tbraraoit ;Is not a.ji ur;~ntic iffacr wras derlr-~in

strat-1 t., nleasurinig the ulptakE Oi suifo58 jl00, rucrose In thA pre.-

ence' of ruranost jnd ucrosei in rni piresence of Irlann; tol,

S*:.eral *::per;brants wrei disigrned to ihot-. the effect of tissur

sllcroser le.*l onr the rate- of jucrose~ upt.j:sk*. The c...periinr~nts jre

sual-i~arizied in Tabic 4. Generall, t.er~e is jn ice~irse: correljtionn be-

rr,.*een thE a~llroviL OI SaICrOfS in rre ririui and the- lc'tE of uprE~.ake Thel

tissue ..as subjected ro:~r~l.. rrious trec,ar. -Lquue~Cr- ce SO jn \ntr,' the

amo~unt of ruirose in theF [iSsuei prior to brjeauringl uptake;. I r.hosi

thra.. case~ narkid 6, ar. azterisk the equncrres were th: sjrne C>r.iept

fo-r the cinoncentrato oa' rugjr during !he firit I hr and in t:=.:h casei

;ucriJse uprake u3r IreaSUred during the fif h hr f rarri, th.- ime the irper; -

me1nt rarted. In thise ijses there ;is jn true-llent ;!ir.*;r*. correlation

t..-twEen [h= aBolount ofi Lissue~ SUCcroe C.'tored jnd rthe rjri ofi uptal.e.

Th~ir is tor t~e e-~pected since- thcre umust be~ somer limr~i to the: jlr~ount ofi

sucrose that cjr. t~e accuuilatedi b; the, slices and one would c.>.pect thi

rare- of uprake to to r-educed js thij liln; t is Tejihid.


G;Is E.iBgL

11h1n yas5 t)change exprCTmnts[ areT carried our usi-ng 3.00 nap of

tiiSFu ir, the \~U~atJur FjLarSk the aliounL Of O, rjiten up tt the clssue

was limritd b, thr rate jt which 1? difluses into3 uner. Thsi h l

by thr: fo~llo\sing: (a) 7).@ rat-s or' 02 co~nsuti~ption noted in thiese experi-

mentrs we~ri cqurl rO the 11rinits of the rate at sshich 32 di fuser into
















Uptake umoles/q br

0.01M1 sucrose 0.01M sucrose

Experi ment 0.01Ml sucrose 0.05M turanose 0. 05M manni tol


1 15.2 10.1

2 15.5 11.7

16.0 12.2

16.5 12.7

3 16.1 11.4 15.6

17.1 14.6 16.6



Slices (1.0 g fr wt) were incubated for I br in water, rinsed
twice, and the indicated solutions were added. The first sample was
taken 15 min after addition of the uptake solution. Uptake was


Table 8

The Effects of Turanose and Mannitol on Sucrose Uptake

















TIssue SiicroSe
le,el up~rjak
Trretnen: sequence umOlFIS.g umoleS'J hr


\lAter 3 Fr, rinse, waerIT br,
ricscoeIhr 50 25::

\ljrcr I r, rini:0, sucrose I hr 68g 17

0.020i Iru~ct:Le 7 hr, rlnje, water~
I br rir.Ee. S crose I r 81 Il

0.III fract;se 3hl. rinse, ueter
3 hr, rinSC, suciJte I hr 914 i

0,lIn fructoje ; he, rinrce, raterT
Ihr. rin:e. scrose I he 125 15;

0.111; fructose : hr. rinse ,
cuirjse I br 133 11



The results. from 3 cropsC of seedling s re riported hire. In each
case the uptakle period ejas I bir, the sucrose fronll r-st..c*, up:3l-e vas
nriasuiied ~as 0.0!11 and~ there was a 15 miir, delal L.etueen ocdainq hth upF-
tjLe scilution ond! taking ther first sanple. Dupl Icat rs amp les ,;re
5ubjiated to the requen~ce as listed and at the beginn~ing of the uiptake
Period onez group rjs k~illed for sucros~e anal,sis rrhile rne other .*.ai
usedto- rmeas~ure c_gar uptale.r


Tjble- 9

Sucro;e Ticsue leverl ilrnd Sucrose Ulptake




65

water (74). (b) W~hen DNP was added under these conditions the amount

of CO2 evolution was increased; however, the amount of 02 consumption

was not. (c) When experiments were run using 100 mg of tissue in water

the 02 consumption increased on a per weight basis and the RQ decreased.

The figures from two typical experiments were: wi th 300 mg tissue, 33

umoles 02/g br, RQ 1.3; with 100 mg tissue, 53 umoles 02/g hr, RQ 0.8.

Since when using 100 mg of tissue, the rate of 02 consumption is well

below the limit and since the RQ is quite low it was assumed that 02

was not limiting under these conditions.

It was not known whether or not 02 consumption was limited under

conditions which prevailed in the water bathi. An experiment in which

sucrose uptake from 0.005M sucrose was measured as a function of the

surface area of the liquid per weight of tissue showed that uptakte was

not l limited by the amount of surf ace area. In this experiment uptake

by 0.2, 0.4, and 1.0 g of tissue was measured. The volume to tissue

ratio was kept the same by starting with 2, 4, and 10 ml of sucrose

solution, respectively, and removing relatively the same amount of

sample for sucrose analysis. The rates of uptake were 11.9, 11.4, and

11..3 .arreiles/9C br t, ?.2, I?.1 4, an 1.0 J oF t;ue. Theic. differences



.uirrj; upt.3|e ase r.or I1I~r..rd L. 0 i b li .








where I fl3Cd, a Luj Seid ..nd 13:i umoles/31 alhlre 4 rwer: used~. !Th so~

t'igures 3re not ns! sucrocse .iccumulated jince thi amountnt Of: tijsu8

suCroSe at Ih: beginning o r ne upraki- period nas not been iiubtracred.)

\Jork byi Hus..phreys arid florrird (701) jnj by thi author hj.e shon,,

that rhe sulcrore content of rcu~telluslr. SliCes is Treducd rher,~ jliiCe

are inrubated in war-r ;ndicating? that rEugar is the jiubscrjite for rre-

pi ratio:n. his; waJ jlso rioted ..hen 0, 25 g of t i 3;ui per f l iSk: ras u~ed.

Hlowvesr, gas eYchdnge StudieS using 100 mg ofi Lis:u+ h e e~l idcjted jr.

P..) ir. water subjstntiall, less than I.0 indicatinq a subjtr. to other

than sugar. This 5 nconj s t ency weiight be a~r la I ned L,< the Con.irsion

of ilcrosee to olrganic 3i;ds.

Figure 12 shows: the anoun[ of Fernientation in \Iater snd in twoo

conccntrrtions oi iucrojsl when j.00 myg O ri.isue u15 used. It i, acjsumed

in present;ng thes fiiures rIha, for tachi uno's af CO2 e .ol\ed in ex~cs~s

of' 0) take..I up orle-h.11f uiinole) of Choxas .-sa; tbeir fe.-,i:nted, Figure 178

repiesents tht 33rs.* e~Perinent laut rhe aiouint of Forilentaririo in the

rwater control haJs been sbtcracted fromn that caus:d b, surose,

fnh the b-si2 Of 0: ptake iexperiments it r -,as clculated that thr

solurions in rheie rI asks w would be dEpleted of surcrose after j b~r in

the~ case of 0.001J:I an~d 4 ;rr ;n the: casei of 0).)035r slucroii.. In the~ case

of 0.0010r >Icro~ r th suga3r-csu jed feri-antation tr .e rh ar

is j dded, incr;ehie in rare, A~id then decrease1 at Frnc (10.9r then the

Jugrl" should all Lakes been tj!.en uo, T.;i sinountr of iiearment t ica caused

b,' 0.0051 juirore. ij about thrE e i~ t r'?j (MC cus:J br ;j.001ft iucrose

andJ as rhow.n eairlierT in Fi3url: 4 the~ raCL of upta're ;s about tlTree

time as rea.' WIh 0;.000.1 juirote thep agreemenr ;n timing bc ..:een

uptakec and ferl-enit.&tn i; not aj c lose, ho..-eST .,lrco care do-.5 begln

ro dzcline at a time~ where1 uptakCe ihouldJ. be coFplem.































Figure 12. Fermentation in later and Sucrose. Each flask contained
300 mg of tissue. The sucrose was placed in the side arm
and added at 30 min to give the concentration indicated on
the gr-aph. Three sets of flasks wrere used, one each for
the 2 concentrations of sucrose and one containing water.
The values were obtained from; CO2 -02/2 (in umoles), in
B the values found with water have been subtracted from the
values found wi th sucrose.







A
60








so oarose;






3 urs jOdddedrs~or





10/





30l



0.00""511 soross


Sucrese odr'd edC

/oO
1 0O -q-q-twM-~

a r~S~:;% ?C~0. 00111 SuCroS C
r $r
.d2 e-I Ir C

Time, hr




69

Figure 13 shows the gas exchange in 0.1M sucrose as compared to

that in water. These experiments were run under conditions in which 02

was not limiting. With water the RQ is less than 1.0 and both 02 uptake

and CO2 evolution proceed at constant rates throughout the experiment.

When sucrose is added there is some depression in the rate of 02 con-

sumption. The rate of evolution of CO2 on the other hand continues to

increase throughout the experiment. Over the period of this experiment

the uptake of: sucrose would proceed at a constant r-ate (Figures 2 and

3). When 0.01M sucrose was added in the same type of experiment (data

not presented) the patter-n of gas exchange was the same; howJever: the

02 consumptioni was not depressed as much and the rate of CO2 evolution

was not as high.

Figure is shr.:si the~ sjare ie pa f U~Ita fo'r 0.1[1 glu~cos. r.ar



resolution of~ C0-. ; grtsll, nrcreased t., [Ihe adds F.(an 0i 1Urj glc li to

sincei. Ibe. F.I during Ith l ear. pr ..d mat-urcd ..jr z7; 115 .,,,.0?. acr..

0. lr.01/1 glu cs !data nres ireset ed)j caused ro:uJh.1, I'te rsame p L~ir r, -,



If Ith .assUmIPLtio is need tha thel d~;liffrtence ir. C02 t\olution ir.

vetrj E It r..u5 thol in sugar ii lui Er.11-I t r e tai;(.S' r r t

is ol~ib~lC to CalCUlateC Ihe reek.-:*r ofi umle!'~C i hCrSid tbeir.9 Fer.T:nited.

Figuret IF shar~~ ~-e thC.- J res lt of uh a icalcula tin. Th sr.lt. r

frir, thC sar~e e~p rir-nts .a Itpo~rted ;rn rigure: 1.. and IL*,

Rate: of~ IIIrrilireatio~ ~U suggested bt [lithe .-.t. d31 t.- ar-conl..ustrar



As ioir-tedJ out crl~ie, 1:F.6 rjte of ftri.,ntation~ ;r higher under icndi-











V00 ;


.p iO. -" arose


1 O, ae



<, 0 ,. i cr'





Sl ,-o~ O,,, water


15 ll


100 L_


,/


C.. ..... .... -L 1.. IJ _1..1. ..L_ .
30 60 90 I20 I50 180 210

Ti rne~, ai n


\:ere run rri lhi 100 cl] L 5ue per i last.
olC'uri.Ior (LOM() was- ajdde f ruin thE Side
of ter re-dilngsr were tregun~.


The sucrose
jrmr 30 rainr









0 C2, glucose 1
300 CO2,water


O 02, gl ucose

y 02, we te r
250




200
















-- .. . L e .

Tij no r
Figu e 1 L 1.ch ~ g. iri'a e a,< 0 11C' ...e cn iE o e
tr e It : i .. F a u e i













C, .0i r1 gluccose

ri* 0. I t avc ros5e















140 //





20 .*








Sugar adde~




L L --. ...
i0 60 90 120 1',0 1 0 210
Timie, min

Figure IS. FcrTer~!a tior, in 5u3ar .elation. odio. n
Figures 13 and IN, F'e ull: \*ere ;alcu;lct d s LO,






73

accumulated is lower under conditions of limited 02 in the water bath.

The rate of glucose uptake from 0.1M glucose was about 87 umnoles/g hr.

The rate of fermentation as indicated in Figure, 15 was 28 umoles hexose/

g hr. This would leave about 60 umoles of hexose available for accumu-

lation, or enough to accumulate sucrose at a rate of 30 umoles/g br.

An experiment was run in which slices were incubated in 0.IM glucose

for 3 hr. Each flask contained 250 mg of slices in 2.5 ml glucose

solution. Sucrose accumulated at a rate of 35 umoles sucrose/g br.


Metal Binding

Experiments were designed to determine the amount of uranyl ion

that would bind to the slices, the effect of other cations on sugar

uptake, and the relationship between the binding of some other cations

and the binding of uraniyl ion.

Table 10 shows the results of two experiments on the effect of

cations on sucrose uptake. In the first experiment the cations were

added to the uptake solutions. In the second experiment the slices

were first treated wi th 0.01N HC! to remove cations attached to the

slices, then treated with metal cations, rinsed, and placed in su-rose.

Uranyl ion reduces sucros~e uptake when it is used as a pretreat-

ment or when it is present in the uptake solution. Cations other than

uranyl ion have little effect on uptake. Notice that the acre~rol,

third column of Table 10, was treated with acid showing tnue rth acidl

treatment does niot ser-iously impair uptake.

The next series of experiments were designed to determine: _Th? quani-

ti ties of the various ions that would bind to the surface- of .nec laces.

Figure 16 shows the amounts of the various Ions that cjn to reno.ed~

..im rhr.01t ~i HI after p~re real~nirr witrh the various metalIs. ea
















Upte..ce in urooles 'q br

Treatment Ul; th upt~l:e Siolution Pr~ e r lle et L


Table 10

Suc-rrose Ilpraic as k.ifcceed b,' Variojus, (io~ns


Coll 2


lInLI2


11.0


li).O


UO2 3 ?


The d)(a~ ill trd riCond Co~lumn sf6 ffrnm ,n experilifnt ir nhi ch
slices were incu!,..ted ;rn warter for I br, rinsed. llith \tearC.r adTI: pl.=sCii
inr 30o1lclons containling 0.005M sucrose anj ii.i0311 cation solution.
Th~e t h Ird iCo lilr., repFrese n 1 a 1 5 -min i ncuta tion in 0].011 H C.I fo lkI..,e.1
by 2 rinses, 50 min in 0.003tt :ar.;or, solution, another rinre jnd i,-
nally the (>.00*,11 suirosE sojlution from r-th~ih uptak~e wass i~eneasred. In
btlUh e~.*periai~nlnt :he uptal:- period wa~s 2' hr b. ig 0 f slices. per flail:







--.I--I-------------- ----- ---- --I----------~-----l"r


I


a


(0.010M)













(0.02MI)

0 M 2


2+
(7 ] 022

W Ca 2+

0 Co2+


SMn24
aa 3+
!:[ Al



Salt solution, N x 103

Figure 16. Metal Finding. The schedule for these e-peria-ncnr
involvedl the following: 1 br in the car~~ .: llr. olud
(or water control), 3 rinses, I hr in !*,<, 3 r~orar
r:i-. ---.' I.r analysiss of th~e respective c nores, tail
i1.0 c..com...ed 1 9 of slices.







points .;an be made. ,iTein..r on ftiecyr eit

searij' rinces and a I-br .r~cubation in eY->rer. (b) A- concentrjttion of

0.0020 .:ation rolution r,:ided enoujh ions5 Eo saturjte the rltes ;rn

all Cases. \c) ApPrCTiiabe quantities o~f the ;oni at 10.-, concentrJ--

rtoi rcr- ur boundl to the slice. In the case ofi ;Jbal: (0.000)11), 1.6

un~clers or the 3 umeles ;I.ji lble wrere bo~und b: rne jlices. (d) fljgne-

jium jppersr to be th-: ;on rhat, normally occupie; the sites. The data

from Fiqure e 1 hou thlE the slici-; incubjtedj in \-d~ear insread oi

mea~l iarion solurionj released3 6.5 umolej ofl 1122'+ aInd 0.?S uni~ls of

Cs- i. (I) G;-earer quait~itiesj of uran,*I ;c7, jrE boul~nd the. ,an OF the

O~lher ions [GE L.d evcept[ r1ig'

In theI caset oi I~renyl ion, twro T.-pZrimenri~ :,rTT de-~;i. d to see- i

rh: cffect in sugjr uotrake paralle~l !he deglre- of catio~n bin~in3 to

the ti res. i re i h a- h re u o h s p ri nt .nd t

can bt: seen that maximum ilhibtr ion i; reached jC concentratacni of

urinel ion tha~t jecrur.te the binding ;irej. TI-e inhibirlor. of glucoC'e

uptcake in this expcrinent was.s greater th~an ruju' !c'.g.s ce Flgu e 9).

IF the- slI;er we~rc first treated --with lICI uni thien incubear~il in

melitpl iolu~~Tions, thF jmouLn*, oir coacTn bihJndin \1as Consideraldl riduced.l.

This is shot-*n in Fi lur- e !2 fo." ursn,*, ;on, 11nl and (C'f. a.1n i

[he jcid treatmcnl I'endered i.oLE of !h's sitri und.alajlblz ror C3Eion

bind ing. M~e..avr, thi EliC; L oo'* up sucroje as ;hanr in Tabls 13 .i Ih

or rithojur replacing th; catinsh. Of TCoorCe the por::bilit C.:.its

thjt the~ sites in.crl ed if. sugarI 'JJ~rj e werre fear-TCapied~ b.' TJa;r; on rom~ri

.cin he 51maliZr I~auanic i. n Urlnyl( "on [I at bind5 after acid

[Ltrel:tmnt vas surficien[ 10 irhibit uitake s jS s sho.an ir. Took 10.

Wlork ,r;th~ 'ICast In..icvtsLda* ll et nll le i ir displaced f..On the binri-


















80






40/


SSucrose

20 .~ 9 Glucose





S2 3 4

Pretreatment uranyl ion concentration, M x 103

Figure 17. Effect of Urany) lon Pretreatment Concentration
on Sugar Uptake. In the two experiments reported
here the schedules were as follows: 1hr in the
designated concentration of uranyl nitrate, 2
rinses, 30 min in later, 2 more riioses, thE- ..pcIIe
solution. The uptakte solution was 10 ml or Ul.0111
sugar in both cases. With sucrose the ayrj;. pi'ned
was 90 min; with glucose the uptake period j; I br.
(Whereas the uptake period mnay affect the Jag~ree Ji
inhibition, in this case the experiment wa; ~sl i 5cja
to emphasize inhibition as a function of J on,I .00
binding and not the amount of inhibition.

























I~ j r- f -n



SI
u 2+
L O
qJ

U j ] ji ~' '
.1 so u io 111 1
ure I.T fla Bi~ ig r le na cd P
'ue:Le rr Tre sl cs'. r st
.:rc ?uj ce o th o lwn
!,r u.: nce s mn i.002 ~ ,I
ir ih 7 .5 l to. ".e o
to- PI h 2 a e r n r is ..0 0
HO re I3 slu,2 '-e c-. eoe

..or ar:1 ,* i .1 c e c t o





79

ing sites during sugar uptake and are again bound after the sugar has

been taken up (53). Sever-a; experiments which involved pre treatment

with Co2+ or M92+ fai led to detect this phenomena ri th slices of the

corn scutellum,

As described in the methods section, corn was normally grown in

tap wa ter, In one experiment corn was grown in distilled water, Su-

crose uptake by slices from this group of seedlings was about the same

as with slices from tap-water-grown seedlings (i.e.,17 umoles/g hr
from 0.01M su~crose). Prtetetwt at fC2+, Mg2+, or KC had

little effect on sucrose uptake into slices from seedlings grown in

distilled water.















From thie ri-rults of this 'tudy the follorinj conclusion jlre

drjwn. (5) SucroseC ;S telken up jcti\ ely~ without in\erdion. (b) Hezasej

3rt tak~en u~P byi tlvo prTO'LCe.Ss operating S;imulter.eo-iUtI,, dillu5;5r 5:rd

3Ct;ic Ironsport. (c) The acLi~ LCu [31 .511( 1801810$ fn $UjrorLe 8"r( 10

hc.srs ari locjted at the plj51male~i m~ma (d) The actise uc;:.i:L mechaj-

r~i ms for b~oth jucross and the hr:..G.5rcEre dlriken toi .0,iol,siia. ()

The charjiracr i st ic o m rE [al b~ndinj 35 relate [d to sugar uptle areCd

quLi re d~i ffrent in thei corln jcur-l lumT .-then ~cmji lre 1 EO (ho~re i*1 ,cjs t



Several line- of evidence hose- IJllCdHunhreis >..3 Garrard (115) to

thr -::niclus~ion tIat jaCrCote iC. taknC~ up vithoUt i I~crsior. b,. th3 Corn

9CUrtlyl. r;SU: 1' cut btained ir. this c-Jork. sul'po t that co~nclusion.

(j) The amount ofi ..xtracellular inversion <*: insufiident~ t, sLpport

u~ptak' it ;he ob C; -ed rate. (b) The tircat~ics of hC:.r~os uptakei d.e

different fr-om the~ ~inedeis ol sucrose uptake (ci. Figure 4 and Ficiure

8). (c) The rate cr ferarranjtition in sucro;F was less than would reo

expcted (Tiable 1) i f inve~rsioi occurred ci ther pr;or- to or during urp-

talke. (d) if inter;on prcu;lsd uprjke, thie attee.: o uranil ;on on

sucrose should I..G simllar to the rFfect on herose rvbich is nrio the

case (ofi. Tablel 4 .and Figure Y). (e) Tjhr rat;O ofr jcCL~Ul3[- d Su~ClO5C

LOr Suga~Tr ken up it ; g. hir Irhenl Cu;Troe iS iUppliced thaln obetn 3lucuse

is cuPplisd (p. 51).

The mj tor.: draa (Table 7) showJ thj; malic.Le is alsor trJ-i-n upF b,

thF LISSue w~itho1~ut hydrob)li .


DISCUS5101J





81

That sucrose uptake is an active process is indicated by the fact

that it is taken up against concentration gradients. Slices that con-

tained 133 umoles sucrose/g (0.13M~ sucrose) will take up sucrose from

0.01M solution (Table 9). Slices incubated in water for I hr contained

68 ucoles sucrose/g (0.068M sucrose) and will take up sucrose from

0.001M sucrose (Figuire 2), The tissue sucrose concentrations given

above are minimum values since an equal distribution of sucrose through-

out the tissue water is assumed; compartmentation of sucrose would in-

crease the ratios. Ihe scutellum is composed of mesophyll parenchyma,

an epithelial layer and vascular tissue. It is assumed that most up-

take occurs in the parenchyma cells since these cells appear to make

up about 80-90%~ of the scutellum.

'The inhibition of sucrose uptake by DNP is consistent ,i th the

idea of an active, metabolic energy-requiring process.

The inhibition of sucrose uptake by turanose (and probably by

lactose) fulfills one of the criteria given for a facilitated diffusion

or an active transport process (I).

The data in Table :0 clearly show that a 15-m~In rinse in 0.01N

HCI does not disrupt the normal functioning of the cells insofar as

uptak~e is concerned. Since this treatment removes uranyl ion from the

cells anid restores the ab iity of the cells to take up sugar (67) it

appears that the effect of uranyl ion is at the cell surfjce. I: ii

concluded that the mechanism for sucrose uptake is located ji the plat-

malemulal and the same argument can be applied to the: activI j.-r."al jr:

glucose uptake (see below). :n contrast to :has..c results, ~is:M-r :ik)

found that uraniyl ion had no effect on sugar uptake. Hum h r: ; I pt, o 1l







suCiroe. i n con tra t prE t,-ca rirtet of i 5ic:1 ; a 1.0.4 aucrosei cauSed

the: slices to 3Cppearj il-cc;d and ienderedi IhemT IsocapillleS ~r :ath~esz-

ingJ sue Isse = hen pi ;_ed i n I!. 1. I Fruc re,L TheSe obse at~t io .5:~ :'uPF:rt

the~ col~tntentio [l,<. the~ .warm~ral R.'mbrjrnc is pFcret~eur. c cc herose. t..a

not [.' ruirose.




ilso0 has an acri.: i ose' transport miechanistll.. The constur nt rtei IJF

?luaSi uptake until ;'u bathi.ng solution h.15 been .Jeacre.)o- (Fi |13)

;;annor be- izploin.1.1 :. .lirfurion alone and ,cr ,i..an darj had bien

aCcumullated to ShlOu rhat Ihi- Cl!Oprl35m is .'ric SFpice U.j C00.~.~. Thi.

combinati o~ CI' diffusion and .in 3cti.C ICr.isport[ m-...aniser ce., i .plaIinr

rthese resulrs. Th: h:t.-use- uptjke j cancincrrcioron cur-:0s mighte ;'so






Is ar~p~aldiffs cuse.It i; ctruc that this curse co:ul.:1 jlro

h the result ofi i ..r;ocss -h~ic follor-0 enz,.r~e hirleic-: prOviGed that

rE.: I'ni is hign in iconsp~rljo n to the concenl rraton.Hss*er hesb

strteL c..ncnttrrration ia,a O.0111 glucoser which -souldJ require quit.-. 3 Yigh






blocking ~.he2 4ctivl porT[; on of gluoseJ Irptake. iht upat.E rtte s Con-

ce~n;..Mrir *:-.cj 'Or thd becoses~ (Figh~ F) jlnd ') do no7. t re-emble




paic-ISS r:-.xpr JFter LT.tret--irt ri te vlrar.,I ;inn in which :;:1 -s losr J

g Ilucc:O ;caus, Irlcai.~L :n I :3. dif ;'? i n 5e al.- re plr f5Cnt rf 0. .3




83

form sucrose or is catabolized. It is assumed that the glucose which

diffuses into the tissue is phosphorylated via hexokinase whi le that

glucose taken up actively is phosphorylated at the plasmalemma. This

is active transport in the sense that uptake is being energetically

driven at thle membrane but it is not active transport in the sense that

glucose is being accumulated against a gradient. The active process

described h~ere would be called group translocation by Roseman (57).

A combination of active transport and facilitated diffusion is

thought to be i involved i n the uptake of glucose by yeast (55). Rei nhold

and Ellam (2.6), working with sunflower hypocotyl, suggested that active

transport operated in the a'osence of DNP but that in i ts presence

sugars diffused into the cells.

The idea that all of sucrose uptake is active whereas half of

glucose uptake is active and half passive is supported by the following

results. (a) At concentrations of 0.01M sugar and 1 x 10~3M phloridzin

the inhibition of sucrose uptake was twice that of glucose uptake

(Table 5), (b) Sucrose uptake wlas inhibited twice as much by DNP as

was glucose uptake. (c) The effect of anerobic conditions (p. 'fl was

10~ irnhib~l ea'ur.:.' e parate [,.ICr i. iiuch "us ?luccee uptall._- h:.-Tae,



_uc.s, inh Ibi io jr; ri ecrose u.rsI; a rt. co l t .









Th: lac: tr t icrmorstjltion ie d-sected c.;i~r. hen 0, ii not lir..iring is

ar. ind ; c'io i on !h-at bL.jn lr[ rati o .Jri.,i; uptke iregrdicr s of' :h~e a jll-










o~F CO-. !o 5 ji!Luration of the le3;pirjtor*, C71 risT. sno h

case rirh J1:te~llum~ 5lic5 inci "hie cojnsumnption ,f On ra; relducId ;n

rlhB pr7eseceii o1: 0.It Sugar.

E.ide~nc;- hjs ben presented to show* thjr ierimenl.Jion dr;t.< suear

upjltae, t: c.-jinr~e hjs beein et~tjirn:J to 3hoes thJt i iPecific. 9l;roI l; i.

Cst'F is responsble, howet~er, !he data ar cjnsist~entr ..ri II- j,: r,.n

5uch as th: phof phot -jnorjes e Ie 5,seiT i n b3,reria w he-:re TE i s thr

enerjv sOurCe for up two Ga3rrard ;rnd Huimphrc,*s (.LS) U~.u-i,ingl iontrol

of gl*,colysse in the minjzi liurcllum foundl no diffriirnces ,n th? r\TP

leittr1 if the pr'eSenic jrid jbienii of fructOse, TIhe ~ir.ount of frucrore-

Pwih:riianslates prsphoffrTuar.lki nrte doubled i n the~ jlresicac of

fruiroie bJt [h.5 rrt; cOnsjiderd iniadequale to, aiccount fiir a ior-fc IJ

in~crea-,2 in th-e rjte of .,liCol,-sis The usr of PEP .0? the suar/~ uprC!;

proces ar ~;ii h tr i gge.r t rm n tajt ion.

Th: .fate ;n Figure & whiic h)\ shu TucO:G upE~he j+ 9 function jF

the concentrrat!Goi in chu bathing silutiorn ilojc!, fit flhes d

;enren cuirse. Tis j .pe of dataJ i of ten pr..:-r crt i,. su!;orr of j



iur-.t.: th:., rhC, J lInes* upiakle ri; h '.ice in .5 bjth of dlecrea~sing icar.-

centrj lion (Fip. 1, i, and 3) are nioc j ..II r,p~ical of rnz,m.. !:;..ic>.

Peg.3rdians of the n~cchainn~ir~a o uptjl:e, La it dififusion or .3 carricr-

ac.0 sted 3c :I.C p'rocu, the rate would~ 'Je pa ~tted rr ~...crejse js the

jug.- cojnirntri atn in th': ,jluti n waj rCduC' 2.5 3 result of upsti-c.

Ait a ;.2n c.)nce~r[ Trade. the ;on4tanL Uptak.e rTeI r.]ht be 61-~

plainrd. js 2. Saturi tion ,f th :Iptae sic..hdn;ism for sucrevez Zut this

tIr`c'lborlio is japparnT~LI na.! Cjaturad unti[l .1a5n--antration o~f aboar







0,4M (64) which does not explain the constant rate at 0.005M (Fi;. :1.

It appears that whereas the substr'ate is not the limiting factor

(Fig. 2) the substrate concentration has something to do wi th the rate

of uptake (Fig. 14). When the sugar concentration is maintained at a

constant level sucrose uptake increases with time~ whereas glucose up-

take does not (c-f. Figs. 5 and 11), A possible explanation for these

phenomena follow. The rate of sucrose uptake is governed by at least

two factors. the external concentration of sucrose and the internal con-

centration of the phosphate donor. When sucrose uptake begins, the








w~hir bi si-Z.:-. .s i.-.ci-:::ir ei rare orf r02 0 .it, H e .c e






Lei r..3 coure.l:; :J t, .r. I;~r..3:re ir ;n[r-t r..1 .isj .. Icl? :s aIC I:;rn`; 1 su



;U..rC;c upro;l e.






L-r.relir s .i., ~.i.por jr .1:nt d jlerine: 1-.e rate: oF CrJ:tr r:p r i r








Th~us :s: the C;co--st.itio dec~linE; inS Sci e proie~s. .: I~j j ijlal.. ..



car..:-c.rr aro ica of L.Y.:.:Phale~ doreT .r : rer~i~l UKl prC e::.; Tne r jIi







re~ul[ ; isi I.0 up..rla- CCate rJij i; almostJ 3 r.ornstinll uniil Ilh' Bla~ocs In




thec 3nmoun: Of e~xt:rnest gliro.,r ; i ConStant t murofd ui

;.COnSrant andl thusl rhe O.srall r.ite GIi uptal.. Irenlj;.5 cons .-.nt.

.4; comisinlar tion of sct;is are) paSrs: e jlutoi C~~~.: uptake netchani e. (ir

<
conc: ntrati[on .*,jljh bic rto i.he i ulllu r: lj 10, the 5rst..m is capable

oi' rL1soIn.)~l~ all of cihe j.J;I-ble glu.cose~ quickly; h3~e..cr w~hcn thi

--ndu',pern l IjllCUm: Is blig'1 the 'cultellun cji.. (;1;C up !lucorei ur. exceI

of the cjp~ci, of th:r ;J=rtl, procrss.

.i; juga- ul tal e~ i5-st~rldr. eri :.,- giJ,col, .;s .lgso fIts Che 1ole 3.

the Lo r~ ur.. Ther x,.lli, to d~i. .- uprjl.*= undEri I~jnited i, 5uPpl .

:lould be of ob..iau. ;.1.tintag toj j s CJ under 3oil canii~r.Jin. sr.r

and- riump;irc rj I.; ')as me..asun-d aj iQ of i wi ch ..holel .calleIcle ;n Air

indij cacingr t1\3 LhE sCu C. 11ucr.~ itseli meight ;imp.'i e conder5n on S o Ilsi r red




I' is ot;iouis 1..1t [hM mu l;l t..iiders. Chara4 cLer St.(. ofl Ir:utL ur..

sli :-sJifer rer, o e of ea=v. TheC tindinj s uran,*l I sn to ;er st

cills -.esonsr to e :u: r. spc~ir'ic to the uprjlid sit. r.53'. ;rt h sc i-

rillUil ;ICi.. noC li.. .,ct of' Ih' Ma~n.' urs.. ion is Csu if lVOL.~1 .3

thei sp[Ske i.r~re~SS aS sh7'~ran 'i! 'he sectucedl bur rill effecc:; .. as.,Irul

J:u ndJ .jfter acidi EreC reent-I (ci. F17r. 16 anld I I, sold T~t.Il: lIC). I




chdl ..randl ion ..a. t~ilun :I L, onr root~ ..issue, -t ....n i o b r s

thei m-~.mbraini surf ac-= If' the 'e~lTaJ F Oi bound icanOn; OiCcurs dancyi

segor uptak.e to sCU[Cll,' n Slice. th-1 as-sun =. 1- Eco,3 Cmil II~ 0. L aleC[':

rith th*- mci;J1 d;S53. pl'O.CJ1ri* uis ...














LITERATURE CITED


1. Ll. D. Stein, The Monvement _lof Molecue Across Cell Membaranes
Academic Press, New York (1967).

2. R. K. Crane, in Comprehepsive Biochemist~ry, ed. by Ri. D. Stotz.
Elsevier Publishiniy Company, Amsterdam, Vol. 17 (1969).

3. A. L. Kursanov, in Advances in Botanical Research ed, by R. D.
Preston, Academic Press, NewJ York, Vol. I (15163).

4. C. E. Hartt, HI. P. Kortschak, A. J. Forbes and G. 0. Burr, Plant
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6. P. c:, \feathe~rley, New Phetol.i, 52, 76 (!953).

7. Libi., 53, 20:1 (locL}

8. Ibid.. Sk, 13 (19551.








12. lbid. 2.0, 565 (19< 1

13. P.' J, Hardy and C. .!:.rtc~r 'I i. ? .:.1. .. 1 I .6








17, P. Krie~demann, Pl r _:. . I

I G r r h . r n i .I








L, F.-int..:.id and 1. E-ht..r, Phon Ph,001,, 0,, 1021 (1). ).

J. L. H:3rh, 11.3 11. 11. lennar..ji, Pioc, P.o. SOC., (,nl. 0, 11,
' 05 I I.-iki,3.

D. P. 't.,rgan ina H. E. Stracr, rnn. Col. fl. .. 23, .9 151.1.

G. 1.. Its....nas and fl. P. \* ir, th'.. Ph tol., 6(. 125 1196 1.

A. E. rou*.;s fl. E. 'r-:.ur.is, and ll. 4. G=-cr. Pla..t C. 11 sird.
10, '.75 (1969).

L. Econr.-alry ar..1 .. Liam, J E-c. B.:,l... IC 29" (1990).

G. t.. P.;.Elesti, Auir. ). Biol. S.:s.. 11, 10?. (19.30).

bid., II, 221 (19001.

Ited., I'". 429 (196 1.1

V. T. Glas.viou, Plant c... ;,al., LE, 895 (1)(0).

It.id., LA, ],tb (1101),

n, 0 tare:I., J. A. Sacher, and I. T. GI..s.:.ou. Pl r.t FI.-ser.,



n. F.I. Hasrb e.1J (.. T C.12nior... PI-.,, lb Col., 3.:. 1' *1963)

J. .A. Sadler, ti Co Hatch and b'. T. Glastacu, Hart Ph, it..I..
MS (1915 ).

fl. G. Flar.h. 6Ioch.-n. J., at C.'I 11960).

J. 5. 11 .,1.. r and :1 0. Hatch, inchem .I.. 99. 101 I1966).

;. 11cad =:ino, I. Beol. Cher... 1.5. ?J. ?"9601.

.;. J. Ms-.1.-r vid II. G. Hatch. th.5 ..*1. II .1,, 10 4' s il96=,).

11. 1. Hare!. -.0 F. T. Glas ion, PI.r... Ph. ol., 19. 180 il9.50.r.

J. A. 5.1 '.er, *:fe Cg;,-,.,-, to 194

2.. H=q aC J. 2. .has i-, F ar.c F.", .il., O 591 (1905'.

it. I IC cohis arid J Ed. non. .'. Ey B-:.E. 2I, E. (197").

. 2. Pa od, Alar.. Fe., 11.,0 Ph ?.el., IE, 15:. 106.1.

F,.*,al. .ard U. ... 1-,s 0, ,j, ra, ,,.. 20', 585 (13 4).







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46, L. F. Leloir and C. E. Cardini, J. Biol Chem., 214, 157 (1955).

47, J. S. Hawker, Biochem. J., 105, 943 (1967).

48. C, P. P. Ricardo and T. ApRees, Phytoc'inem., 9, 239 (1970).

49. A. L. Kurssnov, 5. M. Sok~olova and M. V. Turkina, J. Esp Bot.
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50. A, Rothstein, fi~eRL295&xP 15Biol., 8, 165 (1954).

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52, A. Rothstein and A. D. Hayes, Arch. Biochem. Biophys., 63, 87
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53. J. Vansteveninck and H. L. Booij J. Gen. Phvsiol., 48, 43 (1964).

54, J. VanSteveninck and A, Rothstein, J. Cen. Physiol., 49, 235
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Si. A. Rothstein and, J. VanSteveninck, Ann. N. Y. Acad. Sci,, 137,
606 (1966).

56. H. Wheeler and P. Hanchey, Science, 1ll, 68 (1971),

57. S, Roscman, J, Gen, JIysio, 54, 138 (1968).

58. W. Hengstenberg, J. B. Egan and M. L. Morse, J.Biol~I. Ce, 2113,
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59, J. Edelimn, L. I. Shibko and A. J. Keys, J._ Expg Bot., 10, 178
(1959).

60, T. E. H-umphreys and L. A. Garrard, Phggac-hem., 3, 6ir7 (1964).

61. L. A\. Gar-rard a~nd T. E. Humphreys, Nhatulre, 207, 1095 (119--?)

62. T. E. Hlumphreys and L. A. Garrard, Phytocjaem,, 5, 653 (19*4)!.

63. IbIdJ., 6, 1085; (1967).

Gk bid., 7, 701 (1968).

65. L. A. Garrard and T. E. Humphreys, Phytochem., 7, 1949 ~~i I.

G6. T. E. Humlphreys and L. A. Garrard, Phyt~chemp., 8, 1055 Il?69)i.

67. slbid., 9, 1715 '1l? 0).











70,. 1. C. Memph r, s ,,nd L. Ai. C.irrord, Q gto--hem.'~ , in press (IS'l ),

71. t, la s n o U =m 1 3 7 14 )



:j, r', i., ".ire, in Crl~prgx Larb~oh~drhrre:, ed, b.' E. F. IJessfeld and~
I!, Ginsbulrg. Aa.ii rs ,ue se k(9'6 ,p

/4, 1.. U,. Urn'carcit, r.. Hl. PRurris and J. F. Erjuffer, IV~norneeric Tch-
r l~~, Ru~rge. s, 11i one urspe lls i!1?, 97, p. 28,



17, 110 I19 5)

1,1953),


79. C, H.F.C n r ad LC h r igo ,An g 1 i I .

79, E ,kk.,r l rm t i e er i.to fT a e 1c a i 30.








81. i, I Joihl, And.U Chem c ts, 3..-st 140 f (1943).jn~-j.;l












BIOGRAPHICAL SKETCH


Joseph Henry Whitesell was born August 11, 1936, at Clearwater,

Florida. In June, 1954, he was graduated from Large High School at

Largo, Florida. In June, 1958, ne received the degree of Bachelor

of Science writh a major in Ornamental Horticultulre from Auburn Univer-

sity, Auburn, Alabama. From 1958 until 1960 he served in the Uniited

States Nlavy and was stationied aboard thec USS Cutternut at Long Beach,

Ca lifornia. Following his active duty with~ the Nasvy, he w~orkedi for a

yea"r for the Countiy' of Los Ange-les as a1 marke-t inspector. In 1961

he returned to Florida wh~ere he w~orked as an Assistant County Agent in

Collier County, anld during that time he resided in N~aples, Florida.

In 196j he 2nro~lle in the Graduate School of the University of Florida,

Hea woirkedl as a graduate assistant in the Department of Botany and as

a r~ebeach 5Ssistant at the Pesticid~e Research Labor jor a .l e.

ber, :968, when he received the degren of Mlaster- cai "..:.n- e t ..r~ a1

m~aj or in Botdany. Fromt December, 1968, until the pre star nhna

puirsued his wrork toward the degree of: Occtor of Phi~... -il

Joseph Henry h'h~i teseli is malrried to the former !:.e.n-: 11, ..

Stens-on~ and is the father: of three children.














al..u i.ses n. ...sit .s c ~la di s r ia i r or te de r e 0




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i c.rti., IhjI I h~a.i rE."d this itudrr and that ;ir rt, 1.pinQon .1
onf?-m r r.: acces EDLI~i srandarrdi Ofi shoi jrl prest~ni ot or, ari is f ul l,












i crCif; .r. L E l hs;.:- r:0CS Lhis stu d-, ur. thjt ir, n, opinion ;t
ior f:.i....r.5 to .:p.-ep tlc r.:ji.J-,d~ rds of h y :. pr snI o id fu
adrl' c quite -in* C e jnj q~ujl :v,, ar .; dlsctl Lijnn For r'.* de rs 01


DoctorL'Z C. f~leoh, -



P ychard C, mirn /h


ssiiit .t n. l fes -r f Bc a



:l. .I atJn.:. ..- Src.*e end .uolit,, as 2. dis oraI on[C for~ !Ie JEg:C-a of


Doctor ui *r'd:0(.h,.



Pc.L~ci H-. 8.ggs / /
Frofe nori l of F-rui~ r rr,









Thiis .J;~cirtaion ars sub~mirred to th~e Dejll of the L.ollege of Agricullalne
AIIJ to thei Gradve-ll: Council, andl was- acctepte ;I partial l ulfillmentr of
the requ~iem~in:i for the degree 01' i'c~ctr of Ph losoh,.

Juni, I 1;






Deac., College of Agr icul ture






ean IGraduatre ;ichoo




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PAGE 1

Sugar Transpjort in the Maize Scute! 1 urn By JOSF.PH IIEi.'PvY WHITE SELL A DISGEKTATiON PRESENTED TO THE GRADUATE COUIJCiL OF THE Ul^iiVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQli! RLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 197!

PAGE 2

ACKNOWLEDGEMENTS The author expresses his sincere thanks to Dr. T. E. Humphreys for his guidance throughout the graduate program and for his advice, patience and help in conducting experiments and preparing the manuscript. It is a pleasure to work in his laboratory and share his equipment. The help of Drs. D, S, Anthony, R. C. Smith and R. hi. Biggs as committee, members is also appreciated. The Botany Department generously provided support.

PAGE 3

TABLE 0? CONTENTS Page ACKNOWLEDGEMENTS i i LIST OF TABLES iv LIST OF FIGURES v ABSTRACT vi IKTRODIJCTION 1 LITERATURE REVIEW 3 METHODS A;-;D mTERIALS 75 Preparation of Scutellum Slices 25 Analysis of Sugars 25 Manoin?: try 26 Mstal Analysis 2? RESULTS 29 Kinetics 29 Sucrose Uptake 51 Gas Exchange 62 Metal Binding 73 DISCUSSION 80 LITERATURE CITED 8? BIOGRAPHICAL SKETCH 91

PAGE 4

LIST OF TABLES Table Page 1. Method of Calculating Data 31 2. Glucose Uptake as Affected by Uranyl Ion in the Bathing Solution ^5 3. Fermentation in Water and Sugar Solutions 5^^ k. The Effect of Uranyl Nitrate Pretreatment on Sucrose Uptake 56 5. Inhibition of Sugar Uptake by Phloridzin 58 6. The Inhibition by DNP of Uptake from O.OIM Sugar Solution 59 7. Maltose Uptake 61 8. The Effects of Turanose and Manni tol on Sucrose Uptake ., . 63 9. Sucrose Tissue Level and Sucrose Uptake 6k 10. Sucrose Uptake as Affected by Various Cations 7^

PAGE 5

LIST OF FIGURES Figure Page 1. Sucrose Uptake Vs Time 30 2. Sucrose Uptake Vs Time 35 3. Sucrose Uptake Vs Time 36 k. Sucrose Uptake Vs Concentration 37 5. Sucrose Uptake at Constant Sucrose Concentration kO 6. Glucose Uptake Vs Time k] 7. Glucose Uptake Vs Time k2 8. Rates of Fructose and Glucose Uptake Vs Concentration k'S 9. Glucose and Fructose Uptake as Affected by Uranyl Ion Pretreatment kh 10. Uranyl Ion Pretreatment and Glucose Uptake k8 11. Glucose Uptake at Constant Concentration 50 12. Fermentation in Water and Sucrose 68 13. Gas Exchange in Water and O.IM Sucrose 70 \k. Gas Exchange in Water and O.IM Glucose 71 15. Fermentation in Sugar Solution 72 16. Metal Binding 75 17. Effect of Uranyl Ion Pretreatment Concentration on Sugar Uptake 77 18. Metal Binding Following an Acid Pretreatment .... 78

PAGE 6

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SUGAR TRANSPORT IN THE MAIZE SCUTELLUM By Joseph Henry Wnitesell June, 1971 Chairman: Dr. Thomas E. Humphreys Major Department: Botany Characteristics of the uptake of sucrose, glucose and fructose by maize scutellum slices are presented. Sugars were taken up at almost a constant rate until the bathing solution was depleted even at concentrations well below those which saturated the uptake mechanisms. The effect of DNP, phloridzin, uranyl ion, and anoxia was to inhibit the uptake of sucrose approximately twice as much as the uptake of hexoses. Maltose was taken up without hydrolysis. Turanose was not taken up but slightly inhibited the uptake of sucrose. The following conclusions are dravjn. (a) Sucrose is taken up actively without inversion, (b) Hexoses are taken up by two processes operating simultaneously, diffusion and active transport. (c) The active uptake mechanisms for sucrose and the hexoses are located at the plasmalenvna. (d) The active uptake mechanisms for both sucrose and the hexoses are driven by glycolysis. (e) Metal binding characteristics of the scutellum are different from those of yeast in that binding is not specific to the uptake sites and bound metal ions are apparently not released during sugar uptake.

PAGE 7

INTRODUCTION Much research has been conducted on the movement of sugars in the cells of animals and microorganisms. Stein (l) reviev^s this work in a book which emphasizes the movement of nonelectrolytes across cell membranes. Much less work has been done on sugar transport in higher plants, and yet the problem is central to the ufiderstandi ng of plant physiology. At least witli respect to higher organism's the movement of di saccharides is primarily a plant problem since the gut of animals takes up only nwnosaccharides (2). Sucrose is by far the most common carbohydrate translocated in plants (3). Kursanov says, "A profound understanding of the laws of movement of substance;^ is also of direct importance in agriculture, for it is precisely as a result of the transport of substances that grovjing tissues am provided with food and there is an accumulation of reserve materials in seeds, fruits, roots, and other parts of plants that are of prime importance in crop yields" (3, p. 213). Hartt e_t a_l_. add a quantitative r.ote, "Since sugarcane plants in Hawaii translocate sucrose at the rare of over one million tons per year, translocation studies have an immense economic potential" {k, p. 3C<5) . According to Hartt translocation may form a bottleneck in the total production of photosynthate. Kursanov (3) points cut that the carrier theory which has been developed v;itli respect co mineral .^!lelnents by Epstein and others is probably applicable in the case of the transport of sugars but factu?) 1

PAGE 8

2 materia] to support such concepts is still greatly inadequate. He also states that the very existence of carriers has not been proven. The studies of Eiejeski (5) on the accumulation of sugars by phloem tissue add support to the contention that an active movement of sucrose is probably a part of phloem transport. The work reported herein was undertaken to gain a better understanding of the process of sugar uptake, especially that of sucrose. Several characteristics make the corn scutellum well suited for the study of sugar uptake: (a) The uptake of sugars is rapid allowing relatively short time periods to be used; (b) carbohydrate is stored in the form of sucrose rather than starch; glucose is not -iccumulated; (c) there is no ageing phenomenon during which an adjustment in the respiratory rate occurs; and (e) morphologically, the scutellum is leaf tissue v-;hich functions in the movement of sugar from the endosperm to the developing seedling.

PAGE 9

LITERATURE REVIEW Several systems have been described in which sucrose is taken up without prior inversion. Weatherley (6) measured sucrose uptake by floating leaf disks of Atropa be I ladonna on 10% sucrose solutions and taking dry weights before and after uptake. At times the anxjunt of hydrolysis was very low and the amount of hydrolyzed sucrose in the bathing solution varied independently of uptake. Whereas pH affected the amount of hydrolysis it had very little effect on uptake. The amount of hydrolysis was greater when older leaves were used. Washing disks prior to treatment decreased the amount of hydrolysis. He concluded that sucrose was absorbed as such. He found (7) that the uptake of glucose and sucrose on a per mole basis was nearly identical. Weatherley (8) also found that the uptake of sucrose was reversibly inhibited about 75% under nitrogen. He follovjed the Icjss and uptake of water as well as dry weight and included a discussion of the osmotic situation prevailing in the tissues. He concluded that the uptake of sucrose was probably an active process. Experiiv.ents by Pennell and Weatherley (9) showed that sucrose uptake was inhibited about 50% by 2,^-di ni trophenol (DNP) and not at all by phlorid2:in (both at 1 mM) . In this paper the amount of dry weight increase vias shown to be caused partly by an increase in sucrose, glucose and fructose. The amount of increase not due co thes;e sugars was assumed to be due to starch fcrrration. Bec?u3e a significant

PAGE 10

k amount of weight increase was due to starch it vv/as argued that no "uphill" movement of sucrose need occur. Porter and May (lO) worked with tobacco leaf disks and measured uptake, accumulation and gas exchange in 5% solutions of sucrose and Invert sugar. When invert sugar was supplied, glucose disappeared at a faster rate than fructose. Invert sugar caused a more rapid accumulation of starch than did sucrose. The total uptake of sugar was the same whether invert sugar or sucrose was supplied. Asymmetrically labeled sucrose could be recovered from the leaf disks after being applied in the bathing solution showing that sucrose could be taken up without inversion. They found an RQ. of 0.7^ during incubation of tissue in water, but this increased to about 1.1 when the tissue was incubated in sucrose or hexose. The specific activity of the C0„ evolved was about the same as that of the applied sugar leading them to believe that the observed rate of gas exchange was not the result of a liigb rate of fermentation superimposed on the gas exchange in water. They suggested rather a shift in substrate to sugar when sugars were added to the bathing solution. Vickery and Mercer (II) studied the uptake of sucrose by bean leaf tissue. The bathing solution contained only sucrose and a trace of fructose after incubation, indicating that extracellular hydrolysis was small or nonexistent. When tissue samples were analyzed most of the sugar was sucrose v.^ith small amounts of reducing sugars present. Gas exchange was measured and the RQ. increased from about 0.8 to 1.0 when sucrose was added to the bathing solution. Ihey noticed a linear phase of sucrose uptake with time despite large concentration differences in the bathing solution. The steady rate of sucrose uptake was

PAGE 11

about 3 umoles/g fr \^t hr from 1% (0,028M) sucrose. An inhibition of sucrose uptake of S57o was noted with 2.k x 10~^M OI^P. Uptake was measured over periods of 8 to 26 hr, Vickery and Kercer (12) reported increases in O2 consunipt i on upon the addition of sucrose that were of short duration and independent of concentration. They stated that the rate of C0„ production showed no correlation v/ith the concentration of sucrose in the external solution (and hence, in the free space) but was strongly correlated v/i th concentration of sucrose in the apparent osmotic volume. This is used as an argument that the sites of carbohydrate metabolism are included in the osmotic volume for sucrose and that part, at least, of the cytoplasm is included within the membranes involved in sucrose transport. Sucrose accumulated against a gradient and the initial rates of sucrose uptake follovjed a concentration vs uptake curve adaptive to Michael is and Menten kinetics. After several hr of uptake the rate decreased. This decrease was attributed to inhibition of further uptake by sucrose inside the cell. They argued against the possibility that sucrose pumps occur only at the tonoplast. Hardy and Norton (13) studied the uptake and utilization of Clab^led sucrose, glucose and fructose by slices of potato tubers. Glucose was taken up faster than fructose. All three sugars vjere found vjhen untreated tissue vjas analyzed for sugars. It v;as suggested on the basis of the labeling of various intermediates that sucrose was absorbed unchanged and transported to storage v/here part of i t was hydrolyzed resulting in storage of all three sugars, Sacher (14), using bean pod tissue, found an extracellular or outer space invertase which varied seasonally in its activity. However,

PAGE 12

6 sucrose uptake \jas not dependent on invertase activity as shown by the observation that sucrose was taken up in the absence of outer space invertase activity. Glucose uptake was three times as vast as fructose uptake from 0,03M solution. There was no effect of 10"^ to 10~^M urany! nitrate on uptake of 0,03M glucose or sucrose. The fact that sucrose was taken up as such was also demonstrated by showing that the glucose/fructose radioec ti vi ty ratio was little changed by uptake when using fructose -labeled sucrose. Uniformly labeled sucrobe remained uniformly labeled even in the presence of unlabeled fructose or glucose in the bathing solution. DNP (5 x IO"^M) inhibited uptake from 0.0003M sucrose 96% and from 0.03M sucrose 88%. In freshly cut tissue the endogenous sugars consisted largely of glucose and fructose, and only trace amounts of sucrose. Upon incubation in sugar solution sucrose was stored, apparently in the vacuole, and the sucrose/reducing sugar ratio was high. However, after incubating the tissue for 16 hr in water the sucrose was hydrolyzed and the ratio greatly decreased. Sacher argues on the basis of several lines of evidence that the rate limiting step in hexose uptake lies in the formation of sucrose and that the cytoplasm is free space to the hexoses. Kriedemann and Beevers (I5, 16) worked v/ith castor bean seedlings. In the germinating castor bean, sucrose is synthesized in the eridosperm at the expense of fat. The sucrose is taken up by the cotyledons, and it is transported from them into the developing se^jdling axis. In the castor bean cotyledons, which are functionally analogous to the scutellum, most of the sugar is in the form of sucrose v;! th virtually no free hexoses present. They showed a linear uptake of sucrose with time by the cotyledons and sucrose v;as accumulated against a concentration

PAGE 13

7 gradient. DNP partially inhibited the uptake of sucrose. Sucrose was shown to be taken up without inversion by several lines of evidence including the retention of asymmetry of labeled sucrose applied in the bathing solution. In this tissue sucrose was taken up at a liigher rate than either hexose and, more unusual, the rate of fructose uptake was considerably greater than that of glucose uptake, Kriedemann (17), on the basis of mi croautoradiographs of castor bean cotyledons exposed to labeled sucrose for 20 min, suggests that the cell walls and intercellular spaces provide a diffusion pathv;ay by which solutes can gain access to the vascular system from an external source. Kursanov (3) refers to v;ork in vjhich it v;as shown that the fibrovascular bundles from sugar beet petioles took up sucrose, glucose and fructose but exhibited a much higher affinity for sucrose. Grant (18) studied the uptake of glucose, fructose, and several other nranosacchar ides by carrot and corn root tissue. Some of his results were as follows. Carrot root tissue exhibited a lag of several hr before uptake began. The uptake of glucose under N2 was less than 25% of that in air. He shovyed that glucose entered the cell and accumulated as the free sugar against a concentration gradient. The glucose concentration in the carrot tissue exceeded 0,05M assuming equal distribution v/ithin the fresh weight of the tissue. Uptake of glucose proceeded at a constant rate from O.OOIM solution until the bathing solution was exhausted. He showed that the uptake of glucose and several other sugars follows uptake vs concentration curves according to Michael is and Menten kinetics. He did not seem to be concerned with the fact that a constant rate of uptake with time, in spite of a

PAGE 14

8 declining sugar concentration, is inconsistent with reaction rates £is predicted by Michael is and Menten kinetics. The maximum rates of sugar uptake by carrot discs were from 3 to 10 umoles/g hr and from corn roots were from 7 to 3^+ umoles/g hr (19). It was shown that in carrot tissue the respired COwas derived preferentially from the entering sugars. ApRees and Beevers (20) using carrot and potato slices measured O2 consumption and CO9 evolution in low (0.5 umoles/ml) concentrations of glucose. The RQ. did not vary s i grii f i cant 1 y from unity and the addition of glucose did not induce significant changes in O2 uptake or C0« output. Reinhold and Eshhar (21) demonstrated an active uptake mechanism in carrot root tissue which was capable of accumulating 3-0"methyl gl ucose. The rate of uptake of 3"0-mothyl gl ucose varied with concentration approximately as predicted by Michael is and Menten kinetics. After a period of uptake the tissue v/as rinsed for 30 min and then placed in water and a concentration ratio of 75:1 was maintained betv;een the concentrations in the tissue and water. Chromotography and analysis of C0„ indicated that 3-0-0-^3 thy 1 gl ucose was not metabolized. Harley and Jennings (22) studied the uptake of sugars by beech mycorrhizas. They found that the rate of uptake vs concentration curves for glucose and fructose formed rectangular hyperbolas but the shapes of the curves were different, the maximum rate for fructose being higher than the maximum rate for glucose. In this tissue there is considerable hydrolysis when sucrose is supplied. DNP markedly inhibited the absorption of hexoses. When mixtures of glucose and fructose were supplied, glucose was preferentially absorbed. The addition of sugars caused a respiratory stimulation and from equimolar

PAGE 15

concentrations the stimulation caused by glucose was considerably greater than that caused by sucrose in spite of the fact that a roughly equal amount of sugar on a weight basis was taken up during the measurements. Morgan and Street (23), studying the carbohydrate nutrition of excised tomato roots, found an RQ, of about 0.75 in water and about 1.0 in sucrose. The root segments had been starved prior to measurement and the uptake of Oy v^as stimulated by the addition of sugars. The RQ. of root tips supplied with sucrose, dextrose, galactose, or raffinose was within the range 0.90-0. 96. The endogenous respiration had an RQ. as low as O.7O and in mannose as low as O.6O. Thomas and Weir {2k) measured the uptake of sugar by tomato root segments from solutions of 0,05M glucose and 0.025M sucrose, it was found that more sugar on a weight basis was taken up when sucrose was supplied as compared to glucose. Sucrose is markedly superior to glucose in supporting growth of excised tomato roots. Wiien radish root slices are incubated In sucrose there is a considerable amount of extracal 1 ular hydrolysis (25). It is not known whether or not any sucrose is taken up without inversion. Sucrose at O.O29M and O.O58M both stimulated the evolution of CO2 and it was suggested that this v.-as due to a saturation of respiratory enzymes. Bieleski (5) m.easured the uptake of sucrose by excised vascular bundles or phloem tissues from a variety of plants. In apple phloem and celery vascular bundles about 70% of the uptake from '^C sucrose could be found in the tissue in the form of sucrose. Sucrose uptake, from 0.001 and O.OIM solution, proceeded at a progressively slower rate until the external solution contained about 10% of the original amount of sucrose.

PAGE 16

10 Sucrose vias taken up against concentration gradients of the order of 10-^, The rates of accumulation by vascular bundles or phloem tissue were much higher than rates by parenchyma from the same plant. Vascular tissue accumulated sucrose at rates of 9 to 16 umoles/g fr vvt hr from 0,1M sucrose solution. Rsinhold and Eilam (26) addressed the question as to whether there was a diffusion barrier between external substrate and sites of respiration. They supplied sunflower hypocotyl segments with labeled glucose or glutamic acid in the presence or absence of ONP and measured the total amount of CO2 evolved and its specific activity. They subjected the data to kinetic analysis and concluded that thef-e was not an effective diffusion barrier between the external substrate and the sites at vjhich substrates are respired. An alternaMve is given; "It remains just possible, however, that such a mechanism [active transport^ does operate in the absence of DNP, but that in its presence the molecules are able to diffuse freely inwards owing to a disorganization of the cell membranes" (p. 306), Sugar cane is probably the most studied of higher plants in regards to sugar movements, Bieleski (27, p. 20^) stated the uptake problem as follov/s: "...it was found that disks of sugar cane tissue placed in aerated distilled water lose very litLle of their endogenous sugar to the v.ater. Thus either the tonoplast is extremely impermeable to sugar movement or iihare is an accumulation mechanism in the cell which actively opposes the outv;ard diffusiona] movement of sugar. The first is perhaps the simpler explanation, but raises the problem of explaining how the sugar originally became accumulated behind the impermeable tonoplast."

PAGE 17

n Sugar cane exhibits a large, rapid (1-hr duration, 8-min half time), apparent-free-space uptake followed by a slow uptake which can occur against a gradient and which results in sugar accumulation. The accumulation uptake will proceed over a period of 72 hr. In comparing rates of uptake of various sugars he found that glucose uptake was more than double that of sucrose uptake on a molar basis. Uptake of fructose was similar to that of glucose, Bieleski measured respiration during sugar uptake and found an increased O2 uptake upon the addition of sugar to the bathing solution. He does not mention any change in RQ. associated with sugar uptake, Bieleski (28) found that sugar accumulation was completely inhibited by 10"5m DNP, Phloridzin at 2 x lO'^M caused from 10 to 80% inhibition of the uptake of glucose. V.'hen tissue was prev^ashed in 2 x 10"^M magnesium chloride It caused a 0-20% increase in the amount of glucose accumulated. Double reciprocal plots of sucrose, glucose and fructose uptake rates vs concentration yielded straight lines (29). The Vp^g^ reported for sucrose was 0,7 umoles/g hr. Glasziou (30, 31) suggested that the outer space consists of the cell walls and cytoplasm and is in diffusion equilibrium with the external solution. "Hence the cytoplasm is part of the outer space where outer space is defined as the tissue volume which comes to rapid diffusion equi 1 i br i urn wl th sugars in the external solution (the outer and inner space for this tissue may be quite different for solutes other than sugars)" (31, p, 178). Tracer studies showed that the hexoses in the inner space came from hydrolysis of stored sucrose. Hatch e_t a_l_. (32) reported on some of the enzymes involved. They report characteristics of sucrose synthetase in the direction of su-

PAGE 18

12 crose synthesis. Activity in the reverse direction could not be detected because of the presence of a phosphatase v/hich rapidly hydi-olyzed UDP to UMP. Evidence for the presence of sucrcse-P synthetase was presented, and acid and alkaline invertases viere described. The/ could not find sucrose phosphory 1 ase. Enzymes for the synthesis, i ri terconversion, and breakdown of hexose phosphates viere identified. The amounts of acid and alkaline invertases vary v/i th the growth rate and the sucrose storage rate (33) suggesting a key role for invertase in regulating the movement and utilization of sucrose. Sacher e_t aj.. (3^+) present a scheme for the sugar accumulation cycle in immature sugar cane. Acid invertase occurs both in the outer space and the storage compartment. Sucrose is hydrolyzed prior to uptake and glucose is taken up several times as fast as fructose. Sucrose is released from storage via hydrolysis and diffusion of the hexoses out of storage. Match (35) demonstrated the presence of sucrose-P synthetase in both leaf and storage tissue of sugar cane. He also showed the synthesis of sucrose-P by tissue supplied with glucose. Sucrose v/as stored more rapidly ffcm sucrose than from sucrose-P and more rapidly from fructose than from fructose-P, This is consistent v;i th the proposition that sugar phosphates do not penetrats membranes as easily as do non-pho5phory1ated sugars. Asymmetry of labeled sucrose was lost during storage. While only small quantities of sucrose were stored when sucrose-P was supplied, the asymmetry of label was largely maintained. This was consistent with a scheme in which sucrose-P is formed by the action of sucrose-P synthetase and sucrose is stored against a sucrose cor.centration gradient via the hydrolysis of sucrose-P to yield stored sucrose.

PAGE 19

13 In furtlier support of such a scheme Hawker and Hatch (36) demonstrated the presence of a specific sucrose phosphatase in sugar cane, carrot roots, etiolated barley, oat, and pea seedlings, parsnip root and potato tuber. The enzyme was associated with particles vjhich behaved 1 i ke mi tochondria during differential centr i fugati on, Mendicino (37) had earlier described enzymes in wheat germ and green leaves that included sucrose synthetase, sucrose-P synthetase, and a nonspecific sucrose phosphatase. Hawker and Hatch (38) present a scheme for the mechanism of sugai" storage in mature sugar cane tissue. Evidence v;as presented to shovi that the hydrolysis of sucrose is a prerequisite to storage and a rate limiting step. Mature cane tissue contains an acid, wall-bound invertase and a neutral invertase apparently located in the cytoplasm. The storage compartment invertase found in immature tissue is absent in mature tissue. Sucrose storage takes place more rapidly from hexoses than from sucrose. Uptake of both glucose and sucrose as a function of the concentration of the bathing solution had the kinetic properties of an enzyme-catalyzed reaction, in studies on the localization of enzymes it was found that most if not all of the sucrose synthetase was located in the conducting tissue, and it is pointed out that it may function in the breakdown of sucrose. Hatch and Glasziou (39) presented direct evidence that sucrose is the predominant component of translocated photosynthate in sugar cane. Tlie asymmetry of labeled sucrose was maintained through the vascular tissue or the leaf, sheath, and stem. Randomization did occur during movement into storage, Sacher (40) presented an argument for extracytoplasmi c sucrose

PAGE 20

synthesis in the bean endocarp. He supplied UDPG and labeled fructose and obtained sucrose in which the label was predominately in the fructose moiety. Experiments also indicated the presence of UDPG pyrophosphorylase in the extracytoplasmi c space. When labeled fructose vjas supplied and sucrose synthesis occurred in the cytoplasm the C glucose/' C fructose ratio was approximately one; this sucrose remained in the tissue even after extensive washing. By using acetone-extracted chloroplasts from sugar cane, Haq and Hassid (^1) were able to show the synthesis of sucrose-P from UDPG and fructose-P and the synthesis of sucrose from UDPG and fructose. The preparation contained phosphatases that hydrolyzed sucrose-P and fructose-P. Schoolar and Edelman (kl) measured secreted sugar, CO^ fixation, sugar formation, and starch synthesis by leaf disks of sugar cane floated on various solutions. The amount of sucrose secreted into the bathing solution was increased by 10"^M sodium iodoacetate (lOA). About one-third of the total sucrose synthesized during a i+-day period was secreted. The Inhibitor caused no change in the amount of soluble sugar within the disks and it caused an increase in the amount of total soluble photosynthate produced. Respiration measured in the dark showed an RQ, of considerably less than 1.0 and this was reduced even further by lOA. Other respiratory inhibitors did not elicit similar responses. Many investigations have been made of various enzymes involved in sugar transformations. Only a few v.'i 1 1 be mentioned here. In his review article on sugar transformations in plants, Hassid (^3) discussed the characteristics of sucrose synthetase and sucrose-P syn-

PAGE 21

15 thetase, the two enicymes most likely to be itivoived in the synthesis of sucrose from glucose and fructose or from either hexose alone, Putman and Hassid (kk) studied the transformation of sugars in vacuum-infiltrated disks of Canna leaves, V.'hen labeled fructose or glucose was provided, labeled sucrose was recovered which was labeled in both hexoses; however, no free labeled glucose could be found vjhen labeled fructose was provided and vice versa, an indication tiiat sucrose vias formed via phosphory 1 ated hexose intermediates. When sucrose was provided in the bathing solution there was rapid inversion of the sucrose with the appearance of hexoses in the bathing solution followed by a resynthesis of sucrose v/ithin the tissue. Cardini £t a_K (^5) point out that the equilibrium constant of sucrose phosphorylase lies in the direction of sucrose hydrolysis and that sucrose phosphory 1 ase has not been found in higher plants, A study of the characteristics of sucrose synthetase from a variety of plant tissues is reported. The equilibrium constant, K = (sucrose x UDP)/(UDPG X fructose), varied from 2 to 8 at 37 and pH 7.1^ in different experiments. Leloir and Cardini (^6) studied the properties of sucrose-P synthetase but point out the difficulties caused by the presence of interfering enzymes (phosphatase and sucrase) when working with this enzyme. Hawker (47) found that the ratio of sucrose-P synthetase/sucrose synthetase varied in different plants from 10 to 0.6. Ricardo and ApRees (kS) measured the activity of both acid and alkaline invertases and sucrose content during development of carrot and during ageing of root disks. There was a negative correlation

PAGE 22

16 between sucrose content and acid invertase activity. Acid invertase activity was iiigh during times of high sugar usage and low during times of high sucrose storage. They suggest that high invertase activity prevents sucrose storage and that during periods of lovj hexose demand hydrolysis is due to alkaline invertase v/hich is not associated with the vacuole but located in the cytoplasm. They suggest that the acid invertase is located In the wall and at the tonoplast. Kursanov e_t aj[, (^9) compaied the localization and properties of hexoki nase wi tfi uptake characteristics of conducting tissues from sugar beet. This tissue takes up glucose faster than fructose and the hexokinase associated with the structural elements of the cells has a higher affinity for glucose than for fructose. On this basis they suggest that hexoki nase on the membrane may be part of the uptake process. The uptake or sugars and the metal binding characteristics of yeast have been studied intensively. Rothstein (50) presents several lines of evidence to show that uranyl ion affects the uptake of glucose by yeast due to its binding to the surface and not to an uptake into the cytoplasm of the cell. Rothstein and Meier (51) describe the competition for uranyl ion between the yeast coniplexing loci ar.d various coiTplexinq agents added to the bathing solution. On the basis of this work they concluded that the binding sites on the surface of the yeast cells were polyphosphates. Uranium blocks about 90% of the uptake of glucose in yeast (50). Several other cations, including Co^"*", Mg^"*", Ca^"*", and Hn^"^, bind to the surface of yeast cells but uranyl ion forms a much more stable complex (52). Data were presented to show that whereas the other cations -were bound to the same sites that bind uranyl ion, they did not inhibit the uptake of glucose.

PAGE 23

17 Data showing the amount of various ions bound to the surface of yeast cells as a function of the ion concentration are also presented by VanSteveni nek and Booij (53). They shov'ved that in the case of 2f2+ Ni or Co when glucose was added to the cells the metal was displaced from the surface of the cells and appeared free in solution. When the glucose had been taken up by the cells the metals were again bound. if cells were first poisoned with 1 OA and then supplied with glucose a small amount of glucose uptake occurred but was complete in 15 min. The amount of glucose uptake by the poisoned cells was the same (on a umole basis) as the amount of uranyl ion bound (on a uequivalent basis) by nonpoisoned cells. It was possible by adjusting the growth medium to vary the amount of phosphorus per yeast cell without causing irreversible damage to the cells. The amount of uranyl ion bound and the amount of glucose taken up after poisoning were both reduced in phosphorus deficient yeae^L. There v;as a good correlation between the amount of uranyl ion bound and ti.e amount of glucose taken up by poisoned cells. When yeast vjas poisoned and then provided with glucose the uranyl binding capacity disappeared. The addition of lOA alone caused a SC/i inhibition of cation binding which could be reversed by washing the cells in water. VanSteveni nek and Rothstein (5^) present an argument to show that in yeast, sugar uptake can proceed by facilitated diffusion or by an active uptake mechanism. The facilitated diffusion system can be demonstrated with galactose uptake by uninduced cells and with glucose uptake by poisoned cells. The active transport system is driven by the energy released by glycolysis in spite of the fact that glucose is quickly utilized and does not accumulate in the cells. The two

PAGE 24

18 systems are different with respect to Ni^"*" binding, effect of Ni^"*" on uptake, concentrations of uranyl ion required to inhibit uptake, kinetic parameters, and patterns of specificity. Rothstein and VanSteveni nek (55) summarized work done on uptake and metal binding by yeast cells. It was pointed out that the inhibi2+ tory effects of uranyl ion and Ni are not due to displacement of a required cation. The conclusion is reached that the phosphoryl sites, to which uranyl ion binds, are used continuously in glucose transport and are regenerated continuously by glycolysis. In the yeast system a close correlation is pictured between glycolysis and uptake and glycolytic ATP is assumed to be the energy source for driving uptake. Carrier and glycolytic reactions are thought to be in close gecgrapliic proximity. In their scheme to explain the transport of sugars the carriers for facilitated diffusion and for active transport are considered to be the same. V/hen active transport occurs the amount of carrier available for facilitated diffusion is reduced. Wheeler and Hanchey (56) placed oat roots into 0.1 and 1 , mM uranyl acetate for varying periods of time and then made electron micrographs in which crystals, apparently composed of a uranium complex, could easily be seen. After a 30-min treatment followed by a 30-min desorptlon the uranyl complex was sharply localized in cell walls, Intercellular spaces and secretory products in direct contact with cell walls. With longer treatment times, up to 20 hr and the lower concentration, the uranyl complex crystals could be found in vesicles in the cytoplasm and in the vacuole. Otherwise the cells were normal with no uranyl ion free in the cytoplasm or in cell organelles, Uranyl ion apparently caused a definite dilation of the m,embranes from a normal

PAGE 25

19 widlh of 90 A to a width of from 150 to 200 A. This effect could be seen on the plasmal eninia of treated cells and in vesicles which contained uranium. They concluded that few, if any, free uranyl ions passed through the protoplast and that uranyl ion in addition to being bound to the plasmalemma is bound to cell walls and to secretory products along its surface. Roseman (57) has reviewed the literature on a bacterial phosphotransferase system that is thought to be responsible for the uptake of sugars. The system as it operates in Escheri chi a co_l_i_ consists of three protein fractions: Enzyme 1, Enzyme II, and a low molecular weight protein designated HPr. Phosphoenol pyruvate (PEP) is the phosphate donor, and a variety of sugars including some di saccharides can serve as acceptors. Enzyme ! and HPr are found in the cytoplasm and Enzyme II is associated with the membrane. Enzyme I and HPr are constitutive whereas Enzyme 11 is constitutive with respect to glucose. Most Enzymes II are inducible. The specific sugar requirements of the system are due to Enzyme II, Enzyme I and HPr are common to all sugars phosphory 1 a ted by the system. Enzyme I catalyzes the transfer of phosphate from PEP to HPr which serves as a phosphate carrier. The specific Enzyme II then catalyzes the transfer of phosphate from phosphate-HPr to the sugar being phosphory lated, Roseman presents convincing evidence to show that the phosphotransferase system is the same as the permease systems and is responsible for the uptake of sugars. In most cases exogenous sugars enter the cell as sugar phosphates and this is described as group translocation. When free sugar enters the cell the process is called active transport.

PAGE 26

20 Staphylococcus aureus accumulated sucrose-P when incubated in sucrose and it is thought that the phosphotransferase system is operative in the uptake and phosphorylation (58). Edelman £t £l. (59) worked with scutella, roots and shoots of oats, rye, wheat, and barley. They showed that the scutellum contained a higher ratio of sucrose to hexose than did the root or shoot. Hexose absorption was inhibited by about half when experiments v-jere run under nitrogen. Substantial sucrose formation took place in the scutellum under nitrogen, whereas incorporation into amino-acids, amides, malic acid, and sugar phosphates was considerably reduced. In these tissues fructose is absorbed at about half the rate of glucose. Sugar phosphates, sucrose, glucose, fructose, glutamic and aspartic acids and their amides, malic acid, C0„, and polysaccharides were found to contain label after applying tracer amounts of labeled fructose or glucose. The scutellum was shown to contain much lower levels of hydrolytic enzymes than the roots or shoots. All of the enzymes necessary for the formation of sucrose from hexose were found in the scutellum and a scheme is presented to show the path of sucrose synthesis which involves the enzyme sucrose-P synthetase. Humphreys and Garrard have published a series of papers dealing with the uptake, production, storage, and leakage of sugars by the corn scutellum. They demonstrated that glucose uptake proceeded at a constant rate even though glucose in the bathing solution was largely depleted of glucose as a result of uptake (60). The rate of glucose uptake was shown to vary depending on the conditions and length of the prior incubation of the tissue. Changes in the tissue content of various sugars and sugar phosphates after y/arying periods of time in

PAGE 27

21 water were presented, and it was shown that mannose inhibited the uptake of glucose. Data were presented to show that the corn scutellum accumulates carbohydrate mostly in the form of sucrose, the content of starch and hexose being low. Experiments concerning the glucose-free space of the scutellum, which involved measuring the amount of glucose in the tissue after incubation in various concentrations of glucose in the presence and absence of DNP and mannose and the measurement of glucose exit following transfer into water, indicated that the space vjas intracellular and that a carrier was not involved. Fructose and mannose occupied a space of similar size (61). When incubated in high concentrations of fructose (0.1-0,9M), scutellum slices synthesized sucrose, some of which was stored and some of which leaked into the bathing solution (62). The leakage of sucrose v;as reduced in the presence of Mg*" , Mn or Ca and EDTA increased the leakage from the synthesis compartment (63). When sucrose storage was measured after incubation in fructose or sucrose it was found that the pH of the incubation medium was more important when sucrose vjas the sugar taken up (6'+). The maximum storage occurred at a pH of 'i-.5 with both sugars; at a pH of 7.5 there was about a 20% decrease in stored sucrose v/hen fructose was the exogenous sugar whereas with sucrose in the bathing solution there was 80% inhibition. The authors suggested that the sites of sucrose uptake are in contact with the bathing medium. The amount of sucrose storage was increased when sucrose was added to the bathing solutiori containing optimum amounts of hexose. This would not have been the case if sucrose were being hydrolyzed prior to uptake or during the process of uptake.

PAGE 28

22 The loss from storage was measured by loading the storage compartment with C sucrose and then incubating the tissues at different pH values and with and without "cold" sucrose. More sucrose was lost at the higher pH values and the loss was greater in the presence of "cold" sucrose than in water indicating an exchange between external and stored sucrose. The addition of fructose or glucose to scutellum slices (65) resulted in a strong, aerobic fermentation and the concommi tant production of ethanol. Increased sucrose synthesis upon incubation in fructose accompanied an increase in glycolysis without an increase in O2 uptake. This supported the idea that glycolytic ATP might be responsible for sucrose synthesis. When incubated in water the RQ for intact scutella was about 3 while that for slices was near unity. It was concluded, on the basis of the levels of various phosphofructokinase regulators during different rates of glycolysis, that control of glycolysis in the scutelluiT was exerted through the availability of substrate and the distribution of adenine nucleotides and inorganic phosphate. Pretreatment v;ith tri s(hydroxymethyl )ami nomethane (tris) prevented the storage of exogenous sucrose but the inhibition could be reversed 3+ 2+ 7+ ?+ by hydrogen ion or by AK , Mn , and to a lesser extent Mg and Co . Sucrose storage from fructose was little affected by the pretreatment wi th tris (66) . Pretreatment with uranyl nitrate (67) was similar in its effects to pretreatment with tris in that storage of exogenous sucrose was inhibited and the inhibition could be reversed by H , AH and vo some extent Mn . Uranyl ion pretreatment only slightly inhibited sucrose

PAGE 29

?.3 synthesis from hexose and the Inhibition was not thought to act through the uptake of hexose. It is possible, by incubating slices in high concentrations of fructose, to build up considerable concentrations of sucrose in the synthesis compartment. That this is sucrose and not sucrose-P has been demonstrated. Upon reducing the external sugar concentration and Inhibiting leakage, the free sucrose in the synthesis compartment will be transferred to the storage compartment. Experiments with mannose (68) indicated that whereas mannose inhibited the storage of exogenous sucrose, it did not affect the storage of sucrose which had accumulated in the synthesis compartment. The suggestion was offered that the storage of synthesis compartment sucrose involved a non-nucieotide phosphate doner such as occurs in the bacterial phosphotransferase system. Pretreatment of scutellum slices with HCl (O.OIM) did not inhibit the storage of exogenous sucrose or the synthesis and storage of sucrose from fructose (69) . Recently Humphreys and Garrard (70) suggested that leakage is from the sieve tubes and is the end result of a ssries of events which include intercellular sucrose transport, vein leading and phloem transport. Several compounds, all of which can either displace or form complexes v^i th Ca and Mg , protect the leakage process which is labile at 30 in water. Since some confusion exists in the use of terminology concerning uptake studies, definitions of several terms used in the presentation of data and the discussion are given here. Uptake--This term is used for the disappearance of a substance

PAGE 30

2k from the bathing solution and is not meant to imply a particular mechanism. Some authors would use absorption, Di f fusion--The net movement of molecules as a result of their thermal motion from a region of higher to one of lower concentration. Where a membrane is crossed resistance may be due to the limited number and size of pores in the membrane or to the solubility characteristics of the solute in the membrane. Facilitated di f fusi on--Thi s is a process in which a concentration gradient is the driving force as in diffusion and the process leads to a disappearance of tlie gradient. The process is thought to involve a membrane constituent (carrier) located on or in the membrane which "facilitates" diffusion. Facilitated diffusion of a solute across a membrane requires no input of energy other than that needed to maintain structure, it may show a high degree of specificity, and kinetics are likely to show saturation thus not conforming to Fick's law of diffusion. Active transport--Thi s process involves the use of metabolic energy as a driving force. it is capable of bringing about the accumulation of a substance against its concentration gradient. It is generally characterized by a high degree cf specificity and saturation kinetics.

PAGE 31

METHODS AND miERIALS Preparation of Scute] Uirr, Slices Corn grains ( Zea mays L. , cv. Funks G-76) were soaked in running tap water for 2k hr and then placed on moist filter paper in the dark at Ik-lS for 72 hr. The scutella were excised and cut transversely with a razor blade into slices 0.5 mm or less in thickness. The slices were wasiied in distilled vjater until the wash water remained clear and then viere blotted on filter paper and weighed into groups of from 0.1 to I.O g depending on the type of experiment. During preparation, the slices were thoroughly mixed so that each group of slices was a random selection from 50-100 scutella. This resulted in excellent agreement when measurements of uptake or accumulation were made on duplicate groups of slices from one day's preparation. Results were not as consistent when duplicates were compared f rom di f ferent days' preparations. Unless otherwise noted, incubations were carried out with 1.0 g of slices in each 25 ml flask at 30 in a "Gyrotory" water bath (New Brunsvjick Scientific Company, New Brunswick, N. J.) rotating at approximately 200 rev/mi n. The volume of solution was usually 10 ml. Analysis of Sugars Glucose and sucrose were determined by the glucose oxidase method (Glucostat, Worthington Biochemical Corp., Freehold, N. J.). Sucrose samples were incubated for 2 hr with and without invertase prior to analysis. 25

PAGE 32

26 Fructose and reducing di saccharides were analyzed according to the Nel son-Somogyi copper reduction method (7I , 72) as reported by Spi ro (73). The alternate copper reagent suggested by Somogyi was used. When sampling the bathing solutions for stigars, amounts between 0.1 and 2.0 ml were taken depending on the concentration, and appropriate dilutions were made so that between and I'+O ug of glucose or fructose were used for analysis. Twice this amount was used for disaccharides other than sucrose, Absorbance was read on a Klett-Summerson Model 8005 photoelectric colorimeter. Tissue sucrose was extracted by pouring 20 ml of boiling 80% ethanol over the slices and continuing the boiling for 30 sec. The slices were steeped in the alcohol for 30 min, the alcohol was decanted and the procedure repeated. The slices were then rinsed three times with 5 rnl portions of alcohol. The combined extracting solutions were evaporated almost to dryness on a steam bath. V/ater was added to a volume of 50 ml and 10 drops of O.IN NaOH added to adjust the pH. The resulting aqueous solution was frozen. After thawing the solution was centrifuged for 10 min in a clinical centrifuge. For the determination of sucrose, 0.1 ml of this solution was used. Manometry Experiments were carried out in a Warburg Respirometer at 30°. The direct method for COwas used (7^). The amount of tissue added to the flasks was either 100 or 300 mg. When the slices were prepared they vjere placed without weighing into 25 ml Erlenmeyer flasks in 10 ml of water and incubated for 1 hr at 30°. The water incubation removed leakable sucrose (69). Following the Incubation the slices were blotted

PAGE 33

27 and weighed into Warburg flasks. The sugar solutions were either added at the time the slices were placed in the flasks or added from the side arm during the course of gas exchange measurements. Metal Analysis Uranyl ion was determined by the method described by Rulfs e t a 1 . (75). Absorbance was read at ^00 nm as suggested by Silverman et al . (76). Aluminum was determined by the method of Gentry and Sherrington (77) as reported under procedure A by Sandell (78). The extraction was made at pH 5. The purpurate method of V/illiams and Moser (79) as described by Sandell under procedure A (78) was used for the determination of calcium. Magnesium was determined by the Eriochrome Black T Metnod (80). The permanganate method of Kydahl (81) reported by Sandell (78) was used for the determination of manganese. Cobalt was determined by a modification of the Nitrosc-R salt method of Harston and Dewey (82) as reported under procedure B by Sandell (78). The methods used for uranyl ion and aluminum are not specific; however, since samples from untreated controls showed zero values interfering ions were not present. The methods used for manganese and cobalt are specific. Magnesium, in amounts that would be present in the solutions analyzed in these experiments, is reported not to interfere with the purpurate method for calcium. Copper, iron, and manganese do interfere to some extent. The value obtained for calcium in the control samples was low but not zero; however, some calcium would be expected to leak from the control slices. The method for magnesium is not specific, but the amounts of interfering metals in these experiments were too low to cause significant error. The amounts of calcium and magnesium found by uaing these methods agree very closely

PAGE 34

28 V"/i th those found by atomic absorption spectroscopy in earlier work by Humphreys and Garrard (70).

PAGE 35

RESULTS In the first section kinetic data will be presented to show the rates of upta|
PAGE 36

30 20 15 Time , mi n Figure 1. Sucrose Uptake Vs Time. One g of slices was used In each flask. The slices were incubated for 1 hr in water, given I rinse, and then 10 ml of the sugar solution v;as added. The first sample was taken 1 mi n after adding the solution (time zero on the graph). Samples of 0.5 rnl were taken so that the volume during the last 30 min had been reduced to 8.0 ml.

PAGE 37

3i Table 1 Method of Calculating Data" (O.OO75OM sucrose) Klett uni ts 123456 7 Decrease Due to in sucrose Decrease Concentration Time Without With sucrose due to in column of sucrose min invertase Invertase (3-2) uptake 3 M

PAGE 38

32 Table 1) by ignoring the non-i nver tase-treated sample. In rhis case it is assumed that glucose is coming From extracellular inversion and the sucrose inverted but not taken up is not counted as uptake. The dotted line represents the actual decrease in sucrose of tlie solution (calculated from column 5, Table I). The two methods do not result in large differences in calculated uptake. Table 1 is presented to demonstrate the method of n.easuring sucrose uptake. The i-emainder of sucrose uptake data presented was calculated by the method represented by the sol id 1 i nes. When the rinse vjater v^as withdrawn by suction immediately before adding the sucrose, an unmeasured amount of water adhered to the slices and to the sides of the flask and caused a dilution of tne added sugar solution. There is probably also a free space volume in the slices which causes dilution so that analysis of the first samples indicated a sucrose concentration considerably lower than that which v;as initially added. This decrease in concentration can be accounted for by dilution. In the experiment of Figure 1, the concentration I min after adding the sucrose was O.OO66OM when O.CO75OM sucrose was added and 0,0022511 when O.OO25OM sucrose, was added. These concentrations can be accoi;nted for if it is assumed that there was l.i ml of water and free space in the case of 0.00250iM sucrose. The figure for O.OO75OM sucrose v/ould be 1,4 ml. No effort wos made to determine the amounts of water ond free space. The problem was ignored by measuring upcake from the time the first sample v/as taken. The data are presented to show that there is not a large, rapid phase of uptake when sucrose is first added to the tissue. The rate of uptake increases after the first 30 mir\. Either the

PAGE 39

33 rate gradually increases over the first 30 mi n or there is a delay before uptake begins. in subsequent experiments the first sample was taken 15 min after adding the solution. Notice that the rate of uptake was constant with time in spite of the fact that the concentration of the external solution was continually reduced. In the upper curve the concentration at the beginning of the last 30-min period was 70% of that at the beginning of the first 30-min period. In this same series of experiments a constant rate of uptake was obtained with O.OOIH sucrose (data not presented) in spite of the fact that the concentration of the external solution was reduced to kOYo of that added. More experiments were run to determine the rate of sucrose uptake with time and the effect of sugar concentration on uptake rate. The rate of sucrose uptake with time is shown in Figures 2 and 3. The curves are more unifcrm at the lovjer concent rritions and as the concentrations increase the curves become more erratic owing to the difficulty of detecting small changes in concentrated sugar solutions. A water control showed that the leakage of sucrose amo'jnted to no more than 0.2 umoles per g fr wt over the period of time during which uptake was measured. The best approximation of all of the curves seems to be a straight 1 ine. Figure k shows the effect of sucrose concentration on sucrose uptake. The rate was arrived at by dividing the total uptake by the time period over which uptake was m,easured. As seen by the theoretical curve, the data agree very well with the typical Michael is and Menten hyperbolic substrate concentration curve for which the constants were derived from a Lineweaver and Burk plot of the data.

PAGE 40

Figure 2. Sucrose Uptake Vs Time. In these experiments 1 g of slices was incubated in water for I hr. This was followed by 2-10 m! rinses and then the uptake solution was added to the slices. Ten ml of uptake solution was added and the amount of sucrose removed in tha sampling was taken into account when calculating the data. The first sample was taken 15 min after addition of the uptake solution (time zsro on tlie graph). Samples of 0.5 f^I were taken every 15 r,;in. Each curve represents c'ata from 1 day's experiment.

PAGE 41

35 Time, mi n

PAGE 42

36 Figure 3. Sucrose Uptake Vs Time, Conditions were the same as in Figure 2. Samples were taken every 45 min.

PAGE 43

37 (U

PAGE 44

38 The experiment of Figure 5 shows sucrose uptake when the bathing solution is maintained at a constant sucrose concentration. In this experiment the volume of the bathing solution was reduced to ^f ml for greater accuracy. At the end of each 15-min uptake period the solution was removed and fresh O.OOIM sucrose was added to the slices. The rate of uptake can be seen to increase with time over a period of 2 hr. Figures 6 and 7 show the rate of glucose uptake with time. As with sucrose, the glucose uptake curves roughly represent straight lines with possibly a little more tendency for the rates to decrease vji th time. In the case of 0.005H glucose the concentration of the sugar solution v/as reduced by about half during the course of the experiment, yet the rate of uptake over the last three periods was about the same. Very similar data were collected using fructose but they are not presented. Figure 8 shoves the uptake vs concentration data for glucose and fructose. in the case of glucose and fructose the data do not fit at all when an attempt is made to find the constants Vmax and Km by plotting the data according to a Lineweaver and Burk plot. Neither do the data agree with what v;ould be expected if diffusion were the driving force for glucose uptake. Uranyl ion has been shown to inhibit sugar uptake (67) and to act at the cell surface (55). Uranyl ion caused a partial inhibition of glucose and fructose uptake. This was true both when uranyl ion was added to the uptake solution and when the slices were treated with uranyl ion prior to the uptake period. The dotted lines in Figure 9 show the uptake vs concentration data for glucose and fructose when uptake was measured following a pretreatment with uranyl ion. Table 2

PAGE 45

Figure 5. Sucrose Uptake at Constant Sucrose Concentration, One g of slices was incubated in water for I hr followed by 2-10 ml rinses. 4.2 ml of O.OOIM sucrose was then added and 1 min later a 0.2 ml sample was taken. A second sample v;as taken 15 mi n after the first. Then the entire solution was removed and 4.2 ml of a fresh O.OOIM sucrose solution added. This procedure was continued throughout the experiment.

PAGE 46

ko T i me , m i n

PAGE 47

k\ ^ 16 Figure 6. Glucose Uptake Vs Time. The data from which these curves were prepared come from the same type of experiments as shown in Figures 2 and 3 with the exception that glucose was the sugar taken up.

PAGE 48

42 Figure 7. Glucose Uptake Vs Time. The data from which these curves v^ere prepared come from the same type of experiments as shown in Figures 2 and 3 with the exception that glucose was the sugar taken up.

PAGE 49

43 o o OD Jjq 6/sa(oujn '9>jB3dn 950x39 aJ

PAGE 50

kk U1 tZ iiZ \o

PAGE 51

45 Table 2 Glucose Uptake as Affected by Uranyl ion in the Bathing Solution Glucose concentration Uptake period Inhibition M hr ?^, .001 1 58 .005 1 5^ . 005 2 53 .01 2 kh .01 3 kS .1 3 34

PAGE 52

46 shows the effect of uranyl ion on glucose uptake when uranyl ion was added to the uptake solution. Notice that in both pre treatment and treatment during uptake, the inhibition v/as greater at lower concentrations of glucose. The inhibition was greater when the slices were pretreated with uranyl ion. This is perhaps a result of a long term effect of uranyl ion since in the preLreatment experiments uranyl ion was first applied to the si ices 1013 min prior to the uptake period. The glucose uptake vs concentration curve after uranyl ion pretreatment resembled, at least at low concentrations, a diffusion curve (Figure 9). Since glucose is not accumulated by the tissue but is rapidly used for sucrose synthesis or is fermented (60, 65) the question arises as to whether the kinetics presented are those of uptake, i.e.^ that glucose is limited by diffusion, or those of the hexokinase reaction. Jones (83) studied the properties of hexokinase from the corn scutellum and reports the Km for glucose as 6.5 x 10""M. A Km of this magnitude indicates that diffusion and not the hexokinase reaction is limiting. If diffusion is the driving force for uptake and if it is assumed that the internal glucose concentration remains constant and very low then the uptake should be a straight line function of the glucose concentration. A straight line was, in fact, obtained at concentrations of O.OIM and below. The deviation from linearity at higiier concentrations may be due to higher internal concentrations of ylucosj and to a saturation of the glucose utilization process at the higher concentrations. To check this hypothesis further the rate of uptake into uranyl treated and untreated slices v/as followed with time (Figure lO). In the case of glucose alone the uptake proceeded at almost a constant rate until the glucose in the uptake solution had been depleted to a

PAGE 53

Figure 10. Uranyl Ion Pretreatment and Glucose Uptake. The pretreatment consisted of 1 hr in either water or O.OO3M uranyl nitrate. The slices were then given 2 rinses, 30 min in water, 2 more rinses, and then the O.OIM glucose solution was added. The first sample was taken 8 mi n after the solution was added and samples were taken every 30 min thereafter for a period of 210 min. The curves are an average of 2 determinations. The volume of the uptake solution was 6 ml so that larger differences in Klett readings were noted over short time periods.

PAGE 54

k8 Time, mi n

PAGE 55

level below that of the detection system used for glucose analysis. This curve cannot be explained on the basis of diffusion alone. It might be argued that the rate of utilization of glucose by the tissue is constant and that the curve simply represents the glucose utilization rate. However, as shown elsewhere by both uptake and accumulation data (Figure 7, Table 9), the uptake mechanisms of the tissue are not saturated even at much higher hexose concentrations. In contrast the curve after uranyl nitrate pretreatment is typical of a diffusion curve. If an arbitrary constant is multiplied by the concentration at the beginning of each period of uptake, the curve represented by the dotted line is obtained (P'igure 10), It is postulated that glucose uptake is the total of two processes, one consisting of simple diffusion soon after uranyl Ion treatment and one an active process which Is subject to inhibition by uranyl ion bi ndi no. If this Is true it means that the active component as represented by the difference In the vwo curves In Figure 10 is increasing with time (i.e., since the external concentration is decreasing, diffusion is decreasing and the active component must be increasing In order to maintain a steady rate). With this In mind an experiment was run In which tlie concentration of glucose was kept constant by renewing the solution after each sample as was done with sucrose (Figure 5), The results of this experiment failed to support the hypothesis (Figure 11). Glucose uptake remained constant with time in spite of the fact that the concentration was kept constant. The uptake over a period of 2 hr was very close to the value obtained with declining glucose concentration. When uranyl nitrate was added to the uptake solution In this

PAGE 56

50 4, Uranyl ion added /j-C — "w.. Urany! ion added time zero 30 60 T i tne , m i n J. 90 120 Figure 11. Glucose Uptake at Constant Concentration, Tissue was subjected to 1 hr in water, 2 rinses, and then the 0.005M uptake solutions. The first sample was taken 1 min after adding the solution. Twerity minutes later a second sample v;as taken and the uptake solution was withdrawn and fresh solution added. This procedure v;as continued throughout the experiment. Where indicated on the graph the solution contained 0.003M uranyl nitrate. The uptake volume was h ml.

PAGE 57

51 experiment the rate of uptake declined with time. This may be due to a long term effect of uranyl ion. The amount of inhibition caused by uranyl ion was the same whether it was added at the beginning of the uptake sampling or added after the tissue had been taking up glucose for 80 min. An experiment was run to determine v/hether or not the inhibition of glucose uptake by uranyl ion was parallelled by an inhibition in the amount of sucrose gained by the tissue. The slices were placed in water or 0,003H uianyl nitrate for I hr, rinsed, placed in water for 30 min, rinsed, and placed in .IM fructose or glucose for 3 hr after which the slices were rinsed and killed for analysis of tissue sucrose. A control group was killed after the 30 min water incubation. The net increase in tissue sucrose in the water treated groups was 70 and 73 umoles in fructose and glucose, respectively, and the inhibition by uranyl ion v.'as 5^ and 53%, respectively. Uptake (Figure 9) was inhibi ted 38 and k^%. Sucrose Uptake In order to study sucrose uptake it should be established that sucrose as such 5s the sugar being taken up. Several lines of evidence will be presented to show that sucrose is taken up without inversion by slices of the corn scutellum. The effects of several inhibitors on sugar uptake will also be presented in this section. The amount of inversion of sucrose in the external solution is small as measured by the appearance of glucose in the solution. For instance at a sucrose concentration of O.O5M the maximum amount of glucose noted in the solution v/as O.OOO6M. The amount varied over the uptake period from this value to an amount below the limit of detection

PAGE 58

52 for the amount of sample used. This would mean a maximum hexose concentration of 0.00I2M and an uptake according Lo Figure 8 of approximately 7 umoles of hexose or 3" 1/2 unioles of sucrose per hr which is roughly one-tenth of the rate of sucrose uptake obseived (Figure h) . This argument implies the assumption, justified by Figure 8, that the two hexoses are taken up at about the same rata. This argument has been presented before by Humphreys and Garrard (6U) , The possibility exists that inversion takes place in a position such that hexoses move preferentially toward the point of uptake. Kinetic data on the uptake of sucrose, glucose, and fructose were presented in the first section. The overall shapes of the uptake rate vs concentration curves is considerably different. Whereas hexose uptake increases with concentration over the range shovvn, sucrose uptake approaciies a maximum. The considerable difference in the effect of concentration on uptake is an indication that two different processes are at work. By comparing the data in Figures k and 8 it can be seen that the total amount of carbon taken up from O.IM solution is higher when hexoses are supplied. In experiments measuf-ing tissue sucrose it was noted that the amount of sucrose gained by the tissue was the same or higher when the slices were incubated in sucrose as compared to incubation in hexose. in order to check this effect on the same group of slices an experiment was run in which uptake was allovjed to proceed over a period of 205 mi n from solutions of O.IM sucrose or glucose. Duplicate flasks were run. Uptake was measured over 3 hr of the uptake period. The amount of carbon taken up from gl'icose solution was 110% of that taken up from sucrose solution although the amount of sucrose

PAGE 59

53 gained by the slices was the same in both cases. The slices were incubated in water following the uptake period and then rinsed, killed and analyzed for sucrose content. When gas exchange studies were carried out using 300 mg of tissue in the Warburg flasks it was found that the addition of sucrose, glucose, or fructose to the solution caused high rates of fermentation to occur. It was assumed that the amount of O2 consumption represented sugars being completely respired vyiierees the amount of CO2 evolved in excess of the amount of O2 consumption represented sugars being fermented. It has been demonstrated previously that under similar conditions ethanol is produced in amounts equal to the excess CO^ (65). The data in Table 3 show the amount of fermentation over a 2 hr period caused by the various sugar solutions. In the fermentation experiments the slices were incubated in water for 1 hr in the water bath, blotted, and weighed into Warburg flasks v/hich contained the sugar solutions. The flasks were then attached to the manometers, equilibrated for I5 mi n and the readings begun. No effort was made to regulate the time between adding the slices to the sugar solutions and the beginning of the readings. The rate of respiration was reasonably constant regardless of the solution. The rate of fermentation was higher in fructose or the hexose combination than it was in sucrose. In terms of hexose uptake, the rate of sucrose uptake at O.OIM is 35 umoles of hexose equivalent per hr compared to a rate of 25 umoles for hexose. Uptake of glucose and sucrose from O.OIM solution by the same day's slices gave the following results; sucrose I7 umoles/hr (or 3^umoles hexose), glucose 28 umoles/hr. If the amount of fermentation is a function of the con-

PAGE 60

54 Table 3 Fermentation in V/ater and Sugar Solutions Respiration Fermentation umoles umoles No. of Solution hexose/hr hexose/hr experiments (Data averaged from several day's preparations) Water 5.8 (0.4)" 4.3 (0.8) 5 O.OIM sucrose 4.9 (0.4) 11.6 (1.9) 5 O.OIM fructose 4.9 (0.2) 12.0 (1.7) 4 0,005M fructose & 0.005M glucose 5-3 (O.l) 13.8 (1.2) 2 O.IM sucrose 5.9 28.5 1 O.IM fructose 5.0 (0.1) 36.8 (1.8) 2 O.IH glucose 4.8 40.5 1 'Numbers in parentheses indicate the average deviation. See text, and materials and methods, for experimental detail.

PAGE 61

55 centration of hexose in the fermentation compartment and if sucrose is being inverted, even in the process of uptake, the rate of fermentation in sucrose should be higher than it is. The same argument can be made in the case of O.IM sugar, since the amount of fermentation in hexose solution is proportionately greater than the amount of hexose uptai
PAGE 62

56 Table it The Effect of Uranyl Nitrate Pretreatrnent on Sucrose Uptake Uptake umoles/hr Concentration Uranyl nitrate M Water pretreatrnent pretreatrnent 0.001 k.3 0.3 0,005 1^.3 2.7 0.01 . 21.0 5.^ 0.05 29.0 5.7 0.1 41.0 2,3 Slices (1.0 g fr wt) were placed In either water or uranyl nitrate for 1 hr. The slices were given 2 water rinses, placed in water for 30 rnin, given 2 more water rinses and finally placed in the bathing solutions. Two samples were taken, the first 15 min after the bathing solution was added and the second at the end of the uptake period. Uptake was measured over a period of 60 min with O.OOIM and 0.005M sucrose, 90 min with O.OIM sucrose and 180 min with 0.05 ^nd O.IM sucrose. Rates of uptake in the first three are averages of the results of two experiments, the last two are from one experiment.

PAGE 63

57 Table 5 shows the inhibition of sugar uptake by phloridzin. At 1 X 10~^M this Inhibitor cduseri abouc tv\'ice the amount of inhibition of sucrose uptake as it did with glucose uptake. With neither sugar was uptake inhibited strongly by the low concentrations used with animal systems. Dini trophenol (DNP) also inhibited sugar uptake (Table 6). The inhibition was considerably greater with sucrose than with glucose; the sucrose inhibition approached 100% whereas the inhibition of glucose uptake approached S07o. Sucrose uptake in the presence of 1 x IQ-'+M DNP was linear vn th time for at least 1 hr. The uptake of sucrose was measured in the presence of several disaccharides to determine if the uptake process was subject to inhibition by molecules of similar structure. Several of the di saccharides caused an increase in the noni nver tase-treated samples. This caused confusion in interpreting the results since it was not known whether the increased amount of glucose in solution was coming from hydrolysis of the disaccharide or from an increased hydrolysis of sucrose caused by the presence of the di saccharide. Unless otherwise stated these experiments were run by incubacing first for 1 hr in water and then placing the slices in O.OIH sucrose plus the various di saccharides in concentrations of 0.005, 0.01, and 0,05K. The uptake period was 1 hr. Lactose was not taken up by the tissue as determined by measuring the lactose concentration before and after 1 hr incubations by the reducing sugar method.. When solutions were tested for glucose the amount was found to be insignificant. When sucrose uptake was measured in the presence of lactose there was an inhibition of 33% i i the case of 0.05M lactose if it is assumed that the higher glucose comes from

PAGE 64

58 Table 5 nhibition of Sugar Uptake by Phloridzin Concentration Inhibition of °L_ phloridzi n M Glucose Sucrose 1 X 10"^ 1 X 10"^ n 5 X 10"^ \k 1 X 10"3 13 25 2x10"^ h-] 3 X 10"^ 60 The slices (1.0 g fr wt) were incubated in water for 1 hr, rinsed twice, and placed in a solution of O.OIM sugar and phloridzin as indicated. Results are based on a non-inhibited control. Uptake was msasured over a 2 hr period.

PAGE 65

59 Table 6 The Inhibition by DNP of Uptake from O.OIM Sugar Solution Concentrati on of DNP 1 X 10"5m 3 X 10~5m 1 X 10-^H 3 X 10-^M

PAGE 66

60 the increased hydrolysis of sucrose in the presence of lactose. in other v;ords, the same amount of sucrose was found at the end of the upta|
PAGE 67

Table 7 Mal tose Uptake Concentration of added mal tose

PAGE 68

62 glucose readings were little or no higher than when sucrose was added alone. Turanose at O.O5M caused approximately a 30% inhibition of sucrose uptake (Table 8). The uptake of turanose alone was checked by using the reducing sugar method and was found not to be taken up by the slices. That the effect of turanose is not an osmotic effect was demonstrated by neasuring the uptake of sucrose alone, sucrose in the presence of turanose and sucrose in the presence of mannitol. Several experiments were designed to show the effect of tissue sucrose level on the rate of sucrose uptake. The experiments are summarized in Table 9. Generally tliere is an inverse correlation between the amount of sucrose in the tissue and the rate of uptake. The tissue was subjected to various treatment sequences so as to vary the amount of sucrose in the tissue prior to measuring uptake. In those three cases narked by an asterisk the sequences were the same except for the concentration of sugar during the first 3 hr and in each case sucrose uptake was measured during the fifth hr from the time the experi ment started. In these cases there is an excellent inverse correlation between the amount of tissue sucrose stored and the rate of uptake. This is to be expected since there must be some limit to the amount of sucrose that can be accumulated by the slices and one would expect the rate of uptake to be reduced as this limit is reached. Gas Exchange When gas exchange experiments were carried out using 300 mg of tissue in the Warburg flasks the amount of O2 taken up by the tissue was limited by the rate at which O2 diffuses into water. This is shovn by the following: (a) The rates of 0^ consumption noted in these experi' ments were equal to the limits of the rate at v;hich O2 diffuses into

PAGE 69

63 Table 8 The Effects of Turanose and Mannitol on Sucrose Uptake Uptake umoles/q hr O.OIM sucrose O.OIM sucrose Experiment O.OIM sucrose 0.05M turanose 0,05M mannitol 1 li).2 iO.l 2 . 1 .^^ , 5 11.7 16.0 12,2 16.5 12.7 3 16.1 ll.it 15.6 17.1 1^.6 16.6 Slices (1.0 g fr wt) were incubated for I hr in water, rinsed twice, and the indicated solutions were added. The first sample was taken 15 inin after addition of the uptake solution. Uptake was measured over 1 hr in experiment 1 and 2 hr in experiments 2 and 3.

PAGE 70

Table 9 Sucrose Tissue Level and Sucrose Uptake Gk Treatmenf sequence Water 3 hr, rinse, water 1 hr, rinse, sucrose 1 hr V/ater 1 hr, rinse, sucrose 1 hr 0.02M fructose 3 hr, rinse, water 1 hr, rinse, sucrose 1 hr O.IM fructose 3 he, rinse, water 3 hr, rinse, sucrose 1 hr 0,1M fructose 3 hr, rinse, water 1 hr, rinse, sucrose 1 hr O.IM fructose 3 hr, rinse, sucrose ] hr Ti ssue level umoles/g 50 68 81 125 133 Sucrose uptake umoles/g hr 25* 17 21* 18 15* 11 The results from 3 crops of seedlings are reported here. In each case the uptake period was 1 hr, the sucrose from which uptake was measured was O.OIM and there was a 15 min delay between adding the uptake solution and taking the first sample. Duplicate samples were subjected to the sequences as listed and at the beginning of the uptake period one group was killed for sucrose analysis while the other was used to measure sugar uptake.

PAGE 71

65 water (7^). (b) \vhen DNP was added under these conditions the amount of COj evolution was increased; however, the amount of O2 consumption was not, (c) When experiments were run using 100 mg of tissue in water the consumption increased on a per weight basis and the RQ. decreased. The figures from two typical experiments vjere: with 300 mg tissue, 33 umoles /g hr, RQ. 1,3; with 100 mg tissue, 53 umoles 02/g hr, RQ. 0.8. Since when using 100 mg of tissue, the rate of O2 consumption is well below the limit and since the RQ. is quite low it was assumed that O2 was not limiting under these conditions. It vjas not knovja whether or not 0^ consumption was limited under conditions which prevailed in the water bath. An experiment in which sucrose uptake from O.OO5M sucrose was measured as a function of the surface area of the liquid per v;eight of tissue shov;ed that upt
PAGE 72

66 where 1 flask was used and 139 urnoles/g where h v>fere used. (These figures are not net sucrose accumula ced since the amount of tissue sucrose at the beginning of the uptake period has not been subtracted,) Work by Humphreys and fiarrard (/O) and by the author have shown that the sucrose content of scutellum slices is reduced when slices are incubated in water indicating that sugar is the substrate for respiration. This was also noted when 0,25 g of tissue per flask was used. However, gas exchange studies using 100 mg of tissue have indicated an RQ. in water substantially less than 1.0 indicating a substrate other than sugar. This inconsistency might be explained by the conversion of sucrose to organic acids. Figure 12 shows the amount of fermentation in water and in two concentrations of sucrose v^hen 300 mg of tissue was used. It is assumed in presenting these figures that for each umole of CO2 evolved in excess of Oo taken up one-half umole of hexose was being fermented. Figure I 2B represents the same experiment but the amount of fermentation in the water control has been subtracted from that caused by sucrose. • On the basis of uptake experiments it was calculated that the solutions in these flasks would be depleted of sucrose after 3 hr in the case of O.OOIM and k Ur in the case of 0„005M sucrose. in the case of O.OOIH sucrose the sugar-caused fermentation starts when the sugar is added, increases in rate, and then decreases at the time when the sugar should all have been taken up. The amount of fermentation caused by O.OG5M sucrose is about three times that caused by O.OOIH sucrose and as shown earlier in Figure k the rate of uptake is about three times as great. With 0,005M sucrose the agreement in timing between uptake and fermentation is not as close, however, the rate does begin to decline at a time when uptake should be complete.

PAGE 73

Figure 12, Fermentation in Water and Sucrose. Each flasl< contained 300 mg of tissue. The sucrose was placed in the side arm and added at 30 min to give the concentration indicated on the graph. Three sets of flasks ware used, one each for the 2 concentrations of sucrose and one containing water. The values were obtained from; CO2 O2/2 (in umoles). !n B the values found with water have been subtracted from the values found v-;! th sucrose.

PAGE 74

68 60 50 ko 30 20 30 20 Sucrose added rO^ rr; ^ 0.005M sucrose Sucrose added O.OOIM sucrose U42=££iij; JL X 2 3 Time, hr

PAGE 75

69 Figure 13 shows the gas exchange in O.IM sucrose as compared to that in water. These experiments were run under conditions in which O2 was not limiting. With water the RQ. is less than 1,0 and both 0^ uptake and CO, evolution proceed at constant rates throughout the experiment. When sucrose is added there Is some depression in the rate of consumption. The rate of evolution of CO2 on the other hand continues to increase throughout the experiment. Over the period of this experiment the uptake of sucrose would proceed at a constant rate (Figures 2 and 3). When 0,01M sucrose was added in the same type of experiment (data not presented) the pattern of gas exchange was the same; however, the Oy consumption was not depressed as much and the rate of COo evolution was not as high. Figure ]k shows the same type of data for 0,1M glucose. Again the rate of O2 consumption remains at a fairly constant rate while the evolution of CO2 is greatly increased by the addition of glucose to the slices. The RQ. during the last period measured was 2,3. As with sucrose O.OIM glucose (data not presented) caused roughly the same pattern as 0,1M glucose and uptake proceeded at a constant rate (Figures 6 and 7), if the assumption is made that the difference in COp evolution in water versus that in sugar is due entirely to fermentation (65) then it is possible to calculate the number of umoles of hexose being fermented. Figure I5 shows the results of such a calculation. These results are from the same experiments as reported in Figures 13 and 14. Rates of fermentation as suggested by the above data are consistant with data from experirrjents in which sucrose accumulation was measured. As pointed out earlier, the rate of fermentation is higher under conditions of limited 0^ in the V/arburg apparatus and the amount of sucrose

PAGE 76

70 90 120 150 Time, mi n 180 210 Figure 13. Gas Exchange in Water and O.IM Sucrose. Experiment: v;ere run with 100 mg tissue per flask. The sucrose solution (I.OM) was added from the side arm 30 min after readings were begun.

PAGE 77

71 30 „J

PAGE 78

72 80 . C 0. 1 K cjlucose 0,01 M glucose \f 0. 1 H sucrose ^J7 0.01 h sucrose Figure 15. Fermentation in Sugar Solution. Conditions as in Figures 13 and ]k. Results were calculated as CO2 (in sugar soO'CO^ (in water)/^ (in umoles).

PAGE 79

73 accumulated is lower under conditions of limited 0„ in the water bath. The rate of glucose uptake from O.IM glucose was about 87 umoles/g hr. The rate of fermentation as indicated in Figure I5 was 28 umoles hexose/ g hr. This viould leave about 60 umoles of hexose available for accumulation, or enough to accumulate sucrose at a rate of 30 umoles/g hr. An experiment v/as run in which slices were incubated in O.IM glucose for 3 hr. Each flask contained 250 mg of slices in 2.5 ml glucose solution. Sucrose accumulated at a rate of 35 umoles sucrose/g hr. Metal Binding Experiments were designed to determine the amount of uranyl ion that would bind to the slices, the effect of other cations on sugar uptake, and the relationship between the binding of some other cations and the binding of uranyl ion. Table 10 shovjs the re^iults of two experimients on the effect of cations on sucrose uptake. In the first experiment the cations were added to the uptake solutions. In the second experiment the slices were first treated with O.OIN HCl to remove cations attached to the slices, then treated with metal cations, rinsed, and placed in sucrose. Uranyl ion reduces sucrose uptake when it is used as n pretreatment or wh^n it is present in the uptake solution. Cations other than urany! ion have little effect on uptake. Notice that the control, third column of Table 10, was treated with acid showing that the acid treatment does not seriously impair uptake. The next series of experiments were designed to determine the quantities of the viirious iosis that would bind to the surface of the slices. Figure 16 shows the amounts of the various ions that can be removed with O.OIH HCl after pretreatment with the various metals. Several

PAGE 80

Ik Table 10 Sucrose Uptake as Affected by Various Cations Uptake in umoles/q hr Treatment With uptake solution Pretreatment Control 11.6 9.0 CaCl2 11.5 11. C0CI2 9.1 10.8 MnCl2 12.1 10.1 MgCl^ 10.1 10.0 AICI3 9.7 9.5 U02(N03)p 2.3 2.6 The data in the second column are from an experiment in which slices were incubated in water for 1 hr, rinsed v;ith water, and placed in solutions containing 0.005M sucrose and O.CO3M cation solution. The third column represents a 15-min incubation in O.OIN HCl followed by 2 rinses, 50 mi n in O.OO3M cation solution, another rinse and finally the O.OO5M sucrose solution from which uptake was measured. In both experiments the uptake period was 2 hr by 1 g of slices per flask.

PAGE 81

75 M^"*"**"'^^ (0.01i,M) (0.02M) (0.02M) R Al3+

PAGE 82

76 points can be made. (a) The ions were bound sufficiently to resist several rinses and a 1-hr incubation in water. (b) A concentration of 0.002M cation solution provided enough ions to saturate the sites in all cases, (c) Appreciable quantities of the ions at low concentrations were bound to the slices. In the case of cobalc (0.0003M), 1.6 umoles of the 3 umoles available were bound by the slices, (d) Magnesium appears to be the ion that normally occupies the sites. The data from Figure 16 show that the slices incubated in v/ater instead of metal cation solutions released 6.5 umoles of Mg^'*' and 0.75 umoles of Ca^'' . (e) Greater quantities of uranyl ion are bound than any of the other ions tested except Mg^"*". In the case of iiranyl ion, two experiments v;ere devised to see if the effect on sugar uptake parallels the degree of cation binding to the sites. Figure 17 shows the results of these experiments and it can be seen that maximum inhibition is reached at concentrations of uranyl ion that saturate the binding sites. The inhibition of glucose uptake in this experiment was greater than usual (e.g., see Figure 9). If the slices were first treated with HCl and then incubated in metal solutions, the amounL of cation binding vyas considerably reduced. This Is shown in Figure !3 for uranyl Ion, Mn^'^, and Ca^ . Apparently the acid treatment rendered most of the sites unavailable for cation binding. However, the slices took up sucrose as shown in Table 10 v;i th or without replacing the cations. Of course, the possibility exists that the sites involved in sugar uptake were reoccupicd by caflons from witliln. The smaller Quantity nf uranyl ion that binds after acid treatment was sufficient to Inhibit uptake as is shov/n in Table 10, Work with yaast indicates tba'; metals are displaced from the bind-

PAGE 83

11 .^.^A>. -^ 80 o '". 60 . 40 20 .^ / / V A^ / II 7 Sucrose "v Glucose / » // „1 J ""l " ""2 ' 3 Pre treatment uranyl ion concentration, M x 10-* Figure M . Effect of Uranyl Ion Pretreatment Concentration on Sugar Uptake. In the two experiments reported here the schedules were as follows: I hr in the designated concentration of uranyl nitrate, 2 rinses, 30 min in water, 2 more riiises, the uptake solution. The uptake solution was 10 ml of 0.01M sugar in both ca^es. With sucrose the uptake period was 90 min; with glucose the uptake period was 1 hr. (Whereas the uptake period may affect the degree of inhibition, in this case the experiment was designed to emphasize inhibition as a function of uranyl ion binding and not tlie amount of inhibition.)

PAGE 84

78 T" 2 3 Sal t solution, M x 1 0^ Figure 13. Metal Binding Following an Acid Pretreatment. The slices (1,0 g fr v/t) were subjected to the following sequence' 15 min in 0.01 N HCi , 1 hr in the metal solution, 2 rinses^ water for 1 hr, 2 more rinses, 15 m'" in O.OIN HCI. The MCI solutions were then removed for analysis of the cations.

PAGE 85

79 i rii^ sites during sugar uptake and are again bound artcr the sugar has been talon up (53). Several experiments which involved pre treatment with Co or Mg ' failed to detect this phenomena with slices of the corn scutel \ urn. As described in tho methods section, corn vs/as normally grown in tap water. In one experiment corn was grown in distilled water. Sucrose uptake by slices from this group of seedlings was about the same as with slices from tap-water-grov;n seedlings (i.e., 17 umoles/g hr from O.OIM sucrose). Pretreatment with salts of Ca^"*", Mg^"^, or K^ had little effect on sucrose uptake into slices from seedlings grov;n in di sti 1 led water.

PAGE 86

DISCUSSION From the rer.ults of this study the following conclusionr. are drawn. (a) Sucrose is taken up actively without inversion. (b) Hexoses are taken up by two processes operating simultaneously, diffusion end active transport. (c) The active uptake iiiechani sms for sucrose and the hexoses are located at the plasma lemma. (d) The active uptake meclianisms for both sucrose and the hexoses are driven by glycolysis. (e) The characteristics of metal binding as related to sugar uptake are quite different in the coin scutellum when compared to those cf yeast ce 1 1 s . Several lines cf evidence have led Humphreys and Garrard (65) to the conclusion tliat sucrose is taken up V7i thout inversion by the corn scutellum. Results obtained in this work support that conclusion. (a) The amount of extracellular inversion was insufficient to support uptake at the observed rate. (b) The kinetics of hexose uptake are different from the kinetics of sucrose uptake (cf. Figure k and Figure 8). (c) The rate of fermentation in sucrose was less than would be expected (Table 3) if inversion occurred either prior to or during uptake, (d) if inversion preceded uptake, the effect of uranyl ion on sucrose should be similar to the effect on hexose which is not the case (cf. Table k and Figure y). (e) The ratio oT accumulated sucrose to sugar taken up is higher when sucrose is supplied than when glucose is suppl ied (p. 52) . The maltose data (Table 7) show that maltose is also taken up by the tissue without hydrolysis. 80

PAGE 87

81 That sucrose uptake is dn active process is indicated by the fact that it is taken up against concentration gradients. Slices that contained 133 umoles sucrose/g (0.1 3M sucrose) v\;i 1 1 take up sucrose from O.OIH solution (Table 9). Slices incubated in v;ater for 1 hr contained 68 uroles sucrose/g (0.068M sucrose) and will take up sucrose from O.OOIM sucrose (Figure 2). The tissue sucrose concentrations given above are ;Tiiniaium values since an equal distribution of sucrose throughout the tissue water is assumed; compar tmentation of sucrose would increase the ratios. The scute] lum is composed of mesophyll parenchyma, an epithelial layer and vascular tissue. It is assumed that most uptake occurs in the parenchyma cells since these cells appear to make up Tbout 80-90% of the scutellum. The inhibition of sucrose uptake by DNP is consistent \-/i th the idea of an active, metabolic energy-requiring process. The inhibition of sucrose uptake by turanose (and probably by lactose) fulfills one of the criteria given for a facilitated diffusion or an active transport process (1). The data in Table 10 clearly show that a 15-min rinse in O.OIN HCl does not disrupt the normal functioning of the cells insofar as uptake is concerned. Since tliis treatment removes uranyl ion from the cells and restores the ability of the cells to take up sugar (67), it appears that the effect of uranyl ion is at the cell surface. It is concluded that the mechanism for sucrose uptake is located at the piasmalemma and the same argument ccn be applied to the active portion of glucose uptake (see below). in contrast to these results, Sacher ']k) found that uranyl ion had no effect on sugar uptake, Humphreys (personal communication) has shov/n that slices are not damaged by high concentrations of fructose, When placed tn 2.0M f.Tctose slices will synthebi^.e

PAGE 88

82 sucrose. In contro-jt, pre treatment of slices in l.OM sucrose caused the slices to appear flaccid and rendered them incapable of synthesizing sucrose vjhen placed in O.GIM fructose. These observations support the contention that the external membrane is permeable to hexose but not to sucrose. However, the results of this thesis indicate that the scutellum also has an active 's:;xose transport mechanism. The constant rate of glucose uptake until the bathing solution has been depleted (Fig. 1 O) cannot be explained by diffusion alone and yet many data have been accumulated to shovj chat the cytoplasm is free space to hexosei. The combination of diffusion and an active transport mechanism can e/.plain these results. The hexose uptai
PAGE 89

83 form sucrose or is catabol i zed. It is assumed that the glucose which diffuses into the tissue is phosphorylated via hexokinase while that glucose taken up actively is phosphory 1 ated at the plasmalemma. This is active transport in the sense that uptake is being energetically driven at the membrane but it is not active transport in the sense that glucose is being accuiii'jlated against a gradient. The active process described here v/ouid be called group translocation by Roseman (57). A combination of active transport and facilitated diffusion is thouglit to be involved in the uptake of gli-cose by yeast (55). Reinhold and Eilam (7.6), working v;ith sunflower hypocotyl , suggested that active transport operated in the absence of DNP but that in i tc presence sugars diffused into tlie cells. The idea that all of sucrose uptake is active whereas half of glucose uptake is active and half passive is supported by the following results, (a) At concentrations of O.OIH sugar and 1 x lO'^M phloridzin the inhibition of sucrose uptake was twice that of glucose uptake (Table 5). (b) Sucrose uptake v/as inhibited twice as much by DNP as was glucose ijptake. (c) The effect of anercbic conditions (p. 55) was to inhibit sucrose uptake twice as much as glucose uptake: however, tlie inhibition of sucrose uptake was not complete. It is concluded tiiat the active uptake inechanisms of glucose and sucrose are driven by glycolysis. That glycolysis can drive uptake is indicated by the degree to wliicb uptake proceeded under N2 (p. 55). That 0^ availability limits accumulation of sucrose but does not limit uptaka (p, 65) is a further indication that glycolysis can drive uptake. The fact that fermentation is detected even when 0^ is not limiting is an indication that fermentation drives uptake regardless of the avail-

PAGE 90

8^f ability of . Yoiinis e_t a_|_. (2i)) citcributed on incrensed evolution of COo to a saturation of the respiratory enayrres. This is not the case vn th scutellum slices since the consumption of O2 was reduced in the presence of O.IM sugar. Evidence has been presented to show that fermentation drives sugar uptake. Mo evidence has been obtained to shovj that a specific glycolytic step is responsible; however, the data are consistent with a system such as the phosphotransferase system in bacteria where PEP is the energy source for uptake. Garrard and Humphreys (65) studying control of glycolysis in the maize scutellum found no differences in the ATP levels in the presence and absence of fructose. The amount of fructoseP, which stimulates phosphof ructoki nase, doubled in the presence of fructose but this was considered inadequate to account for a four-fold increase in the rate of glycolysis. The use of PEP in the sugar uptake process might trigger fermentation. The data in Figure h vjhich show sucrose uptake as a function of the concentration in the bathing solution closely fit a Hichaells and Menten curve. This type of data is often presented ip support of a carrier uptake mechanism that follows enzyme kinetics. Hov/ever, the curves that show a linear uptake with time in a bath of decreasing concentration (Figs. 1, 2, and 3) are not at all typical of enzyme kinetics. Regardless of the mechanism of uptake, be it diffusion or a carriermediated active process, the rate would be e;cpected to decrease as the sugar concentration in the solution was reducv^d as a result of uptake. At a given concentration the constant uptake rate might be explained as a saturation of th>e uptake mechanism for sucrose but this mechanism is apparently not saturated until concentrations of about

PAGE 91

85 O.^M (6^+) which does not explain the constant rate at O.OO5M (Fig. 2). It appears that whereas the substrate is not the limiting factor (Fig. 2) the substrate concentration has something to do with the rate of uptake (Fig, k) , When the sugar concentration is maintained at a constant level sucrose uptake increases with time whereas glucose uptake does not (cf. Figs. 5 snd il). A possible explanation for these phenomena follow. The rate of sucrose uptake is governed by at least two factors, the external concentration of sucrose and the internal concentration of the phosphate donor. When sucrose uptake begins, the phosphate donor is limiting but as uptake proceeds fermentation generates phosphate donor v/hich in turn increases the rate of uptake; thus the uptake of sucrose is autocatalyti c. This is supported by data which show an increasing rate of CO2 evolution Vv'i th time in sucrose (Fig. 13). The straigiit line of sucrose uptake in declining concentration may be the result of a decreasing outride concentration of sucrose being countered by an increasing internal rate of glycolysis and thus an increased iriternal concentration of the phosphate donor which drives sucrose uptake. With glucose uptake the amount of diffusion of glucose into the synthesis compartment determines the rate of active transport. There is a competition in the cytoplasm for phosphate donors, ATP or PEP, between hexokinase and the active uptake process. As the external concentration of glucose declines, the amount of diffusion declines thus allo'wing more phosphate donors to be available for the uptake process. Thus as the concentration declines the active process has available a lower external concentration of glucose for uptake but a higher interfial concentration of phosphate dor.or to drive tiie process. The overall

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86 result is an uptake rate that is almost constant until tlie glucose in the bathing solution is ilupleted. At constant glucose concentration the amount of external glucose is constant, the amount of clii'fusion is constant and thus the overall rate of uptake remains constant, A combination of active and passive glucose uptake mechanisms fits well with the role of the scutellum in germination. When the glucose concentration available to the scutellum is lov-j the system is capable of removing all of the available glucose quickly; however, when the endosperm glucose is high the scutellum can take up glucose in excess of the capacity of the active process, A sugar uptake system driven by glycolysis also fits the role of the scutellum. The ability to drive uptake under limited 0, supply vjould be of obvious sdvantage to a seed under soil conditions, Garrard and Humphreys (65) have measured a RQ. of 3 with whole scutclla in air indicating that the scutellum itself might impose conditions of limited 0^ aval 1 abi 1 i ty. it is obvious that the metal binding characteristics of scute] luni slices differ from those of yeast. The binding of uranyl ion to yec':st cells seems to be quite specific to the uptake sites (53). V/i th scutellum Slice,-, however, most of the bound uranyl ion is superfluous to the uptake process as shown by the r-educed but still effective amount bound after acid treatment (cf, f'igs. 16 and 18, and Table 10). This is in agreement with the results o^ Wheeler and Hanchey (56) who showed that uranyl ion -vjaz bound by oat root tissue at many sites other than the membrane surface. If the release of bound cations occurs during sugar uptake by scutellum slices the amount is too small to be detected with the metal assay procedures used.

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LITERATURE CITED 3. h. 5. 6. 7. 8. 9. 10. 11. !2. 13. 15. 16. 17. !8. 19. W . . Stein, Th e Movement of Molecules Across Cell Membranes . Academic Press, Mew York (I367). R, K. Crane, i n C o ni p r e h e n s i v e Bio c h e m [st ry . ed. by R , D. Stotz. Elsevier Pub li shiny Company, Amsterdam, I'ol . I 7 (1969). A. L. Kursanov, in Advances in B ota nical Research , ed, by R. D. Preston. Academic Press, New York, Vol. 1 (1^63). C, E. Hartt, M. P, Kortschak, A. J. Forbes and G. 0, Burr, Plant fil^lLL^l-. 33, 505 (1963). R, L. Bieleski, PTtir.t Phyi. iol. . k] , hkl (I966). P . 1-: , V.'eo th e r 1 oy , 2iSw_£iv:!i2i' . 5 ^ , 76 ( ! 953 ) . JLPJA. 53, 20^f (195^0. JJllcl.. . 5^, 13 (1955). G. A. Pe.mell and P. E. Weatheriey, jMiLilt'X^^' . 57> 326 (I958). H. K, Porter and L. H. May, J. Exp. 3ot . . 6, ^+3 (1955). R. S. Vickery and F. V. Mercer, Aust. J. Biol . Sci . , !/, 338 (iJo'-O JJ2id. , 20, 565 (1967). P. J. Mardy and G. Norton, New P hytol . , 67, 139 (1966). JA, Sacher, flant_ Ph_v s !_oJ_ , , ^1, 181 (1966). P. Kriedemann and H. Seevers. Plant Fitys j£^; . . hi., !6i (I967), Ibid., hi, \-/h (i367). P. Kri edema nn, Pla.n_ta, 73, 175 (I967). G, R. Grant, Ph.D. Thesis, Purdue University (1962). B, R. Grant ard I!. Seevers, Plant Pjiy^ioj . , 39, 78 (196^0. T. ApRees and H. Se^rvers, i;jJl.Lt_^ijXSJ_oX, , 35, 839 (i960). 87

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21. L. Reinhold and Z. Eshhar, Plant Physiol . , ^3 , 1023 (1968). 22. J, L. Harley and D, H. Jennings, Proc. Roy. Soc . . London B. 148 ^03 (1^58). 23. D. R. Morgan and H. E. Street, Ann . Bot. N. f, . , 23, 89 (1959). 2k. D. R. Thomas and N. R, Weir, N ew Phyto l . . 66, 125 (196"/). 25. A, E. Younis, M, E. Younis, and H. A. Gabr, Plar^t Col 1 Physio' 10, 575 (1969). ~ ' 26. L. Reinhold and Y. Eilam, J. Exp. Bot. , 15, 297 (I96i|). 27. R. L. Bieleski, A ust. J. Biol . Sci . . 13, 203 (I960), 28. ibid. , 13, 22! (i960). 29. Ibid. , 15, i+29 (1962). 30. K. T. Glasziou, P lant Physiol . . 35, 895 (I96O). 31. .Ibid., 36, 1/5 (I96I). 32. M, D, Match, J, A, Sacher, and K. T. Glosziou, Plant Physiol . 38, 338 (1963). ~ 33. M. 0. Hatch and K, T. Glasziou, Plant Physi ol . . 38, 3V^ (1963), 34. J. A. Sacher, V\. D, Hatch and K, T. Glasziou, Pl ant Physiol , . 38 348 (1963), 35. M, 0. Hatch, Biochcm. J, . 93, 521 (1964). 36. J, S. Hdwker and M, D. Hatch, Biochem J . . 99, 102 (I966), 37. J. Heiidicino, J , Biol , Chen . . 235, 334/ (i960). 38. J. S, Hawker and M, D, Hatch, Physiol. Plant .. 18, 444 (I965). 39. M. D, Hatch and K. T. Glasziou, Plant Physio] , . 39, I80 (1964). 40. J. A, Sacher, Li fe Sciences . 3, 1053 (1964). 4!, S, Haq and W. Z. ilassid, Pl ant Physiol . . 40, 591 (I965), 42. A. I. Schoolof and J, Edelman, J. Exp. Bot, . 21, 49 (1970). 43. W, Z, Hassid, Ann, Rev. Plan t PhysJoi . , 18, 253 (196/), 44. Futinan and W. Z. Hassid, J. Bioi , Cham, . 20/, 8S5 (1954),

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89 kS. C. E, Cardini. L, F. Leioir, end J, Chiriboga, J. 3 iol . C hem . . 21'+, 1'49 (1955). 46. L. F. Leicir and C. E. Cardini, J. Bio) . Chern. , 2] if, 157 (1955). ^7. J. S, Hawker, B iochein. J.. i05, 9'+3 (I967). kQ. C. P, P, Ricardo and T. ApRees, Phy coc'riem. . 9, 239 (1970). kS. A, L. Kursanov, S. M, Sokolova and M. V. Turkina, J. Exp. Bot .. 2i, 30 (1969). 50. A. RoLhstein, Symp. Soc. Exp. Biol . . 8, I65 (195^). 51. A. Rothstein and R. C. Meier, J. Cel I . Comp. Physiol . . 38, 2'45 (1951). 52. A, Rothstein and A. D. Hayes, Arch. Biochem. Biophy s.. 63, 87 (1956). 53. J. VanSteveninck and H. L. Booi j , J. Gen. Physiol . . kS, k3 i]SGh) 5'^. J. VanSl:eveninck and A. Rothsiein, J. Gen. Physiol . . '+9, 235 (1965). 53. A. Rothstein and J. VanSteveni nek, Ann. N. Y. Acad. Sci . . 137, 606 (1966). 56, H. Wheeler and P. Hanchey, Science . I7I, 68 (1971), 57. S. Roscman, J. Gen. Physiol . . 5^, 138 (I968). 53. W. Hengstenberq, ,1. B. Egan and M. L. Morse, J. Biol . C hem. . 2^3, 1881 (I9C8). 59. J. Edel-nan, L. I. Shibko and A. J. Keys, J . Exp. Bot. , JO, T/B (1959). 60. r. E. i-lumphreys and L. A. Garrard, Phytochem. . 3, 6^7 (196if). 61. L, A. Garrard ?,-.d T. E. Humphrey?, Nature. 207, 1095 (1965). 62. T. E. riumphreys and L. A. Garrard, Phytochem . , 5, 653 (I966). '^3. ibid., 6, 1085 (1967). (-''"" ,ibj..4. . 7, 701 (1968). 65. L. A. Garrard and T. E. Humphreys, Phytochem . , 7, 1949 (1968). G6. T. E. Hiiniphreys and L. A. Garrard, Phytochem. . 8, 1055 (1969). 67. ibii., 9, 1715 (1970).

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90 68. L. A, Garrard and T, E. Humphreys, Fhytochem. . 8, 1065 (I96s). 69. Ibid. , 10, 2'(3 {'<971). 70. T, v.. Humphreys and L, A. Garrard, Phy tochem . . in press (1971), 71. N. Nelson, J. Biol. Chem. . 153, 375 (19^^). 72. M. Soniogyi, J, Biol. Chem. . 195, 19 (1952). 73. '^. (i. Spiro, in Complex Carbohydrates , ed. by E. F. Neufeld and V. Ginsburg. Academic Press, New York (1966), p. 7. 7^. W, W. Umbreit, R. H. Burris and J. F. Stauffer, Manometric Tech ni ques . Burgess, Minneapolis (1957), p. 28. 75. C. L. Rulfs, A. K. De, J. Lakritz and PI J. Elving. Anal. Chem.. 27, 1B02 (1955). 76. I., Silverman, L. Moudy and D, V/. Hawby, Anal . Chem . , 2^, 1369 (1953). 77. C, H. R. Gentry and L. G. Sherrington, Analyst . 71, k}! (19^16). 78. E, B. Sandell, C olorimetric Determination of Trace Metals . 3 ed. Interscience Publishers, New York (1959). 79. M, B. Williams and J. H. Moser, Anal. Chem .. 25, l^^U^ (1953). 80,. F. H. Pollard and J. V. Martin, Analyst . 81, 351 (1956). 81. F. Nydahl, An al. Chem. Acta . 3, l•f (19^9). 82. H. R. Marston and D. W. Dewey, Aust. J. Exp. Biol. Med. Sci .. 18, 3^3 (19^0). 83. H. C. Jones, Pfi.D. Thesis, University of Florida, Gainesville (1965).

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BIOGRAPHICAL SKETCH Joseph Henry Whitesell was born August II, 1936, at Clearwater, Florida. In June, 195^+, ho was graduated from Largo High School at Largo, Florida. In June, 1953, ne received the degree of Bachelor of Science vjith a major in Ornamental Horticulture from Auburn University, Auburn, Alabama. From 1958 until '960 he served in the Urtited States Navy and vjas stationed abo-ird the USS Putte-nut at Long Boach, California. Following his i^ctiva duty witii the Mavy, he worked for a year for the County of Los ArKjeles as a market inspector. In 1961 he returned to Florida where he worked as an Asr^ista^t County Agent in Collier CcuPt/, and during that time he resided in Maples, Florida. 1.1 i-j66 he enrolled in the Graduate School of the University of Florida, He. worked as a graduate assistant in the Oepartment of Botany and as a renearch assistant at the Pesticide Research Laboratory until December, !968, v^hen he received the degree of Master of Science witii a (rajor in Botany. From December, I968, until the present time he nas pursued his work '-oward the degree of Doctor of Philosophy, Joseph Henry V/hiteseli is r.arr'tnd la the former Jeanne Marie Steniop and is ihe father of three children. 9i

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I certify thac 1 have read this study and that in my opinion it conl-orr,!'; i:c acccptuble standards of scholarly presentation and is fully adcqu:-*!'.; , in scope and quality, as a dissertation for the degree of Doctor of Philosopliy. Thomas E, Humphreys, Chairms/n Professor of Botany I certiTy that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy, ^^.^ /£i David 5. Anthony Associate Professor of Botav// I certify that I hava read this study and that in my opinion it conforms to acceptable standards of scholeily presentation and is fully adequate, in scope ana quality, as a dissertation for the degree of Doctor of Phi losophy. Rychard C. Smi th Assistant Professor of Botany I certify that I have read this study and that in my opinion it confotms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor ot Pni losophy. Professor of Fruit C/rops

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This dissertation was submitted to the Dean of the College of Agri cul ti.re and to the Graduate Council, and was accepted as partial fulfillment of the requi reiiiants for the degree of Doctor of Philosophy. June, 1971 College of Agriculture Dean, Graduate School