VOL. XI, No. 2 APRIL, 1934

THE ABSORPTION AND ACCUMULATION OFSOLUTES BY LIVING PLANT CELLS

VII. THE TIME FACTOR IN THE RESPIRATION AND SALTABSORPTION OF JERUSALEM ARTICHOKE TISSUE

(HELIANTHUS TUBEROSUS), WITH OBSER-VATIONS ON IONIC INTERCHANGE

BY F. C. STEWARD AND W. E. BERRY.

(Department of Botany, University of Leeds.)

(Received 20th May, 1933.)

(With Four Text-figures.)

Discs cut from plant storage organs have long been favourite material for experi-ments upon salt absorption. A recent series of papers (Steward, 1932 a-c, 1933;Steward, Wright and Berry, 1932) which refers especially to potato tissue indicatesthat in the past the attention directed to the respiration of these tissues was far tooscanty. With the development of an adequate technique (Steward, 1932 b) thegeneral principles operating in the case of potato tissue became increasinglyapparent, and the occasion thus arose to extend the investigations to other storageorgans. A survey of several storage organs (Berry and Steward, 1934) revealed thatdiscs cut from artichoke tubers combined a conspicuous ability to absorb the saltstudied (KBr) with a characteristic respiratory behaviour which presented featuressomewhat different from those of potato. The present paper records an attempt toascertain whether the observed behaviour in respiration is reflected in salt absorptionin the manner anticipated and to determine further details of the absorption process,with particular reference to potassium bromide and artichoke tissue. A matter whichreceived some special attention was to ascertain whether the absorbed salt replacesions already within the cell, or accumulates, against a concentration gradient, in thevacuole where it exists in true solution without displacing other ions. Since theywere obtained, the results here presented have gained added interest in the light ofthe results published by Briggs and Petrie (1931) and, more particularly, in view of atheoretical interpretation of absorption by cells (Briggs, 1932) based upon ionicexchange.

EXPERIMENTAL METHOD.

The experimental technique was designed to ascertain:(a) Whether the tissue showed a falling rate of salt absorption with time

corresponding to the march of carbon dioxide production, to be expected in the.light of the previous results.

jBB-xiii 8

104 F . C. S T E W A R D andW. E. B E R R Y

(b) The effect, if any, of the added salt (KBr) upon the respiration of the tissueand its behaviour in time.

(c) Whether the absorption of a particular salt (KBr) was merely due to exchangefor ions already present in the vacuole or was unaccompanied by any loss of solutefrom the tissue.

To this end it was desirable to obtain a complete record of the respiration andsalt exchanges of tissue continuously in contact with (a) distilled water (see columns 3and 4, Table I), (b) dilute potassium bromide (see columns 5 and 6, Table I) duringthe whole experimental period. For both of these cases the total carbon dioxideproduced during successive 24-hour intervals, which together comprise the wholeexperimental period, was determined. Determinations of the electrical conductivityof the external solution made daily revealed the corresponding changes with respectto time of the total electrolyte content of the outer solution (see columns 3 and 4,Table II and Fig. 2). Saps1 were expressed from a comparable batch of the initialtissue2 and from that exposed during 138 hours to distilled water and potassium bro-mide. Upon these, bromide, total halide3 and conductivity determinations were made.

In order to ascertain whether there was a drift of salt absorption similar to thatof respiration, different, but comparable, batches of tissue were exposed to dilutepotassium bromide only during short periods (24 hours) occupying consecutiveportions of the respiration/time curve. The six periods (which except in the case of1 and 2 do not overlap) of actual contact with potassium bromide solution togethercomprise the whole period of approximately 140 hours. During the period precedingcontact with the salt the tissue remained in 2 litres of stirred, aerated distilled water(unchanged). The desired salt concentration (0-00075 M) was always obtained, atthe appropriate time, by addition of 15 c.c. of a standard bromide solution to the2 litres of distilled water in which the tissue had been respiring during the previousperiods. Since the addition of potassium bromide might interfere with the normalmarch of respiration with time, carbon dioxide determinations were made for the24-hour periods preceding and during contact with the salt solution. When the datafor the six batches of tissue are assembled this corresponds to retraversing therespiration/time curve in two ways. From the respiration data for the six tissuesamples for the period preceding bromide addition one may compile a respiration/time curve for tissue respiring in water (columns 9 and 10, Table I). This curve,though a composite one, is in every way comparable to that obtained from the tissuesample continuously in water during the whole period of 138-2 hours (columns 3 and4, Table I). From the data obtained from the six tissue samples during contact withthe bromide solution one can compile a corresponding, composite curve whichrepresents the respiration drift of tissue which, though in contact with the saltsolution, is never in contact toith it for longer than 24 hours. In this way one canevaluate the effect of the addition of the salt solution at six successive stages of therespiration/time drift.

1 After the customary procedure of rinsing, blotting and weighing (Steward, 1933 b).1 Initial tissue = tissue cut and washed for a brief period in running tap water.1 For methods see Steward (1932 b).

Absorption and Accumulation of Solutes by Living Plant Cells 105

Table I.

Period

( 1 )

I2

3456

Hr.

( 2 )

18-2523-5824-0824-1724-0824-17

Tiaaue in continuous contact with

Distilled water

Total CO,mg.

(3)1090125-068-i5O-546-33 8 0

mg. CO,gm. x hr.

(4)0-21580-1914O-IO2IO-O7540-06950-0568

KBr solution

Total CO,mg.

(5)115-0126-06 9 55O-347-o40-3

mg. CO,gm. x hr.

(6)0-2268019320-1042007510-07050-0602

Period

(7)1234

6

Hr.

(8)242424242424

Tissue in contact with KBr for 24

Tissue in distilled water

Total CO,mg.

(9)155-0*122-06655i-545-837-7

mg. CO,gm. x hr.

(10)0-23270-1831OIOOO0-07550-06900-0568

IT. only

Tissue in bromide solution

Total CO,mg.

(11)170-0136-083-553-450345-2

mg. CO,gm. xhr.

(12)

025550-20480-12560-08030-07560-0680

• Recalculated from a period of 18 hr.

Table I I . Conductivities of external solutions.

(All data in mhos x io"1.)

Period

(1)1234

6

Tissue in continuouscontact with

Distilledwater*

(2)1-4070-6700-5010-476o-5570-478

KBrsolution*

(3)n-669097936-45689618

Tissue in contact withKBr for 24 hr. only

Beginning ofabsorption

(4)10-312-1io-8io-6107107

End ofabsorption

(5)10-648288-8i9-079-429-71

Distilled water 0-462. KBr solution 10-3.

• Determinations at end of each period.

Conductivity determinations upon the external solution made (a) immediatelyafter addition of the bromide aliquot, and (b) at the end of the subsequent 24 hoursperiod of absorption (see Table II) measure the gross change in electrolyte content;

8-2

106 F. C. STEWARD andW. E. BERRY

this may then be compared with the absorption of potassium bromide determined(both analytically and by conductivity methods) on the sap expressed from therinsed and blotted tissue.

In all cases each bottle contained 40 discs, 0-085 c m - thick and 3-40 cm. indiameter in 2 litres of distilled water to which was added 15 c.c. of o-i M potassiumbromide solution as required. Variables other than time and salt concentration(temperature, aeration, stirring, etc.) were strictly controlled throughout the experi-ment by the procedure previously described (Steward, 1932 b), and were maintainedat the values previously adopted for the work upon potato (temp. = 23-2° C , rate ofair flow for aeration 16 litres per hour, paddle speeds 100 R.P.M.). The discs werecut from selected large tubers of Sutton's White Jerusalem Artichoke (supplied byMessrs Sutton and Sons of Reading) so that irregularities due to the proximity ofbuds were reduced to a minimum. It was most convenient to withdraw smallsamples of the external solutions for the conductivity determinations and to makethese, not at the temperature of the experiment, but at 250 C. The determinationswere made by a dipping form of electrode, using a slide wire bridge with a calibratedresistance box and a suitable valve oscillator which produced a steady note ofcontrolled frequency. All the conductivity data are calculated for 250 C. and thenecessary constants for potassium bromide at this temperature obtained fromLandolt-Bornstein. The cell constant was determined, using a solution of potassiumbromide, since all the data were calculated in terms of this salt.

EXPERIMENTAL RESULTS.

Respiration.

The respiration data in Table I, and Fig. 1 confirming the data previously pub-lished, show that at 23-2° C. the respiration of thin, immersed discs of artichoketissue decreases rapidly with time from a high initial rate (0-22 mg. CO, per gm.per hour) to much lower values (0-06 mg. CO, per gm. per hour). There is everyindication that an even lower respiration rate would have been reached with theelapse of a still longer period. Other work (Steward, 1932 a~c, 1933; Steward,Wright and Berry, 1932) has shown that the most important variables in the respirationof immersed potato tissue are oxygen concentration and temperature and that thetissue is able to maintain almost indefinitely (140 hours) the high rate of respirationconsonant with these two environmental factors. We know (Steward, Wright andBerry, 1932) that in the case of potato the high rate of respiration which is main-tained, is accompanied by progressive depletion of the insoluble carbohydratereserve (starch), though its high carbohydrate content would be adequate to main-tain for a very long period the highest rates yet reported. The artichoke is likewiserich in polysaccharide (inulin) and were the whole of this1 readily available it wouldsuffice for a similarly protracted period of maintained high respiration. Fig. 1

1 The inulin (total carbohydrate) content of artichoke tissue is of the order of 70 per cent, dryweight or 18-5 per cent, fresh weight (Tincker, 1938) or 160 gm. per litre of expressed sap (Tanret,1893). For further information on the nature of the polysaccharide see Schlubach and Knoop (1933).

Absorption and Accumulation of Solutes by Living Plant Cells 107

indicates that this is not so, and, although we shall subsequently report strikingeffects of both oxygen and temperature upon the respiration of artichoke, it is clearthat some other internal factor, which is conditioned by time, here determines therespiration. It is possible that this difference in behaviour between potato andartichoke may be determined in part by the failure of the latter tissue to utilise thebulk of the carbohydrate reserve1. In this connection the old observations ofGreen (1887-8, 1893, 1899) which suggest that dormant artichoke tubers beforegermination do not contain inulase, but only an inactive zymogen, are clearly sugges-tive and would repay confirmation, especially with reference to tissue treated as heredescribed. Though other and more recent workers (Thaysen, Baker and Green,

0-32

0-30

0'28-

0-26

0-24-

o 0-22-

&a 0-18-

? 0-16

8 0-12-

^ > 0-10 -

0-08-

0-06-

0-04-

0-02-

0

IN DtspiOATiotH DATE.

TIUUC IMMNC MOMCC A&3OAPTION

• n MCTLP POIOO TO «weonoN

m CONTMUOUXV ft VttTtB-

•X— •• • • KbP SOLUTtOrt

0 24 48 72 % 120 144

Hours

Fig. i.

1929) found that during the rest period inulin slowly gave place to more solublecarbohydrate, they state that "attempts made to confirm experimentally the assump-tion of the presence of inulase in the tubers did not meet with very great success."Another inulin storing organ—Dahlia tuber—only yields a very weak inulase extract(Pringsheim and Hensel, 1931). Accordingly there is still some doubt whetherslices cut from dormant tubers could readily utilise the main carbohydrate reserve.

We have had occasion to stress (Steward, Wright and Berry, 1932; Steward,1932 c) that a relatively small number of cells at the surface of immersed aeratedpotato discs contributes most of the carbon dioxide. Unpublished work suggests a

1 Reducing and total sugar estimations (after acid hydrolysis) showed that loss of sugar to theexternal solution was quite negligible.

108 F. C. STEWARD andW. E. BERRY

similar conclusion for artichoke tissue. Further work will be necessary to evaluatethe surface effects in respiration in relation to changes with time. The data in thispaper may be adequately interpreted for our present purpose on the basis of wholediscs, although we are quite clear that neither respiration nor salt absorption is strictlyuniform throughout discs of this thickness.

Anatomical explanations for the respiratory behaviour with respect to time,based upon the production at the surface of the discs of a layer impermeable tooxygen, although possible, do not appear to be entirely adequate. Unpublishedresults obtained in this laboratory do indicate that artichoke (which, like potato,heals in air at a cut surface after preliminary suberisation) suberises, though notheavily, at the cut surface of discs in aerated water, but it fails to develop a meristemor show any sign of cell divisions under these conditions. We are not certain atpresent how much significance, if any, is to be attached to the suberisation as aprimary cause of the observed decrease of respiration with time.

Previous work (Steward, 1933) with potato indicated that the respiration rate wasunaffected by the dilute solutions of potassium bromide commonly used in thiswork. No great significance was attached to some slight, apparent effect of theconcentration of this salt. Comparing columns 3 and 4 with 5 and 6 of Table I, it isevident that except in the first period the differences are too small to be significant.It is equally evident, however, that the data of columns 9 and 10 compared with nand 12 of Table I, show a significant increase due to the presence of the salt. Thisslight effect superimposed upon the behaviour in distilled water apparently occursif the bromide is added at any point along the respiration/time curve1 (Table I andFig. 1) though it does not persist much longer than 24 hours, after which the tissuereproduces almost exactly the respiratory behaviour it would have had in the com-plete absence of potassium bromide (compare columns 5 and 6 with 3 and 4, 9 and10, Table I). Dilute potassium bromide (0-00075 M) therefore causes a transientincrease in respiration which rapidly disappears after 24 hours.

Loss of electrolytes by the tissue and subsequent absorption.

It is well known that storage tissues, after the customary technique of cutting,washing, blotting, etc., when placed in distilled water or dilute salt solutions loseelectrolytes (Stiles, 1927; Steward, 1932 a). There is little doubt that both theinitial loss and the subsequent re-absorption of these solutes are dependent upon theexperimental conditions (and particularly upon aeration). Artichoke tissue indistilled water behaves in this way2 though the magnitude of the initial loss ofelectrolytes is apparently much less than that for potato under identical conditions.Table II (columns 3 and 4, which correspond to columns 1-6, Table I), gives theresults of conductivity determinations made at intervals upon the distilled water anddilute potassium bromide solution (0-00075 M) which contained identical quantitiesof tissue. The data show the reality of the initial loss of solutes by the tissue both to

1 There is some indication that the absolute increase of respiration due to addition of bromide issmaller the later the addition is made.

1 In relatively strong solutions (o- iN NaCl) the preliminary loss was not observed by Stiles (1924).It was presumably masked by absorption.

Absorption and Accumulation of Solutes by Living Plant Cells 109

distilled water and dilute salt solution, and also that in both cases after a relativelyshort period (18 hours) absorption of solutes predominated over their loss andcontinued smoothly for the remainder of the experimental period (see Fig. 2).

One of the most recent papers on this question (Stiles, 1927) describes an experi-ment upon artichoke. The experimental technique is in many respects not comparableto our own and, as one would anticipate, the changes in conductivity of the externalsolution are not in entire agreement with those here reported. Adopting the presenttechnique, the magnitude of the initial loss of solutes by the tissue appears to be

CwANCt m LXTLBNAL CONDOCTIVITV

Q - Tusut COMTuiuotULy IN K&r solo

144

much smaller and the subsequent re-absorption is completed more rapidly, whilsta second very pronounced and protracted loss of solutes, which had commenced inthe experiment of Stiles before 100 hours, is entirely absent. It is suggested that thedifferences between these two curves (which are indeed foreshadowed by the effectsof agitation and casual aeration to which Stiles refers) are most probably associatedwith the aeration and stirring technique adopted in the experiments here described.A similar experiment by Briggs and Petrie (1931), using carrot tissue in distilledwater provided with slow aeration, revealed similar time curves for the externalconductivity and respiration. The tissue, however, required 8 days to reabsorb the

no F. C. STEWARD andW. E. BERRY

solutes lost in the first day (or less) and to restore the external conductivity to thevalue of the initial distilled water.

The conductivity data in Table II (columns 4 and 5) illustrated in Fig. 3 revealthe amount of absorption which took place in different periods of 24 hours each.The five points on the upper curve (from column 4, Table II) represent the con-ductivity of the external solution immediately after the addition of the aliquot ofpotassium bromide. They reflect the curve for the change of conductivity of dis-tilled water containing tissue (lower curve, Fig. 2) with the addition, at each of the

CONDUCTIVITY •*• CJKTLBNAL ^OLUTHDNS

—O O— Ejtjd of Ab*orbiioo \*rw4

24 48 72 % 120 144

Hours

Fig. 3-

five points on the time axis of a constant conductivity value, namely that of theadded salt. The lower curve comprises five points (from column 5, Table II) each ofwhich corresponds to one point in the upper curve (joined by dotted line), andrepresents the conductivity of the same solution after absorption had proceeded for24 hours. The uniformly upward tendency of the lower curve, and the regularlydecreasing slope of the dotted lines as time increases both indicate clearly that thetissue can absorb during successive intervals of 24 hours a steadily decreasing amountof salt.

It will be observed that the first period of 24 hours is not included in Fig. 3.This period is abnormal (though quite intelligibly so) principally because the tissue

Absorption and Accumulation of Solutes by living Plant Cells i n

first loses salt, and then after a certain lag period commences to reabsorb it alongwith some of the added potassium bromide. Whilst we have recorded a figure forthe external conductivity after 24 hours we cannot compute the amount of absorp-tion by comparing this with the corresponding value at zero time, because in themeanwhile the conductivity of the external solution was much increased by loss fromthe tissue itself. Furthermore, the undoubted presence of an initial period duringwhich loss predominated over absorption renders this absorption period incom-parable with the rest.

The data obtained (Table III) by analysing the expressed saps from the differentbatches of tissue at the end of their respective absorption periods are entirelycomplementary to those from the external solution.

Table III.(All cones, in equiv. x io~* per litre of sap.)

Period

0

12

3456

1-6

Cl + Br

17-3824-8035-3732-3829-2328-3937-396l-75

Br

o-o6 4 9

17-3414-6810-6910-530-31

38-03

Cl

17-3818-3118031770i8-5417-86180817-69

Conductivitymhos

0-010490-012460-013150-012540-011740-011700-01130

0-01387

On the basis of (1) the conductivity determination upon the expressed sap,(2) total halide determination, (3) bromide determination, it is clear that moreabsorption took place in the second 24 hour period than in any subsequent one. Assuggested by the conductivity data upon the external solution, less absorption tookplace in the first period than the second, though again this appearance is somewhatillusory since the tissue has both lost and regained solute quite apart from the addedsalt (KBr). The close parallelism between the curves for sap conductivity, bromideand total halide content prove that after the first period of 24 hours the tissueabsorbed bromide without loss of chloride and apparently the changes in sapconductivity reflect this absorption.

A comparison of Figs. 1 and 4 will show that in periods 2-6 the relationshipbetween the respiration and absorption is clear. A high rate of respiration is asso-ciated with a high rate of absorption. Period 1 presents an apparent exceptionhaving highest respiration but not the highest bromide absorption1. This exceptionis, however, only apparent, for during this period more than in any other, the tissueis absorbing in preference to bromide, other salts previously lost from it, and ifcomplete allowance could be made for this, there is no reason to suppose that thetotal absorption in period 1 would not exceed that of period 2. A comparison of thefigures for the bromide content of saps (periods 2-6, Table III) and for respirationin the same periods (column 12, Table I) will show that the relative decrease with

1 Unpublished results of Rosenfels (University of California) show an almost identical behaviourfor the respiration and bromide absorption of Elodea.

i i 2 F . C. S T E W A R D and W. E. B E R R Y

time of salt absorption is not identical with that of respiration but that the respirationfalls off relatively more rapidly than the salt absorption. In other words, the ratio

36-

34

CHANGE IN SAP COMPOSITION- o D - Total halide

— A - Conductiultij

-O O - Bromide

144

HoursFig. 4.

of total carbon dioxide produced to salt absorbed (either from actual analyses orconductivity data) decreases with the elapse of time.

It is interesting to compare the total absorption of bromide obtained when asingle batch of tissue was exposed thoughout the whole period of 138-3 hours to the

Absorption and Accumulation of Solutes by Living Plant Cells 113

bromide solution, with the aggregate1 of the concentrations attained during the sixshort periods of absorption which, though referring to different tissue samples, to-gether comprise an identical period. Provided no internal condition tending to re-strict absorption develops after the 24-hour period one might anticipate that the finalbromide content of the sap of the tissue in continuous contact (Table III, period 1-6)would represent the summation of the concentrations attained in the six tissuesamples which had only shorter absorption periods. The figures (38-03 mg. equiv.bromide per litre of sap for the continuous contact method and 67-40 mg. equiv.bromide per litre of sap for the "discontinuous" method) clearly show that after24 hours of continuous absorption the influence of some factor which tends toreduce absorption becomes very evident. This factor is to be clearly distinguishedfrom the normal drift of absorption with time to which reference has already beenmade. There are two possible explanations of this behaviour.

(1) In all probability this is merely another case (Hoagland, Hibbard and Davis,1926) where previous absorption of a solute retards the subsequent accumulation,i.e. the absorption during the earlier periods has a retarding effect in the later ones.Other work with potato tissue (Steward, 1933) yielded the unexpected result that insolutions of this order of concentration absorption proceeded for relatively longperiods at an approximately constant rate despite the internal concentrations ofbromide attained. Apparently this is not so in the case of artichoke.

(2) We have already shown that artichoke, like the other tissues examined bythese methods, exhibits higher salt absorption whenever the respiration is increased.We have also referred to the transient, but significant increase of respiration whichmay be observed only in the first 24 hours of contact with bromide solution but whichappears in some degree irrespective of its location relative to the normal march ofrespiration with time. By the method of six separate 24 hour periods of contact thiseffect is apparently much magnified, so that, whilst the tissue in continuous contactwith bromide solution produced during 138 hours 448 mg. of carbon dioxide, thecorresponding amount for the same length of time obtained from batches of tissue,no one of which was in contact with bromide solution for longer than 24 hours, was496 mg.—a difference of 48 mg. The difference between the total respiration of thetissue in continuous contact from the beginning of the experiment with distilled waterand of that in potassium bromide, which corresponds to a single application of thetransient stimulating effect mentioned, was only 11 mg. carbon dioxide. Whilst thereis no proof that this is the sole cause of the greater absorption obtained by the "dis-continuous" method, it seems legitimate, and consistent with the other results, toregard it as a contributory factor.

The observed correlation between bromide accumulation in the sap (Table III,and Fig. 4) decrease of conductivity in the external solution (Figs. 2, 3, and Table II)and increased conductivity in the expressed sap (Table III, and Fig. 4) stronglysuggests that the principal feature of the absorption of bromide is the absorption ofthis ion accompanied by potassium without loss of any ions by the tissue. The

1 Periods 1 and 2 overlap by 6 hours—so that the concentration obtained in period 1 should forthis purpose be taken as 486 approx.

ii4 F - C. STEWARD and W. E. BERRY

analytical data show that there was no significant replacement of chloride by bro-mide but the conductivity data may be still more closely analysed to test this point ofview. One may convert the decrease of conductivity observed after 24 hoursabsorption from 2 litres of external solution (columns 4 and 5, Table II) to a cal-culated total amount of potassium bromide absorbed by the tissue. One must thenassume that this is distributed throughout all the water contained in the tissue(final fresh wt. x water content)1 and thus calculate the minimum bromide concen-tration to be expected in the expressed sap. These results are given in Table IValongside the observed concentration of bromide. It is not to be expected that thedetermined concentration of bromide would equal exactly the value calculatedfrom the conductivity change upon the above assumption since there can be noquestion that all the water which maybe expelled from the cell at ioo° C. cannot becontained in the phase receiving bromide (the vacuoles). The approximate agree-ment between the observed and calculated bromide content makes it unlikely thatexchange of ions is the principal mechanism involved, since this would result inmuch smaller changes in external conductivity than those observed. This pointmay be checked independently and perhaps more satisfactorily as follows. From theobserved content of potassium bromide in the expressed saps one can calculate theexpected increment of sap conductivity upon that of the initial tissue on the assump-tion that the bromide enters as potassium bromide without loss of any ions inquantity. The calculations only refer to those cases where the salt initially lost bythe tissue is completely reabsorbed and retained. The agreement between the ob-served increments of sap conductivity and those calculated from the bromideanalyses is sufficiently close2 to justify the main assumptions.

Table IV.

Period

2

3456

1 - 6

By analysis

Equivalent bromideper litre of

expressed sap

0-01730-0147001070-01050-00930-0380

Calculated fromchange in external

conductivity

Equivalent bromideper litre of water

in tissue

00205o-oioo0-0080000630-005300271

Observed

Change ingap conductivity

mhos

+ 000266000205000125O-OOI2I0-00081000340

Calculated frombromide content

of sap

Change insap conductivity

mhos

+ 000 2420002050-00150000149000134000510

Since investigators have so frequently emphasised the unequal absorption of thepositive and negative ions by cells in general, and by storage tissue in particular,some might take exception to calculations which are based upon equal absorption ofboth ions; though it is also necessary to recall that the discrepancies between the

1 The percentage water content of similar discs to those in question was 88-57 P*1' cent.1 Any effects of the salts already present in the sap upon the conductivity due to the added

bromide or effects upon the conductivity due to metabolism are of course neglected in these calcula-tions.

Absorption and Accumulation of Solutes by Living Plant Cells 115

absorption of anion and cation tend to disappear in dilute solution (Stiles, 1924).For the case in point equality of anion and cation absorption is not a mere assump-tion. The artichoke tissue used in the experiments of the preceding paper (Berry andSteward, 1934) absorbed bromide until the expressed sap contained 20-2 x io~s

equivalents per litre. Simultaneously it gained in potassium content by 21-68 x io~s

equivalents per litre1. These figures correspond to almost equal absorption of anionand cation, and it is further significant that the slight discrepancy (if real) is of thesame kind {i.e. K> Br) as observed for potato (Steward, 1932 a).

It is evident therefore that all the data are consistent with the conclusion that thetissue absorbs from the external solution bromide accompanied by potassium2 (withan appropriate decrease in external conductivity) and the absorbed salt reappears inthe expressed sap where it contributes approximately the expected amount to theconductivity. Exchange of electrolytes between the cell and its environment canonly play a very minor role in these phenomena. This result is very similar to thatobtained by one of us with potato tissue (Steward, 1932 a) which certainly absorbssimultaneously both potassium and bromide without very extensive loss of otherions from the cell.

It is not suggested that ionic exchange plays no part. Such a conclusion demandsan accuracy of technique which has certainly not been realised. In fact there is astrong suggestion (see period 1-6, Table IV) that with prolonged continuous contactwith bromide solution the importance of exchange (though anions other than chloridemust be concerned) may become greater3. It is interesting to speculate whethertissue which, by the elapse of an even longer time would presumably have an evenlower respiration rate, might not be reduced entirely to an exchange mechanism forbromide absorption—if indeed it retained at all the capacity to absorb this salt.However, in so far as the still actively respiring tissue which retains the capacity toaccumulate bromide readily from dilute solution is concerned, it seems that, as withpotato so with artichoke, both anion and cation are simultaneously absorbed, andthere are no experimental facts inconsistent with the view that they reappear in thevacuole without displacing other ions in equivalent amounts. Furthermore, it isvery questionable whether any conspicuous salt absorption by storage tissue has yetbeen obtained, except in cells showing this enhanced respiration in some degree.The writers are convinced that the absorbed salts reside almost exclusively in thevacuole. Some may challenge the homology of the expressed sap with the vacuolesolution. We are aware that in recent writings, referring especially to storage tissues,Briggs (1932) appears to envisage the cytoplasm as distinct from the vacuole sap asan important phase in which the exchanges of salt between cells and external solutionoccur and he even suggests that it may tolerate within it high salt concentrations(Briggs, 1932, p. 131), though the evidence for them is as yet entirely lacking. It is

1 Initial sap: K content = 92-82 x io~3 equivalent per litre. Final sap: K content= 114-5 x io~'equivalent per litre.

• The writers are aware that Stile* found (1924) that artichoke tissue absorbed from 01 NaClabout three times as much Na as Cl, due, perhaps, to base exchange in the wall.

' Compare the case of Nitella (Hoagland, Hibbard and Davis, 1926) in which previous accumula-tion of chloride causes subsequent absorption of bromide to depend on exchange to a greaterdegree.

n6 F C. STEWARD and W. E. BERRY

well to recall that in all these parenchyma tissues (and in potato and artichoke inparticular) there is remarkably little cytoplasm and vacuoles occupy the bulk of thecell. There can be little question that these vacuoles are the principal source of theexpressed sap, and there seems little doubt that in them the bulk of the absorbed saltis located. An expressed sap becomes unavoidably somewhat contaminated withcytoplasmic contents, but with due regard to the scanty amount of cytoplasm and thebulk of the vacuole it seems inconceivable that the magnitude of such contaminationcould vitiate these results. We do not suggest that the method of expressed saps isentirely free from objection, but the discrepancy between the sap and the vacuolesolution may be overestimated; it does not appear to be serious for the purpose ofthese investigations.

GENERAL DISCUSSION.

The data here presented are in complete agreement with the view stated else-where (Steward, 1932 a, c, 1933 ; Steward, Wright and Berry, 1932) that the abilityof the cells of storage tissue to accumulate salts from dilute solution is determined bytheir metabolism. It has been shown that at constant temperature both the saltabsorption and respiration of discs of potato tuber are conditioned by the oxygensupply, and if this is adequately maintained both processes may proceed at anapproximately uniform rate for relatively long periods (Steward, 1933). In the caseof artichoke, however, the initial high respiration rate is not maintained, apparentlyowing to the increasing operation of an internal factor (see p. 107) which limits therespiration. The consequent falling rate of respiration is accompanied by a similarlydecreasing capacity to absorb salt.

Concerning the relationship between respiration and salt absorption there areseveral possibilities. The most obvious suggestion is that the entering anions replaceissuing bicarbonate ions—a process which has formed the basis of recent theoreticalspeculations upon the mechanism of salt absorption (Briggs, 1930). This, thoughpossible, does not appear to be the true relation between respiration and salt ab-sorption in this case, for the respiration of tissue in distilled water, which onlyabsorbs a little solute previously lost, is almost identical with the respiration of tissuein a solution of a readily absorbed salt. A small but transient increase of respirationcaused by the salt solution itself was observed, but it is evident that salt continues toenter after this has expired (Fig. 2, and p. i n ) . This temporary initial increase inrespiration represents an additional amount of bicarbonate ions (total COg) which iseven greater than the total bromide ions absorbed during the same period, butfurther work, particularly with reference to the effect of various concentrations ofsalt upon absorption and respiration and a comparison of the effect upon respira-tion of potassium salts with but slowly absorbed anions (e.g. KjSO^), is desirablebefore one can contemplate with any confidence the idea that the role of respirationis merely to provide bicarbonate ions which may be replaced by entering bromideions.

A more indirect role of respiration seems to be more plausible. In other papers(Steward, 1932 c, 1933; Steward, Wright and Berry, 1932) we have emphasised that

Absorption and Accumulation of Solutes by Living Plant Cells 117

a limited number of cells at the surface of discs of storage tissues under the experi-mental conditions here adopted exhibit intensified vital activity, the energy for whichis derived from their increased respiration. There can be no question that in the caseof immersed artichoke this recrudescence of vital activity is arrested and not main-tained as long as it is with potato, where it may even culminate in cell division. Thespecific behaviour of this tissue is at present under investigation, but at least in oneparticular, namely an increased activity of protoplasmic streaming1, the two organsare similar. It is suggested that in the case of artichoke, as for potato, the saltabsorption demands the expenditure of metabolic energy, the source of which is theaerobic respiration of carbohydrate substrate. The striking parallelism between thedecrease with time of total respiration and of salt absorption is adequate indicationof the connection between them, but the fact that the relative rates of decrease arenot identical2 (p. 111) warns against a too direct relation between the two processes.

This is not the place for a detailed discussion of theories of absorption, but somebrief reference to recent papers is permissible. Briggs (1930) has published aningenious theoretical scheme which purports to explain accumulation of solutes inthe sap. From the writers' standpoint the most interesting part of this scheme is theemphasis laid upon respiration. Whilst the details need not concern us here, thegeneral conclusion is that high respiration rate leads to high salt absorption—a resultwith which the writers fully agree.

However, the same author, when considering the case of storage tissue (largely onthe basis of old results (Briggs, 1932) and two additional experiments (Briggs andPetrie, 1931)), is led to postulate an entirely different mechanism. The postulatedeffect of respiration in this case is that it determines an equilibrium between cytoplasm(not sap) and the external solution, the characteristics of which seem to be that ahigh rate of respiration is associated with a high salt content in the outer solution andvice versa. One of us (F. C. S.) has ventured to suggest that the data in question admitof another interpretation (Steward, 1933). In any event it is obvious that the resultsin this paper, consistent with those previously reported, indicate that a high rate ofrespiration is associated with a high rate of absorption and vice versa—i.e. apparentlythe reverse of the effect observed by Briggs and Petrie (1931) if we read them aright,and more in harmony with the general scheme suggested by Briggs (1930) foraccumulation in the sap. Furthermore Briggs (1932) is so impressed with thenecessity for a special mechanism to meet the case of storage tissue, i.e. maturecells, that he has devoted most of a recent paper to a suggested interpretation ofabsorption experiments with this material on the basis of ionic exchange. The detailsof the suggested system which are of immediate concern are as follows:

(a) A cell sap phase capable of exchanging anions but not cations with the externalsolution. (A considerable degree of cation impermeability is postulated, especiallyin dilute solutions.)

1 Observations by Miss E. Chamberlain (unpublished).1 This may be due to differing magnitude and extent of the surface effects in absorption and

respiration. Perhaps one ought to compare the salt absorption only to "surface respiration." (SeeSteward, 1932 c.)

n8 F. C. STEWARD and W. E. BERRY

(b) A cytoplasmic phase which for the reasons stated may exchange both dif-fusible cations and anions with the external solution.

(c) Superimposed upon the above "there may perhaps be a slight accumulationof both anions and cations in the sap, but the facts suggest that even with potassiumsalts, this is likely to be slight, and with others negligible over short periods of time "(Briggs, 1932, p. 306).

The properties of such a system are discussed at length but, as clearly stated(p. 315), they include that "the net results of placing the tissue in the solution shouldbe to leave the concentration (in terms of normality) unchanged, except in the casewhere impenetrability of cations is impaired in strong solutions1. The conductivityof the external solution would then be affected only in as far as the ions coming outof the tissue had different mobilities from those absorbed by the tissue."

It will suffice to state that it is difficult to reconcile these postulates with theresults in this and previous papers (Steward, 1932 a, c, 1933 ; Steward, Wright andBerry, 1932) on storage tissues. Under the experimental conditions adopted, ionicexchange seems to play but a minor r≤ accumulation in the "sap " of both anionand cation without loss of any solutes (which is summarily dismissed by Briggs)seems to be the most important feature of the process and the observed conductivitychanges appear to be in general agreement with this view. The writers have not over-looked the fact that the scheme in question is suggested for the mature cells of whichit is assumed that the discs of tissue are composed. Inasmuch as under the experi-mental conditions here adopted it is evident that the cells which absorb salt (cer-tainly for potato and in the light of unpublished work apparently so for artichoke)are embarking upon a renewed period of vital activity, they may not legitimately betermed "mature" within the meaning of the scheme referred to. However, it hasbeen repeatedly stated in other papers (Steward, 1932 a, c, 1933 ; Steward, Wrightand Berry, 1932), and is reiterated here that the mechanism of rapid accumulation ofsolutes from dilute solution by living cells of storage tissue is definitely a part of thoseincreased vital activities in the surface cells in which the less active, "mature" cellswithin the disc do not participate. Unfortunately few workers with storage tissuehave endeavoured by microscopical investigation of the tissue or other methods totrace the extent or intensity of these processes. In fact it is greatly to be questionedwhether any of the data concerning salt absorption yet obtained with storage tissueare completely independent of these effects. If exchange of ions is ever a predominantfeature of absorption by storage tissue then we suspect that it refers only to cellswhich, for lack of aeration or other causes, have not at their disposal the intensesupply of energy characteristic of the rapidly metabolising cells at the surface ofadequately aerated tissue. There is a possibility (see p. 115) that exchange of ionsmay become increasingly more important with artichoke tissue after the rate ofrespiration has declined from its initial high value.

1 The writen cannot tee in the results in this paper, in others (Steward, 1932 a-c, 1933 ; Steward,Wright and Berry, 193a), or in their unpublished results any evidence of impermeability to cations indilute solution.

Absorption and Accumulation of Solutes by Living Plant Cells 119

SUMMARY.

1. The rapid decline in the respiration (CO, production) of immersed, aeratedartichoke discs is confirmed.

2. The decreasing respiration is not associated with any known external factor.3. Dilute potassium bromide solutions cause a temporary, increased respiration

which quickly disappears after 24 hours. This effect appears at various points onthe respiration/time curve.

4. By the technique used, washed, blotted artichoke discs immediately after re-immersion release electrolytes to both distilled water and dilute salt solutions butre-absorption, which very rapidly supervenes, is extensive after 18 hours and iscomplete after 48 hours.

5. Decreasing respiration is accompanied by a decreasing ability to absorbinorganic ions (KBr). The reductions with time of total respiration and salt ab-sorption are similar but not identical.

6. Absorption of potassium bromide from the external solution is accompaniedby a decrease of conductivity in the outer solution and an increase in that of theexpressed sap. During short periods (24 hours) it appears that the mechanism ofionic exchange can only play a minor role. Bromide does not replace chloride.Apparently potassium and bromide are simultaneously accumulated.

7. Apparent irregularities in the first period may be satisfactorily explained.8. Some factor associated with long, continuous contact of artichoke tissue with

dilute bromide solutions tends to reduce absorption compared with a series ofshort periods. This may be due to accumulation in the earlier periods which reducesabsorption in the later ones or may admit of an explanation based upon the transienteffect of addition of bromide.

9. The general relation between salt absorption and respiration for artichoke isestablished. An indirect role is preferred to a direct one. Respiration is assumed tomaintain vital metabolic processes which supply the energy necessary for an ab-sorption process in which work must be done.

REFERENCES.BERRY, W. E. and STEWARD, F. C. (1934). Ann. of Bot. 48, 1-16.BRIGGS, G. E. (1930). Proc. Roy. Soc. B, 107, 248-69.• (1932). Ann. of Bot. 46, 301-22.

BRIGGS, G. E. and PETRIE, H. K. (1931). PTOC. Roy. Soc. B, 108, 317-26.GREEN, J. R. (1887-8). Ann. of Bot. i., 223-36.

(1893). Am. of Bot. 7, 83-137.(1899). The Soluble Ferments and Fermentation. (See Chapter vi.) Cambridge University Presa

HOAGLAND, D. R., HIBBARD, P. L. and DAVIS, A. R. (1926). Journ. Gen. Pkysiol. 10, 121-46.PRINGSHEIM, H. and HENSEL, W. G. (1931). Ber. d. deuttchen chem. Ges. 64 (2), 1431-4.SCHLUBACH, H. H. and KNOOP, H. Liebig't Ann. der Chem. 497, 208-34.STEWARD, F. C. (1932 a). Protoplasma, 15, 29-58.

(1932 b). Protoplasma, 16, 497-516.(1932 c). Protoplasma, 17, 436-513.

• (i933)- Protoplasma, 18, 208-42.STEWARD, F. C , WRIGHT, R. and BERRY, W. E. (1932). Protoplasma, 16, 576-611.STILES, W. (1924). Ann. of Bot. 38, 618-33.

(1927). Protoplasma, 2, 577-601.TANRET, CH. (1893). Comptes Rendus, 117, 50-3.THAYSEN, A. C , BAKER, W. E. and GREEN, B. M. (1929). Bioch. Journ. 23, 444-55.TINCKHR, M. A. H. (1928). Ann. of Bot. 42, 101-40.

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