THE DETERMINATION OF THE SALT RELATIONS OF THE

CYTOPLASMIC PHASE IN CELLS OF BEETROOT TISSUE

By M. G. PITMAN*
received December 17, 1962]
Summary
An analysis of the rate at which isotope diffuses out of disks of beetroot tissue
shows that there are at least two components of the non-free space. As tlwse com-
ponents are not due to differences in cell type within the tissue, it is suggested they
are due to a cytoplasmic phase in the parenchymatou's cells, and to the vacuoles.
Evidence is given to support the suggestion that, as in the characean cell,
these phases are in a series with the free space, so that ions pass to the vacuole in
salt uptake through the cytoplasmic phase.
Using this serial model for the cell it has been estimated from the amount of
isotope diffusing out of labelled tissue that the cytoplasmic phase contains about
3 m-equivjkg K+ or Na+, and 0-1-10 m-equivjkg Br-, when the tissue is brought
to equilibrhun with potassium or sodium bromide solution whose concentration is
10-50 m-equiv/l. The time for 50% exchange of K+ in this phase is about 2 hr at
20, and for Br- about 40 min. At 250 the exchange of both K+ and Br- is some
three times faster.
The fluxes into and out of the cell hiwe been estimated for K+ and Br- when
either the concentration in the -solution or the content of the vacuoles was varied.
It was shown that "salt saturation" of the tissue was mainly due to an increase in
efflux as concentration in the vacuole increased. The permeability of the boundaries
of the cytoplasmic phase to I{+, estimated from the fluxes, was about 10-
8
em/sec,
and it is suggested these boundaries are due to the plasmalemma and the tonoplast
membranes respectively.
1. INTRODUCTION
It has been suggested by MacRobbie and Dainty (1958) that as far as the
movement of ions between the solution and the vacuole is concerned, the cell of
Nitellopsis obtusa behaves as a system of three phases in series. These phases 'are
the free space and two components of the non-free space, namely, a cytoplasmic
phase with a relatively rapid turnover, and the vacuole, containing the major part
of the salt in the cell and equilibrating only slowly with the solution. The contents
of these phases were estimated from an analysis of the isotope diffusing into a series
of non-radioactive solutions from tissue that previously had been in a radioactive
solution. In this method the cytoplasmic phase was detected as a component with a
rate of equilibration intermediate between that of the free space and the vacuole,
* Botany Department, University of Cambridge; present address: Botany Department,
University of Adelaide.
Aust. J. Bioi. Sci., 1963, 16, 647-68
648 M. G. PITl\iAN
and sensitive to changes in temperature. The serial model has subsequently been
supported by Diamond and Solomon (1959) working with Nitella axillaris, and the
cell wall has been shown to be part of the free space in Ohara australis by Dainty and
Hope (1959). The location of the cytoplasmic phase is not well defined but by a
process of elimination it must be part of the cytoplasm, for the bull;;:: of the nonfree
space with a slow turnover is in the vacuole, and the cell walls are part of the free space.
This paper gives the results of similar experiments carried out with beetroot
disks (i.e. analysis of the rate at which isotope diffuses out of labelled tissue). In
general, these resuits are similar to those found for the Characeae, that is, three
phases can usually be detected; a free space component, a cytoplasmic phase whose
tempo of equilibration is much more rapid at 250 than at 2e, and a slowly exchang-
ing component from the vacuoles. These phases are not due to any difference in cell
type within the beet disks (cf. p. 660) and they are considered to be characteristic of
the salt relations of the parenchymatous cells that make up the major part of the tissue.
It has been shown elsewhere (Briggs, Hope, and Pitman 1958a) that. the free
space in beet disks 1 mm thick has the exchange properties associated with a Donnan
phase occupying about 20;0 of the tissues, and containing 10 m-equiv/kg of non-
exchangeable anion, together with a "water free space" in ,vhich both anions and
cations have the same concentration as the solution, and occupying about 20% of
the tissue. This DOlman space has since been shown to be located in the cell ,valls,
while the water- free space is mainly due to cut cells (15%) and to intercellular spaces
(5%) (Pitman, unpublished data). Thus, as in the characean cell, the only available
region in which to locate the component with the intermediate tempo of exchange
is the cytoplasm.
There is, however, no a priori reason for equating the cytoplasmic phase with
the whole, or any particular region, of the cytoplasm, and two extreme configurations
can be devised. In one the phases form the series free space-cytoplasmic phase-
vacuole but in the other the cytoplasmic phase is connected to the vacuole only via
the free space, Le. is in "parallel" with the vacuole. The first arrangement would
occur if the cytoplasmic phase were the whole of the cytoplasm, so that salt taken in
to the vacuole had to pass through the cytoplasmic phase. The second arrangement
would apply if the cytoplasm as a whole were free space, and the "cytoplasmic phase"
some particulate component (e.g. mitochondria, plastids) within the cytoplasm. To
emphasize the relation of the cytoplasmic phase to the vacuole the two models are
called "serial" and "parallel" in what follows.
If the isotope diffusing out of the tissue is to be used to estimate the content
and fluxes in the cytoplasmic phase, it is necessary to know which of these models
applies to beet tissue. For example, the proportion of isotope in the cytoplasmic
phase that diffuses to the- solution depends on the ratio of the fluxes to solution and
vacuole, and may be much smaller if the phases form a series than if they are in
paralle1. Evidence is given below to show that the phases behave as if in series, but,
for convenience, is presented after the description of experimental procedure and the
methods of estimating the K + or Br- content of the cytoplasmic phase. The relation-
ship between the cytoplasm and the cytoplasmic phase is then considered.
SALT RELATIONS OF CYTOPLASmc PHASE IN BEETROOT TISSUE 649
II. EXPERIMENTAL DETAILS AND PROCEDURE
(a) Material
Disks 1 mm thick and about 15 mm in diameter were cut with a hand-
microtome from cylinders of beet tissue. The cylinders were cut with a cork-borer
in the general direction of the vascular bundles, and were taken as far as posElible
from the parenchyma between the rings of vascular bundles. The disks were rinsed
in distilled water and kept in continuously aerated distilled water for a period of
about 7-10 days; during this time the ability to take up salt by the tissue had
developed to a maximum.
(b) Isotopes
The isotopes used were 42K, 22Na, 24Na, and 82Br. The 42K and 24Na were
obtained from the Atomic Energy Research Establishment, Harwell, as irradiated
carbonates which were converted to chlorides by titration with hydrochloric acid,
and boiling the solution to remove the carbon dioxide. The 82Br was also obtained
from Harwell but as irradiated NH4Br. This was used as a solution of NH4Br and
diluted with KBr solutions to the required activity. In this way the concentration of
the NHt ions in the labelling solutions was not appreciable.
These three isotopes were irradi8,ted for only 24-48 hr to reduce radioactive
impurities to a minimum compatible with adequate isotope production. Irradiation
for 48 hr produces 93% maximal activity of 42K and 89% 24Na.
22Na was obtained from the Radiochemical Centre at Amersham as a solution
of Nael, and was used in this form, with suitable dilution with carrier NaC!.
(0) Methods of Estimation
Radioactive concentrations were determined by counting the pulses from
about 10 ml of solution with a Mullard MX 124 Geiger-Muller tube. Where possible
the solutions were counted for a period sufficiently long such that the error due to
random variation in the rate of isotope disintegration was less than 1 %: for some
solutions oflow activity it was not practicable to count more than 1000 disintegrations.
Concentrations of sodium and potassium were determined with an "EEL" flame-
photometer. The accuracy of the determinations was better than O 02 m-equivjl
when the scale was set to 1 m-equivjl KCl or NaCl, and 001 m-equivjl when set
to 02 m-equiv/l potassium in 5 m-equivjl NaC!. There was no detectable interference
by sodium with potassium determinations.
Bromide and chloride were determined by potentiometric titration with silver
nitrate solutions. A silver-silver halide electrode was used. The accuracy of these
determinations was between 1 and 2%.
(d) Experimental Procedure
The experimental procedure can be divided into three parts: the pretreatment
of the tissues, the uptake of isotope, and the subsequent elution and measurement
of the loss of isotope. The purpose of the pretreatment was first to remove the divalent
cations from the free space and to equilibrate the free space with the solutions to be
650 M. G. PITMAN
used in later stages, and then to bring the fluxes in the system to steady values under
the conditions of the experiment. To this end the tissue was put into a 50 m-equiv/l
KCI solution at 2'C for three periods of about 30 min, followed by three similar
treatments with solution or-the same content as the labelling solutions:, The tissue
was then left in' aerated solutions of this concentration for about 7 hr with occa-
sional changes.
The tissue and unlabelled solutions were then brought to the appropriate
temperature for uptake onsotope. When disks are accumulatiug labelled salt but
losing salt at a lower specific activity from the vacuoles, the specific activity of the
free space towards the centre of the disks 'will be lower than that towards the surface,
because of the greater length of the diffusion path to the solution from cells in the
centre. This difference in specific activity, and in isotope uptake, will increase with
the flux iuto the cell,but will be much lower at 2'C than at 25'C, so the temperature
of isotope uptake was 20 wherever possible.
The ratio of solution to tissue was relatively large during isotope uptake to
reduce the changes in specific activity to a minimum compatible with determination
of the net salt uptake .. At the end of about 15-18 hr the l.issue was removed from
the solution, lightly blotted, and put into a series of non-radioactive solutions for
elution of isotope. Samples of about 2 g of tissue (10 disks) were used in a volume of
solution varying from 100 ml at the start of the elution, when . there was a lot of
isotope in the free space, to 10 or 12 ml duriug the later stages of the elution, when
the rate of loss from the tissue was small. For similar reasons, the length of each
period of elution was varied from about ,20 min at the start of the elution to I hr or
more towards the end. Throughout the experiment, the solutions were stirred and
aerated by a stream of moist air. Samples were taken at the end of the periods of
elution by removing as much as possible of the solution, and replacing it with an
equal amount of unlabelled solution at the same temperature. All experiments
were at the same temperat.ure throughout, with the exception of the determination
of potassium content at 250. In these experiments there was a series of elutions at
20 lasting 2 hr an'd then the temperature was changed to 250 for the non-free-space
elution. In this way, during the first 2 hr, the isotope in the free space was reduced
to about 01% but only 10-40% had diffused out of the cytoplasmic phase; at 25'C
a similar elution would have removed the same amount of isotope from the free space,
but 60-90% from the cytoplasmic phase.
At the end of the experiment the disks were removed from the solutions,
blotted, weighed, and then treated to extract the isotope and salt in the tissue by
first boiling with water to remove as much bromide as possible, and then boiling
with 5 ml O 5N nitric acid to remove the rest of the sodium and potassium. These
two extracts were kept separate. The concentrations of isotope, and of sodium,
potassium, and halide were determined by the methods described above.
(e) Experimental Errors
Primary determinations were measured to within 1-5%, depending on the
concentrations of salt or isotope involved. As duplicate samples were used, a more
useful value was the mean difference between the duplicates. For the apparent con-
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROOT TISSUE 651
tent of the cytoplasmic phase and for the net uptake rate (<p), when <p was
relatively large (1-2 m-equiv/kg/hr), this difference was between 5-lO%. For the
content of the cytoplasmic phase (Qo1, for'lower values of,p, and for the fluxes into
and out of the cytoplasmic phase, it was about lO-15%.
III. THEORETICAL BASIS OF ESTIMATION OF THE PROPERTIES OF THE CYTOPLASMIC
PHASE
(a) Symbols Used
A;, Uamount" of isotope apparently in the cytoplasmic phase (counts/min/
20 ml/g);
C, concentration (m-equiv/l): Co in solution, C
c
in the cytoplasmic phase,
C v in the vacuole;
E, potential (m V);
F, Faraday's constant;
ke, rate constant for exchange of the cytoplasmic phase
Q, amount of an ion (m-equiv/kg): Qe in the cytoplasmic phase, Q, in the free
space, Qv in the vacuole; '.
Q"', "amount" of isotope (couhts/min/20 ml/g), sUbscripts as in preceding
defiuition;
R, universal gas constant;
s, speciflc activity [(counts/min/20 ml)/(I'-equiv/20 ml)]: Se in the cyto-
plasmic phase, 8v in the vacuole, 8 inside, 80 outside, or in the solution;
T, absolute temperature (OK);
t, period in solution (hr);
t
i
, time for half-exchange of the cytoplasmic phase (hours or minutes);
,p, flux (m-equiv/kg/hr): <p", from free space to cytoplasmic phase; <Pes, froID
cytoplasmic phase to free space; 4>cv, from cytoplasmic phase to vacuole;
<Pve, from vacuole to cytoplasmic phase; ,p (no subscript), net flux or net
uptake rate.
(b) Estimation of Content., of Cytoplasmic Phase
The amount of isotope in the tissue throughout the elution can be estimated
from the series of measurements of isotope diffusing out of the tissue, and from its
content at the end of the experiment. In general three stages can be distinguished:
(1) A period in which the isotope diffusing out of the tissue is mainly from
the free space, but also from the cytoplasmic phase and the vacuole, and
in which the rate of exchange is limited by the diffusion in-the free space.
(2) Eventually isotope lost from the tissue is limited by the exchange between
the cytoplasmic phase and the free space, and so has the tempo of exchange
of the cytoplasmic phase. In this stage the isotope comes both from the
vacuole and the cytoplasmic phase.
(3) Finally when the specific activity in both free space and the cytoplasmic
phase has fallen to a valne intermediate between tbat of the vacuole and the
solution, isotope diffusing out of the tissue is limited by the flux out of the
vacuole, and all the isotope appearing in the solution is from this region.
652 M. G. PIT:MAN
When these phases can be separated, the analysis of the isotope diffusing out
of the tissue can conveniently be made by the method described by MacRobbie and
Dainty (1958). Briefly, the logarithm of the amount of isotope in the tissue is plotted
against the time from the start of elution. The vacuolar contribution is then -the
final straight part of the graph and its extrapolation to t ~ O. Subtraction of these
values from the total amount of isotope in the tissue gives estimates at different times
of the amount of isotope in the free space and cytoplasmic phase together. These
5
w
~
"
v
~

~
S
4
L

"
~
'""TW"'
v
~
w
"

,

~
" 0
,
0
~
~ 2
.,-
x ~
~
0

o 2 3 4 5
,
7
HOURS
Fig. I.-Amount of isotope estimated to be in the free space and
cytoplasmic phase together plotted semi-logarithmically (see
text) . Disks at 2C throughout. X Disks changed from 2C to
25C at t = 233 hr.
two phases can also be separated by a semilogarithmic plot, but now the final straight
part is characteristic of the exchange of the cytoplasmic phase (Fig. I). During the
first part of the elution the tissue was at 20, but the temperature of one set of
samples was changed at t = 233 hr to 250: the two straight lines from this stage
onwards represent isotope lost from the cytoplasmic phase at the two temperatures.
From these analyses of the experimental results, the following quantities can be
estimated, as well as the content of the free space and the rate constant for its
exchange with the solution:
(i) The rate constant for exchar1{fe between the cytoplasmic phase and the
solution, estimated from the slope of the straight part of the graph (cf.
Fig. 1, when k, ~ 033 at 2'C, and 085 at 25'C).
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROOT TISSUE 653
(ii) The amount of isotope estimated to have been in the cytoplasmic phase
at the start of the elution (A:) from the extrapolate to t = 0 (in Fig. I,
8500500 counts/min/20 m! at 20).
(iii) The amount of isotope in the tissue at the end of the elution, and its average
specific activity.
(iv) The rate of loss of isotope from the tissue at the end of the experiment, when
loss is predominantly from the vacuole.
(v) Other quantities that can be determined are the specific activity of the
labelling solution, and, from the change in concentration, the net uptake
rate of salt.
These quantities can be related to the fluxes into and out of the phases and
their contents, but the details of the relationship depend on the spatial arrangement.
In what follows only the serial model is considered in detail, and Figure 2 shows the
Q, Q,
Q
v
."
,
."
,
."
/
.YO
CO
'0 sc;(sm OR Sf)
'v
SOLUTION PLUS CYTOPLASMIC VACUOLES
FREE SPACE PHASE
(5,0) (e) (v)
Fig. 2.-Schematic representation of the serial model of
free space-cytoplasmic phase-vacuoles.
quantities involved. The conditions of the experiments were arranged so that several
simplifying assumptions can be made. Firstly, the net uptake was reduced to small
proportions by using tissue that was at 20 or salt-saturated; as a result, the content
of the vacuole increases by not more than 10% over the course of the experiment,
and the fluxes into and out of the cytoplasmic phase and its content are assumed to
be steady. Furthermore there will be little change in the concentration in the free
space due to the diffusion path at different distances from the surface.
Secondly, it is assumed that the specific activity of the free space in all parts
of the tissue is equal to that of the solution SQ, so that the equation for the rate of
change of isotope in the cytoplasmic phase is
dQ;/dt = " . so+v, . sv-s,(,,+,v). (I)
During the period following transition from one solution to another of different specific
activity, the specific activity in the free space will not be equal to that in the solution
and the equation will not be true. As the time for half.exchange of isotope in the free
space is much less (c. 10 min) than that in the cytoplasmic phase (60-100 min). this
error has been ignored.
654 1\1. G. PITMAN
More serious eITors could arise when dQ;/dt is small. On the assumptions of
equation (1), the net flux of isotope across the outer cytoplasmic phase boundary will
be (r/>sc' 8Q-4>08 .8
e
) inwards. If the cell is losing salt from a region of different
specific activity while taking it up from the solution, the specific activity adjacent
to the cytoplasmic phase will be some value intermediate between that of the solu-
tion and the phase, and determined by the diffusion path to the solution, the fluxes
into and out of the phase, and the content of the free space adjacent to the phase.
The difference between 80 and the relevant specific activity in the free space will he
larger for anions than for cations, as there is less in the free sp'ace. For example,
when Co = 5 m-equiv/I KBr, the free space will contain about 0,5-10 m-equiv/kg
of Br- and 12-13 m-equiv/kg of K+; if the region adjacent to the cytoplasmic phase
were the eel] wall containing the Donnan free space, then the difference would be
greater, about 01 m-equiv/kg Br- to 12 m-equiv/kg K+. This source of error is 'not
considered to be important for K + at 20 and possibly not at 250, an assumption
that is supported by the good agreement between the observed and calculated values
of the final efflux from the tissue (cf. p. 655 and Table 2). For Br- there is some
evidence that the specific activity in the free space during elution is appreciably
larger than so; one method of for this is to put a. Se as the relevant
specific activity, where a is a constant for a particular set of fluxes and
tions. Equation (1) then becomes
dQ;/dt = 1>" . .1>,,)s,. (2)
In this case Se can no longer be taken as an approximation for the net isotope
flux out of the cytoplasmic phase but (1),,-1>,, . a) s, may be used instead (cf. p. 661).
Using the symbols given in Figure 2, and assuming that equation (1) is true, it can
be shown that the quantities determined in the experiments have the following
relationships:
(i) The rate constant for exchange of the cytoplasmic phase, k" estimated from
the slope of the graph in Figure, 1 is
k, = (1),,+1>,v)/Q,. (3)
The time for half-exchange of the cytoplasmic phase, t
l
, equals O 694/k,.
(ii) At the start of the elution the amount of isotope in the cytoplasmic phase is
Q; and equals Qe . Se. If the time in the labelling solution, h, is more thari
about five times t
i
, then the value of Se at tl is more or less steady at
sm = (1),,80 +1>vo8v)/(1>,,+1>,v).
(4)
At the end of the elution will have fallen to Qe . Sf where, as So is very
nearly zero, under the conditions of the experiment
8f = 1>vo8v/(1),,+1>,v). (5)
The apparent content of the cytoplasmic phase, A;, is the extrapolate to
t = 0 of the straight part of Figure I, and is related to Q, by
A; = Q,(Sm-sf)1>,,/(1>,,+1>,v). (6)
SALT RELATIONS OF CYTOPLAS:MIC PHASE IN BEETROOT TISSUE 655
(iii) The specific activity of the tissue as a whole at the end of the experiment when
nearly all the isotope is in the vacuole is Q:/(Q,+Qe+Qv). As Qs is
estimated separately in the experiment, and as Qe is small compared with Qv,
an approximate value of Qc can be used to estimate Sv = Q:/Qv. ,The amount
of isotope in the vacuoles, Q;, is related to the fluxes and specific activity
in the cytoplasmic phase by
Q: fev(J:' Se dt)-fve(J:' 80 , dt),
(7)
where t2 is the time at the end of the experiment. If <pvc Sv is small, and
if the period of labelling (t,) is about the same as the period of elution
(t2-tr), then equation (7) is very nearly
Q: feo(sm- 8f)tr. (8)
(iv) The rate of loss of isotope from the tissue .at the end of the experiment, or
the "apparent efflux" is
fes . Sf = foe. Sv f,,/(f,,+fev), (9)
(v) The net uptake rate, estimated from the change in concentration of the
solution, is
f = (f,,-f,,) = (fev-fvc). (10)
From equations (8), (6), and (3), (fes+fe,,)(sm-Sf) can be determined,
and as
(fcs+fev)(Sm-Sf) = f" . s,
= (f+fcs)s"
(4,5)
(10)
<Pcs can be calculated if <P is known. By substitution in other equations, values of the
other fluxes and the content of the cytoplasmic phase can be estimated. From these
quantities a value for the apparent efflux (eqn. (9)) can be calculated and compared
with the value found experimentally (from the rate of loss of isotope at the end of
an elution experiment when the specific activity of the _ cytoplasmic phase is at a
steady value intermediate between that of the free space and the vacuoles, Sf). This
comparison has been made for potassium in Table 2.
IV. EXPERIMENTAL RESULTS
(a) Pota8sium and Sodium in the Cytoplasmic Phase
To ensure that So has reached the value Sm given above, the tissue must be in
the isotope solution for at least five times t
j
Figure 3 gives values for the apparent
isotopic content of the cytoplasmic phase; A;/so, when tissue is eluted varied
in the labelliI?-g solutio"u, showing it reaches a or steady value.
The line is calculated from the final (t = 20i hr) and, is in reasonable
agreement with the other determinations. Similar values for the apparent uptake,
Q:/so are also given. These results are not taken to differentiate between the two
models but only to show that the serial model is capable of explaining the observations.
656 M. G. PITMAN
Most of the determinations described below have been made with potassium
instead of sodium for two reasons. Firstly, tissue that has been in sodium chloride
solution for periods as long as are used in the experiments sometimes loses some of
the red pigment from the vacuoles, and can no longer be assumed to be in the same
state throughout the experiment; secondly, at 250 the turnover of sodium in the
cytoplasmic phase is too fast to make separation of this phase from the free space
practicable. For example, the time for 50% turnover of sodium at 20 is about
,-,
'-0
(;' 1"5
<
.,
s
i
o
...
*.t 1'0
T

1 ---1
6
1 ~
T
A;/,o/ 1 /1
1 1 ~
1 yT 1 '4
II I ?
o
* 3 8
Q,,/so i
T /
*J
2
V 10
o 5 10 15 20
HOURS IN SOLUTION
Fig. a.-Values found for the apparent content of the cytoplasmic
phu.se, A; /so (.), and for the apparent uptake, Q ~ / s o (x), after
varied periods in the labelling solution. The line gives values cal-
culated from the uptake at 20t hr.
1-1-25 hr but only 20-30 min at 25'C; the time for 50% exchange of the free space
is about 10 min for I-mm thick disks at both 2 and 25'C, but as the content of
the free space is some 10 times the apparent content of the cytoplasmic phase, the
two phases are not separable at 250.
The effect of temperature on the fluxes of potassium in the system is shown in
Table 1, together with some results for fluxes of sodium at 20. There are two sets
of results for potassium fluxes: one when the tissue was salt-saturated and net uptake
was low, the other when the tissue contained less salt and showed appreciable
potassium uptake to the vacuole. In both cases the turnover of potassium was
faster at 250 than at 20, due to the increase in the fluxes CPeB and cpcv. The temper.
ature coefficient for these fluxes is about 1 5-1 9 and is about the same for each
--------
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROOT TISSUE 657
flux. This is smaller than the tempemture coefficient of net uptake (25-3 0), but q,
is the difference between the fluxes into and out of the vacuole, and so these may well
have different temperature coefficients.
TABLE 1
COMPARISON OF THE CONTENT AND lo'LUXES IN THE CYTOPLASJ)[lC PHASE FOR SODIUM AT 2C AND
FOR POTASSIUil-I AT BOTH 2C AND 25C. AT HIGH AND LOW VACUOLE CONCENTRATION
CO = 5 mequivjl; duplicate samples
Solu- Temp.
A:jso Q,
t,

." ."
Q,
tion ("0)
(m-equiv/ (m-equiv/
(h')
(m-equiv/ (m-equiv/ (m-equiv/ (m-equiv/
kg) kg) kg/h,) kg/h,) kg/h,) kg)
NaCl 2 033 22 125 02 033 090 35
KOI 2 072 29 2'25 01 040 050 70
KOI 25 070 29 075 12 11 1-65 85
KCI 2 19 40 20 0 092 0-45 205
KOI 25 2-3 43 07 0 295 140 205
The net rate of salt uptake by beet tissue increases with external salt concentra-
tion until it reaches a maximum at room temperature of about 4 m-equiv/kg/hr when
the external concentration is about 30-50 m-equiv/I (KCI) (cf. for example Briggs,
2'0
(.)
"
,,0
(b)
-'
2'0
,
d"
"0-
--
r
1'5
--
, ... ,... -a
>
,!oc 0,'
Qc, ..... "
3
",
"5
.," >
0
. -'
.-- ...... _... 3 a S

. .-
.! 1-0
", <$c. ,..-of
.,...... 8'

.... 0 .. + ......
"0
i
w
0' ......
'"' "
x
0
-'
2' 0
0'5
+"",. J. .. __ !-j,-----J.
......... ..0 __ ... --0 -----0 0
0 .....
0
-
"' ..........
0'5

+-- +----.... -- .....
/
O' 5 10
'0 '0
400510
Co (M-EQUIV/L)
20
'0
40
Fig. 4.-Effect of potassium chloride concentration (Co) on pot,assium fluxes
into and out Qfthe cytoplasmic phase, and on (Jc. Temperature 2"C. (a) Net
uptake rate (A) and the fluxes across the outer boundnry, <Psc (0) and
<PCB (+). (b) Content of the cytoplasmic phase (.), and the fluxes at the
inner boundary, rpcv (0) and <Pvc (+).
Hope, and Robertson 1961, p. 134). At lower temperatures the net uptake rate also
rises to a maximum but to a lower value. Figure 4 gives the results of an experiment
at 20 to measure the fluxes into and out of the cytoplasmic phase and the potassium
content in t.he cytoplasmic phase, when the external potassium chloride concentration
658 M. G. PITMAN
was varied from 5 to 40 The concentration of potassium in the vacuoles*
was 60, 63, 67, 68, and 69 m-equiv/kg, so in effect the only factor varied was the
concentration in the solution. Over this range the amount of potassium in the
cytoplasmic phase increased from 2'4 to 38 m-equivjkg, and, as may be expected,
the fluxes across the outer boundary increased as well (Fig. 4). The net uptake
to the vacuole also increased with concentration, but at the inner boundary this was
50
i
..,.
3
o 4.0


cl
30
05
0'4
I
"f
>-- 0'3
3
?
::E 0'2


x
3 0'1

II
0</
11_ /11
/I
'0
.---.---------.
... +

01 1
75 100 125 150 175 200
Q .. (M-EQU1V! KG)
Fig. of Qc (iJ.), and 4>vc (+) for potas-
sium at different levels of potassium in the vacuoles (Qv).
Temperature 2C.
mainly due to an increase in the flux The efflux from the vacuole, <pvc, showed a
small decrease from 035 to 025 m-equiv/kg/hr, although the concentration in the
vacuole increased from 60 to 69 m-equiv/kg.
In spite of this decrease in efflux, measurements made with tissue that had
been allowed to take up varied amounts of potassium chloride from a solution
containing 5 m-equiv/l showed an increase in effiux (r/>vc) with increasing vacuole
content (Fig. 5). At the highest content the net uptake has been reduced to a small
proportion of that when Qv = 88 m-equivfkg and this decrease in 1> was due mainly
to an increase in ,pvc, but also to a decrease in rpcv. Qc also increased with internal
(vacuolar) concentration.
* The vacuoles occupy about 0-70 ml/g fresh weight of disks, so I m-equiv/kg is approxi-
mately equal to 14 m-equiv/l.
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROO'J' TISSUE 659
The apparent efflux, v,. 8
v
. d(,,+ov) (cf. eqn. (9)) can be measured
directly in these experiments and compared with the value calculated from the
other fluxes (cf. p. 655). Table 2 gives values of the calculated and observed apparent
effiux from the results of Figure 4, showing that there is a reasonable agreement.
TABLE 2
OBSERVED AND CALCULATED VALUES OF THE APPARENT EFFLUX OF POTAS-
SIUM OVER A RANGE OF EXTERNAL POTASSIUM CHLORIDE CONCENTRATIONS
Potassium
Chloride
Concn.
(m-equiv/l)
5
10
20
30
40
Duplicate samples
Apparent Potassium Efflux (m-equiv/kg/hr)
Observed Calculated
O'IS 016
014 016
013 016
013 017
013 015
Disks cut transversely from a beetroot contain sections through vascu]ar
bundles and so are not strictly homogeneous in cell type, i.e. the cytoplasmic phase
could be due to this cellular difference rather than a difference in subcellular organiza-
tion. Table 3 gives results of an experiment to show that the cytoplasmic phase
TABLE 3
AMOUNT OF POTASSIUM ELUTED BY SODIUM CHLORIDE SOLUTION FROM BEETROOT DISKS, FROM
VASCULAR REGION OF DISKS, AND FROM THE PARENCHYMA ONLY
Sample Free Space
Potassium
Tissue Eluted Weight Potassium
Q,
Efflux from
Ig) (m-equiv/kg)
(m-equiv/kg)
Vacuoles
(m-equiv/kgJhr)
Beetroot disks:
Sample I 225 !l2 29 032
Sample 2 2 15 !l1 31 031
Vascular region 1'77 !l7 35 043
Parenchyma 269 102 25 023
is a property of the parenchymatous cells, by comparing the behaviour of:
(1) whole disks of tissue (two samples);
(2) tissue cut from two samples of disks so as to contain mainly vascular tissue;
(3) the remainder of the tissue from the two samples, which was entirely
parenchyma.
660 M. G. PITMAN
The samples had first taken up 42I( and were then eluted with a sodium chloride
solution. Each sample showed the same pattern of salt relations, but there was a
small difference in absolute amounts. For example, each elution gave free space,
cytoplasmic phase, and vacuolar components, but the cytoplasmic phase and efflux
from the vacuole for sample (2) are larger than for sample (3). This difference is
probably due to the smaller size of the parenchymatous cells near the vascular
bundles, which have a larger surface area per volume of tissue than the larger cells
in the regions bet\veen vascular bundles.
0"
l' 0'4
"0-
X
-,.
3
o
w
,
::;: 0-3
~
X
j
"
,
Cl 0'2
X
-,.
3
g
,
>
cJ 0'1
o
. // .
/
/",,,/.1'
/
////
.c /// ... x
//e //
/ //
/ /
/ /
// Qc ....... X ....
~ .... //
// .... /
/ /,X
X / ...... ""
.'", ______ x'/ ~ 1 l _ 1 l
1 l ~ 1 l
+----
'0
--+--
___ + _ 4>vc
---+------+
20 30
Co(M-EQUIV/Lj
_0
'0
Fig. 6.-Effect of variation in potassium bromide concentration (Co)
on bromide fluxes and content of the cytoplasmic phase at 20. Qc ( x );
"" (e); "" (+); and" (6).
(b) Bromide in the Cytoplasmic Phase
Although good agreement is found between observed and calculated values for
the apparent efflux of potassium (Table 2), this is not always so for anion exchange
when the observed efflux may be 30-50% of the calculated value. These observations
apply to chloride, iodide, and bromide, but bromide has been used in these experiments
as its isotope has the most suitable half life and energy of radiation. As discussed
above, under some conditions the specific activity adjacent to the cytoplasmic phase
could be much larger than that in the solution, and the isotope diffusing out of the
tissue would then not be rpcsSc, but (o/cs-o/sc . a)sc. This effect leads to underestima-
tion of o/cs and overestimation of Qc. and may be expected to be greater at 250 (when
the fluxes are larger) than at 2'C.
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROOT TISSUE 661
Allowance can be made for the underestimation of by assuming that the
observed effiu-x from the tissue is in fact
(</>"-'Ps' . a)s, = </>;8 S,
and using this relationship together with the other measurements given above to
estimate </>;8. As </>" equals (l/so)(sm-St)(</>;,+</>,v) [cf. eqn. (2), (4), and (5)] and </>"
equals can be estimated and compared with For the tissue
used at 2'0 (Fig. 6) there was little difference in observed and calculated efllux,
presumably as the influx was small, but for other tissue (Tables 4 and 5) when 0
0
was only 5 mequivjl, and was larger, in some cases cpsc . a was about 50% of CPcs.
TABLE 4
EFFECT OF TElI1PERATURE ON THE BROMIDE CONTENT AND FLUXES IN THE CYTOPLASMIC PHASE
OF BEETROOT TISSUE
Duplicate samples of tissue placed in potassium bromide solution of concentration 5 m-equiv/l
Temp. Q,
f!
Q,
('a) (mequiv/kg) (mequivjkg)
2 83 006 035
25 92 040 100
Temperature
coefficientt 23
t For a temperature rise of 10 degC.
:\: Units: m-equivjkgjhr.
f,,!
fd
f,,! .pIlC:\:
f;'!
012 006 019 013 006
060 020 135 095 011
20 17 23 23 13
Table 4 gives some values for Qc and for the fluxes in the system at 20 and at
250, when 0
0
was 5 m.equiv{l, estimated by making allowance for the difference
between observed and calculated efflux. As for potassium, the exchange of bromide
in the cytoplasmic phase was more rapid at 250 than at 20, and the temperature
coefficient for net was about 2 5. The temperature coefficient for was only
about 1 3, but if allowance is made for the diffusion effect, i.e. by estimating the
temperature coefficient for all the fluxes was about 2, as shown in Table 4. The
change in the fluxes with increased temperature was accompanied by a threefold
increase in Qc of about 0 65 m.equiv{kg. If the same increase in potassium content
had taken place in the cytoplasmic phase, it would have been detected with difficulty,
as it is only a little larger than the experimental error in Qc for potassium.
The bromide content and the fluxes into and out of the cytoplasmic phase when
the concentration of the potassium bromide solution was varied are shown in Figure 6.
The net uptake of bromide is generally much smaller than the uptake of potassium
(Fig. 4) although the vacuolar content of each was about the same in both experi-
ments. In these results the vacuolar content of bromide increased from 13 to
662 M. G. PITMAN
17 m-equivfkg, and of potassium from 66 to 77 m-equivjkg as C
Q
was increased
from 10 to 50 m-equiv/l KEr. The pattern of results is generally similar to that in
Figure 4, but Qc was much smaller for bromide than for potassium, and increased
more with increasing C Q. Again the fluxes across the outer boundary, and CPcv,
increased \vith increasing 0
o
, but <Pvc showed a small decrease with rising net uptake.
In comparison with Figure 5, Table 5 gives some results of the effect of varying
the bromide content of the vacuole. As with potassium, increase in Qv decreased the
net uptake, mainly due to an increase in the flux from the vacuole, CPvc. The increase
in Qv was accompanied by an increase in Qc from 075 to 125 m-equivJkg. Values
of Qc for potasF:lium are also given in the table.
TABLE 5
EFFECT OF BROM:IDE CONTENT OF VACUOLE ON THE BROMIDE CONTENT AND FLUXES IN THE
CYTOPLASMIC PHASE OF BEETROOT TISSUE
Tissue placed in potassium bromide solution of concentration 5 m-equivjl. Temperature 2C
Q,t
fJ Qct ~ s t ~ s t ~ v t ~ v t f;'J
Q,t
(Br-) (Br-) (K+)
32 040 075 050 010 063 023 005 86
95 015 125 025 010 072 057 004 150
t Units: mequivjkg. t Units: m-equivjkgjhr.
V. SPATIAL RELATIONSHIP OF THE FREE SPACE, CYTOPLASMIC PHASE,
AND THE VACUOLE
(a) Experiments with Radioactive Sodium
Q,t
(K+)
28
36
Tissue was allmved to take up salt from a sodium chloride solution of concentra-
tion 5 m-equiv/l, and which was labelled with 22Na, until a relatively high concentra-
tion was reached in the vacuole. It was then put into solutions of inactive sodium
chloride of the same concentration to reduce the specific activity of the free space
and the cytoplasmic phase to as Imv a value as possible. Mter a suitable period it
,vas transferred to a sodium chloride solution labelled with 24Na and then, after a
period long enough for the cytoplasmie phase to have reaehed a relatively steady
specific activity, the radioactive sodium was eluted from the tissue by inactive
sodium chloride solutions as described above. 22Na and 24Na concentrations were
estimated by counting the solutions at two different times, and the ratio of
[22Nal/[24Nal determined in the eomponents of the free spaee, cytoplasmie phase,
and the vacuole. In the free space this ratio was about 1/6000; in the tissue at
the end of the experiment (and in the efflux from the tissue at the end of the
experiment) the ratio was only 1/4, but in the cytoplasmic phase it was between
1/30-1/50. This low ratio eould have been maintained only if there was a large flux
between the cytoplasmic phase and the vacuole, i.e. as if the phases ,vere in series.
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROOT TISSUE 663
(b) Experiments with Radioactive Potassium
Similar experiments with radioactive potassium are not easy to carry out
as the potassium isotopes other than 42l( that are available at low specific activity.
An alternative approach is to examine the effect of different treatments on the
apparent content A ~ ) of the cytoplasmic phase, i.e. on the amount of isotope diffus-
ing out of the tissue as if from the cytoplasmic phase. For example, tissue was
allowed to take up 42K from a labelled potassium chloride solution of concentra-
tion 5 m-equivJl, and then eluted with either potassium chloride or sodium chloride
at 2 or 25C. Table 6 gives the amounts of potassium and its isotope that diffused
out of the non-free space during a 9-hr period of elutions, following elution of the
TABLE 6
AMOUNTS OF POTASSIUM AND OF POTASSru:r.[ ISOTOPE DIFFUSING FROM THE NON-FREE SPACE OF
BEETROOT DISKS IN 9 HR INTO SOLUTIONS OF POTASSIUM CHLORIDE OR SODIUM CHLORIDE AT
2 OR 250
Tissue allowed to take up 42K+ from a labelled potassium chloride solution of concentration
5 m-equiv/l prior to elution with sodium chloride or potassium chloride solution
Elution with Elution with
Sodium Chloride Potassium Chloride
2C 25C 2C 25C
Amount of 42K + diffusing from tissue
(arbitrary units) 58 101 19 26
Amount of potassium diffusing from
tissue (m-equiv/kg) 355 100 - -
Specific activity (arbitrary units) of:
Labelling solution 50 50 50 50
Potassium in vacuoles at end of
elution period 045 045 037 038
Minimal estimate of Q; 46 62
free space at 2C for 2 hr (of. p. 650). The specific activity of the labelling solu-
tions epd of the pota5sium in the vacuole were also measured. It is evident that
the soq.ium is having some effect other than simply replacing potassium in the non-
free space, and furthermore there is a large contribution from some other region
than free space and the vacuoles, as the average specific activity of potassium diffusing
into the sodium chloride solution is higher than that in the vacuoles. A minimal
estimate of the contribution of the cytoplasmic phase can be made by assuming that
the specific activity in this phase is as high as that in the labelling solution (an extreme
value). Even this conservative estimate is some 25 times the amount of isotope
diffusing out of the tissue to the potassium chloride solutions from both parts of the
non-free space together.
This difference can be expl&ined simply on the serial model as sodium inhibits
the uptake of potassium to the vacuoles, making the ratio 4>,,/(4),,+4>,,) more nearly
equal to unity. As a result the amount of isotope diffusing to the solution, A;, becomes
664 :1\1. G. PITMAN
more nearly equal to Q ~ As an example of this inhibition, uptake of potassium
from a mixture of sodium and potassium chloride solutions (conen. of each 5 m-equiv 11)
is negligible until the sodium concentration has fallen (as a result of sodium uptake) to
about 40% of the original concentration (Briggs, Hope, and Pitman 1958b).
The inhibition of potassium influx also has an effect on the apparent efflux
from the tissue (eqn. (9)). For example, Table 7 gives values of the apparent efflux
measured by loss of potassium or sodium isotopes from tissue in a range of mixtures
of potassium chloride and sodium chloride, of total chloride concentration 5 m-equivjl.
As the sodium concentration increased, the apparent efflux of potassium became
larger.
TABJ.E 7
AI'PARENT EFFLUX OF PO'L'ASSIUl\I AND SODIUM FRO:i\J TISSUE ACCU:i\HJ"LATING SALT FROM SOLUTIONS
CO."TAINING v AR1ED RATIOS OF POTASSIUM TO SODIUi\1 BUT WITH TOTAL CHLORIDE OONOENTRATION
OF 5 M-EQUIVjL
Solution Conen. Content of Yaeuoles Apparent Efflux
(m-equivjl) (m-equivjkg) (,u-equivjkgjhr)
Sodium Potassium Sodium Potassium Sodium Pot,assium
5 0 29 90 25 620
4 I 26 92 24 400
25 25 22 95 22 310
I 4 21 98 24 270
0 5 16-5 98 18 170
-- ---- --_.
The establishment of the higher efflux when tissue was transferred from
potassium chloride to sodium chloride solutions of the same concentration was rate-
limited by some process much slower than the equilibration of the free space, and
with a temperature coefficient for kc of between 2 and 3. For example, Figure 7
shows the pattern of increase in 42K diffusing out of the tissue when the solution was
changed from potassium chloride to sodium chloride of the same concentration at
either 25 or 16C. The tissue, which had taken up the isotope from a labelled potas-
sium chloride solution of concentration 5 m-equiv/l, was put into a series of unlabelled
potassium chloride solutions until the efflux of isotope fell to a steady value, which
is sho-wn in the figure. The apparent efflux is plotted as a percentage of the final
steady value in sodium chloride, which at 250 'vas 13 m-equivjkg/hr, and at 160
was og m-equiv/kg/hr. The eqUilibration of the free space is shown on the same
scale, and can he taken as the same at each temperature.
The inhibition of potassium uptake could increase the isotope diffusing out- of
the tissue if tho diffusion path to the solution was such that a Ia,rge proportion of
the isotope diffu:sing out of cells at the centre of the disk ,vas accumulated by cells
nearer the surface. This eff-ect can be estimated only roughly as both the diffusion
path and the diffusion coefficient of potassium in the free space are somewhat
SALT RELATIONS OF CY1'OPLASl\ITC PHASE IN BEETROOT TISSUE 665
indeterminate, but taking a low value of the diffusion coefficient (lO-7cm2sec-l), and
an influx of 1 m-equivjkgjhr, the underestimation would be only about 15% when
the tissue was in potassium chloride solutions of the concentrations used. It is
considered that the results given above support the suggestion that the tissue behaves
as if the three phases are in series, rather in parallel.
'00
x
o
"
.00
"
, ___ -'
w
" !
25
0
C
Z I
i
< .
& 60 r-...J
i ;-.......... ---.-'
j 1
C I
j
40! r.M -------.>
[
Z ,
w ,
U ,
20 ____ ... _ .... . !f ;-.- --.-.---.J
KCI NaCI
I I
o 2 3 4 5 6
TIME (HR)
Fig, 7.-Apparent potassium efflux from tissue at 25 and 160 when
transferred from a potassium chloride to a sodium chloride solution of
the same concentration, 5m.equiv/1. --- at 250; --- at
160; -'-'- equilibration of the free space. Values are plotted as
a percentage of the final steady values reached at each temperature.
VI. DISOUSSION
The first object of this paper has been the demonstration that beet tissue
contains two non-free space components-the cytoplasmic phase and the vacuoles-
and that these phases are in series. It is suggested that the phases detected in salt-
uptake studies are due to differences in the salt relations of parts of the cells and
not to a plurality of cell type within the tissue, As used in these studies, beet disks
contain both vascular tissue and parenchyma. The main argument in favour of
relating these phases to parts of the cell is that the three-phase system can be demon-
strated in beet disks cut so as to contain only parenchymatous cells (Table 4).
On these assumptions the isotope diffusing out of labelled tissue can be analysed
to calculate the fluxes into and out of the cytoplasmic phase if some assumptions
are also made about the uniformity of labelling in the tissue, It is realized that these
fluxes are "average" vaJues for all the cells in the tissue, but the comparison of the
fluxes when conditions are varied can give some qualitative information on the
relation of the cytoplasmic phase to salt uptake to the vacuoles.
666 l\f. G, PITMAN
The cytoplasmic phase must be located in the cytoplasm but may be the whole
of it, bounded by the plasmalemma and the tonoplast membrane, or a smalI part
cut off by, say, the endoplasmic reticulum and the tonoplast membrane. However, a
reasonable working hypothesis is that the cytoplasmic phase is the whole of the
cytoplasm. This assumption has the advantage that there are obvious boundaries,
which have a suitable organization to account for the high resistance to ion diffusion
found for the cytoplasmic phase boundaries.
The cytoplasm in beet cells comprises about 5% of the tissue, showing in
electron micrographs as a layer of about the same thickness as the cell wans
(Chambers, unpublished data) and, as far as can be estimated, contains about 5-10%
of mitochondria. Beet disks 1 mm thick have about 15-20% of cut or damaged
cells, and 5% of intercellular space all of which acts as the water free space. The
cell ,valls occupy about 5% of the tissue and contain the Donnan free space in about
half this volume, the rest being solid material or water free space. There is therefore
about 7 0 ~ r i available for the vacuoles.
The cytoplasm is certainly not a homogeneous phase, and whatever the location
of the cytoplasmic phase, the concentrations of potassium and bromide are not likely
to be simply Q,j5%. Regions within the cytoplasmic phase but with a turnover
morc rapid than that of the phase as a whole ,vauld contribute to Qc but would not
be detectable from the rate of exchange. For example, uptake of bromide to the
mitochondria could account for a large part of Qc; a reasonable concentration in
mitochondria might be about 100 m-equivjl, which if there are 10% mitochondria
in the cytoplasm, would be equivalent to 05 m-equiv/kg. This is not an extreme
estimate, and shows that care must be taken in attempting to locate active transport
at either boundary from arguments based on Qc and an estimated volume of the phase.
This criticism does not apply to the flux determinations which are independent
of the distribution of Qc, and so may be used to investigate the properties of the
boundaries of the cytoplasmic phase. The temperature coefficients of the fluxes
across both boundaries are between I 7 and 2 . 3, i.e. much higher than the temperature
coefficients for diffusion in solution. The high value is not taken to show that these
fluxes are active, but only that the t,vo boundaries may have the same "activation
energy" for penetration. The similarity between the membranes is also sho-wn by
estimation of the permeability, assuming that the potassium fluxes r?sc and r?vc are
not active. The diffusion flux across the outer boundary, assuming uniform potential
gradient, is
1>" ~ _p. zFEjRT
{I exp(zFEjRl')}' 00,
(11)
,vhere
p ~ uRTjFl,
u being the mobility of the ion, and l the thickness of the boundary. The relevant
value of E is not known, but it is likely to be about -58 to -116 mY, whenP would
be 2 X 10-
8
cm/sec or 11 X 10-
8
cm/sec, respectively, if r?sc is taken as 0 75 m-equiv/
kg/hr* when Co is 5 m-equiv/l (Fig. 4). Taking the same values of E, and again using
* 1 m-equiv/kgjhr = 0-3 p-equivjcm2/sec for beet tissue_
SALT RELATIONS OF CYTOPLASMIC PHASE IN BEETROOT TISSUE 667
the data of Figure 4, where Qv = 60 estimates can be made from 4>vc
giving p= O'49xlO-
s
or 27xlO-
S
cm/sec. If the membrane were about IOO!
thick the va.]ue of the diffusion coefficient would be 10-
14
cm
2
/sec, and so about that,
found for lipoprotein membranes.
The similarity in resistance of the two boundaries can also be seen from the
dependence of Qc on both Co and on Ov. If one boundary had been very much more
permeable than the other, Qc would have been related more closely to the concentra-
tion in the solution separated from the cytoplasmic phase by the lower resistance.
If the influx, o/sc. is assumed to be passive, the relationship between o/sc/Co
and 0
0
gives an estimate of the concentration of anions of about
100 in the membrane, or, if the negative charge were on a surface, ahout
10-
6
coulombs/cm
2
This charge density is about that found for many organelles,
such as chloroplasts, mitochondria, or blood cells. A negatively charged membrane
(or surface) would thus not be unlikely and would be convenient to explain the low
permeability of the cytoplasm to divalent anions, and the stimulation of uptake
of chloride ions by divalent cations unpublished data).
As there are SO many different ways of distributing potassium and bromide
ions within the cytoplasmic phase, there does not seem to be much point in using
these results to attempt to locate active uptake of bromide or potassium at either
boundary, as they can be made to support many model, by suitable choice of a volume
for tbe cytoplasmic phase and distribution withiJl the cytoplasmic phase.
The demonstration that the tissue behaves as a system means that
in some cases the estimation of fluxes suggested by Briggs (1957) and used by Briggs,
Hope, and Pitman (1958b) and by Van Stevenick (1962) will not be valid. This
derivation was based on a two-phase system and
<P' = K,(dsi/dt)/(so-s,),
where 4>-& is the influx, 8t, 8
0
are the specific activities inside and outside, and ](-& is
the concentration in the vacuoles. In fact, 80 should be replaced by 8c, if 4>1. is made
c/>cv; as 8-& is about 5% of 8
0
in most cases, and Sc may be about 50-70% of So (parti-
cularly when there is a net loss of salt from the vacuoles), this difference may lead to
underestimation of the influx to the vacuole by 45/95 to 65/95, depending on the
value of 8
c
.
VII. ACKNOWLEDGMENTS
I am grateful to Professor G. E. Briggs and to other colleagues in the Botany
Department, University of Cambridge, for their helpful comments.
VIII. REFERENCES
BRIGGS, G. E. (1957).-Estimation of the flux of ions into and out of the vacuole of a plant cell.
J. Exp. Bot. 8: 319.
BRIGGS, G. E., HOPE, A. E., and PITMAN, M. G. (1958a).-Exchangeable ions in beet disks at low
temperatures. J. Exp. Bot. 9: 128.
BRIGGS, G. E., HOPE, A. B., and PITMAN, M. G. {1958b).-Measurement of ionic fluxes in red
beet tissue using radioactive isotopes. In "Radio-isotopes in Scientific Research." Vol. 4.
p.319. (Pergamon Prf1Ss: Oxford.)
668 1\1:. G. PITMAN
BRIGGS, G. E., HOPE, A. E., and H,OBERTSON, R. N. {l961).-"Electrolytes and Plant Cells,"
(Blackwell Scientific Publications: Oxford.)
DAINTY, J., and HOPE, A. B. relations of cells of Ohara australis. 1. Ion exchange
in the cell wall. Aust. J. Biol. Sci. 12: 395.
DIAMOND, J. M., and SOLOMON, A. K. (1959}.-Intracellula.r potassium compartments in Nitella
axillaris. J. Gen. PhY8iol. 42: llOS.
MAcRoBBIE, E. A. C., and DAINTY, J. (1958).-lon transport in Nitellopsis obtusa. J. Gen. PhYBiol.
42: 335.
VAN STEVENICK, R. '(1962).-Potassium fluxes in red beetroot tissue during its "lag phase".
Physiol. Plant. is: 211.