Download Intracellular-volume measurements of wheat

BIOCHEMICAL SOCIETY TRANSACTIONS
850
high concentrations of NEM are required to observe this
effect. At these concentrations NEM appears not only to
induce K + permeability but also has an inhibitory effect
upon malate oxidation.
In summary, the results presented in this report are
consistent with a H+/O stoichiometry of 9, 6 and 2 for
electron flow from NAD+-linked sbustrates, succinate and
cytochrome c respectively. Since a similar stoichiometry is
observed with succinate and NADH, even though they are
on opposite sides of the membrane, it may be deduced that
with succinate, uniquinone is reduced by matrix protons,
whereas protons must be derived from the medium when
external NADH is the substrate.
This work was supported by a grant from the S.E.R.C.
Alexandra, A,, Galliazzo, F. & Lehninger, A. L. (1980) J . Eiol.
Chem. 255, 10721-I0730
Brand, M. D., Reynafarje, B. & Lehninger, A. L. (1976) J. Eiol.
Chem. 251, 5670-5679
Brand, M. D., Harper, W. G., Nicholls, D. G . & Ingledew, W. J.
(1978) FEES Lett. 95, 125-129
Mitchell, P. (1976) J. Theor. Eiol. 62, 327-367
Moore, A. L. & Cottingham, I. R. (1983) in Metals and Micronutrients; Uptake and Utilisation by Plants ((Robb, D. A. &
Pierpoint, W. S., eds.), pp. 170-240, Academic Press, London
Moore, A. L. & Proudlove, M. 0. (1983) in Isolation of Membranes
and Organelles from Plant Cells (Hall, J. L. & Moore, A. L.. eds.),
pp. 153-1 84, Academic Press, London
Pozzan, T., Miconi, V., Di Virgilio, F. & Azzone, G. F. (1979) J.
Biol. Chem. 254, 10200-10205
Villalobo, A,, Briquet, M. & Goffeau, A. (1981) Eiochim. Biophjs.
Acta 637, 1 24- 129
Wikstrom, M. K. F. & Krab, K. (1980) Curr. Top. Eioenerg. 10,51101
Wikstrom,M.K. F.&Krab,K.(1981)Ann. Rec.Biochem.50,623654
Intracellular-volume measurements of wheat-leaf mesophyll cells and protoplasts
KAY L. M. VALLES,* MICHAEL 0. PROUDLOVE,*
FRANCIS A. WILLIAMSON,t
R. BRIAN BEECHEYt and ANTHONY L. MOORE*
*Department of Biochemistry, University of Sussex, Falmer.
Brighton BNI 9QG, U.K., and ?Shell Research Ltd.,
Sittingbourne, Kent ME9 8AG, U.K.
Protoplasts isolated from mesophyll tissue are considered to
be the closest analogue to the leaf cell. Of increasing
importance is their application to studies concerning the
interaction and compartmentation of metabolic processes
between mitochondria, chloroplasts and cytoplasm (Hampp
et al., 1982; Stitt et al., 1982, 1983). We have investigated
the distribution of a lipophilic cation complex
(TPMP+/TPB-) as a probe for intracellular membrane
potentials in wheat mesophyll protoplasts. By following the
uptake of the radiolabelled complex into the cell, the net
accumulation ratios achieved reflect the distribution of the
complex throughout the cell, according to the electrical
potential across each internal membrane system (Valles et
al., 1984). Quantification of such membrane potentials
requires an estimate of the steady-state concentration of
cation in each cell compartment, hence their respective
volumes must be measured. Accordingly, as the relevant
morphometric data are unavailable, we have used conventional stereological techniques (Weibel, 1969) to assess the
volumes of intracellular compartments. A comparative
study with wheat-leaf mesophyll cells was undertaken, since
it is uncertain whether protoplast morphometry differs to
any appreciable extent from that of the tissue.
Central to the analysis is the Delesse Principle which
states that the areal density of component profiles on microscopical sections is an unbiased estimate of the volume
density of the component. Since volume density measurements are dimensionless and therefore independent of the
absolute volume of the cell, values for tissue and protoplasts
can be compared directly. The results are shown in Table 1.
No differences were observed between the two populations
when the cytoplasmic organelle volume densities were
expressed on percentage cell basis. The exception is the
vacuole compartment. This is significantly larger in the
tissue sample compared with that in isolated protoplasts.
Abbreviations used: TPB-, tetraphenyl boron; TPMP+,methyltriphenyl phosphonium.
Table I . Fractional cell volumes in isolated protoplasts and tissue
sections from wheat leaf
Results are expressed as percentage cell volume density
fS.E.M. *Mitochondria and microbodies pooled because of
limited resolution at magnification used.
Cytoplasm
Vacuole
Cytosol
Chloroplasts
Mitochondria
Microbodies
Nucleus
Tissue
35.6 f2.3
64.4f2.3
8.9f 1.3
24.4 f2.2
}
1.3f0.2'
l.Of0.4
Protoplasts
43.4 f I .5
56.6 f I .5
13.2k 1.5
27.1 f2.3
1.2f0.1
0.1 f0.05
1.820.5
Since protoplasts act as osmometers this difference may
be explained by a preferential loss of vacuolar water,
resulting in a net shrinkage of the protoplast upon isolation.
This is supported by recent data (Komor et a[., 1982) which
indicate that protoplasts suspended in 0.4~-mannitol(cf.
0.41~-sorbitolused in this work) had only 75% of the
volume of the sugar cane cells (non-plasmolysed) from
which they were isolated.
The relative volumes for vacuole and plastids found in
this work differ from those reported by Hampp et al. (1982).
Using young oat leaves they found the vacuole to comprise
78% of the mesophyll protoplast. This was however
calculated from isolated vacuoles which may have suffered
osmotic volume changes on isolation. Additionally, their
value of 10.8% for the plastid compartment estimates only
the stromal volume, again calculated from measurements on
isolated organelles. Using the sterological method, fractional volumes are measured in situ and, whilst we include the
intermembrane space, this will constitute only a small
fraction of the total volume in uivo.
In conclusion, although there are no gross changes in
organelle volume densities during protoplast isolation,
account must be taken of a possible stress condition. In
particular, under photosynthetic conditions, the activity of
stromal enzymes may be impaired due to increased
concentrations of internal solutes (Kaiser et al., 1981).
Hence, a degree of caution must be exercised if one is to
interpret metabolite distribution data as representing the
situation in the whole leaf.
1984
607th MEETING, LONDON
This work was supported by an A.R.C. grant (A. L. M.) and an
S.E.R.C. C.A.S.E. award (K. L. M . V.).
Harnpp, R., Goller, M. & Ziegler, H. (1982) Plant Physiol. 69,448455
Kaiser, W. M., Kaiser, G . , Prachuab, P. K., Wildrnan. S . G . &
Heber, U. (1981) Planro 153. 416-422
85 1
Kornor, E., Thorn, M. & Maretzki, A. (1982) Plant Phjxiol. 69.
1326-1330
Stitt, M., McC.Lilley, R. & Heldt, H. W. (1982) Plant Physiol. 70,
971-977
Stitt, M., Wirtz, W. & Heldt, H. W. (1983) Plant Physic)/.72, 767774
Valles, K . L. M., Proudlove, M. O., Beechey, R. B. & Moore, A . L.
(1984) Biochem. SOC.Trans. 12, 851-852
Weibel, E. R. (1969) Int. Rer. Cjtol. 26, 235-296
Membrane potential measurements in wheat-leaf mesophyll protoplasts
KAY L. M. VALLES,* MICHAEL 0. PROUDLOVE,*
R. BRIAN BEECHEYt and ANTHONY L. MOORE*
*Department of Biochemistry. Unicersity of Sussex. Falmer.
Brighton BNI 9QC. U . K . ,and tShell Research Ltd..
Sittingbourne, Kent ME9 8AG. U .K .
120,
fa)
The lipophilic cation TPMP+ has been used to assess the
degree of mitochondrial energization in intact wheat
protoplasts under illuminating and non-illuminating conditions. Uptake of T P M P + , which will accumulate in cell
compartments which have an inside negative relative to the
outside, has been compared with that seen for 8hRb+,a
cation which will equilibrate specifically across the plasma
membrane.
Uptake was measured as described previously (Valles et
al., 1983). Accumulation of both cations was found to be
time-dependent, the steady-state distribution of Rb+ occurring after 30-40min. that of TPMP+ after 2-4h. The
equilibrium ratio of Rb+ was found to be the same under
/
illuminating and non-illuminating conditions, indicating
that the plasma membrane potential (-54mV) does not
change under these conditions. This potential was depolarized by S O ~ M - K C Ithe
, final value of -20 to -25mV
representing the plasma membrane Donnan potential.
Uptake of TPMP+ by wheat protoplasts was found to
exceed that of Rb+ and inclusion of greater than equimolar
-concentrationsof TPB- were found toenhance both the rate
of uptake and the accumulation ratio of TPMP', equilibra0
3
6
9
12
tion being reached in 10-15min. The [TPB-] which gave
Time (min)
maximum accumulation ratios was, however, found to vary
between protoplast preparations (50-90 ,UM-TPB- at 1 p ~ TPMP'). The role of TPB- is at present poorly understood
although it is thought to form a neutral ion pair with
fbl
TPMP' (Stark, 1980), this complex having a far greater
lipid solubility than either ion alone.
The effect of illumination on TPMP+ ( + T P B - ) and Rb'
accumulation by protoplasts, compared with dark controls,
is presented in Fig. la. From this one may see that in the
light there was an initial, significant increase in the TPMP+
(+TPB- ) accumulation ratio. After I2min, however, the
value had fallen to that found in the dark. This diminution
may be due to an adverse effect of TPB- on chloroplast and
mitochondrial activity since this anion inhibits COzdependent oxygen evolution by wheat protoplasts, water
splitting in isolated chloroplasts (Khanna et al., 1983) and
oxygen uptake by isolated mitochondria. Illumination and
TPB- had no effect on the plasma membrane potential.
Taking the data for cell compartment fractional volumes
(Valles et al., 1984). the value for the plasma membrane
potential and the maximum accumulation ratios for Fig. I . The efjhct of light and dark on the time-dependent
B - IOpM-Rh'
TPMP' (Fig. l a ) and substituting them into an expansion accumulation O J ' I ~ M - T P M P+' ~ O ~ M - T P and
of the Nernst equation (Valles et al., 1983) gives a predicted by wheat protoplasts (a) and their eirect on mitochondrial and
mitochondrial potential of -132mV in the dark and chloroplast membrane potentials, dericed.from an e.ypansion of'
the Nernst equation (h)
The
maximum
accumulation
ratios (ARAT) in light and
Abbreviations used: TPB-, tetraphenyl boron; TPMP'. rnethyldark were used to calculate organelle potentials.
triphenyl phosphoniurn.
2ol
Vol. 12
.