Plant Cells - Plant Physiology

4 downloads 0 Views 364KB Size Report
Sep 7, 1971 - The work was performed at Ar-gonniie Nationial Laboratory tinder tile auspices of tli-t-Inite(I States Atomic Energy Comumission andl funded, ...
Plant Physiol. (1972) 49, 1019-1020

Gravity and Intracellular Differences in Membrane Potentials of Plant Cells Received for publication September 7, 1971

B. ETHERTON Department of Botany, University of Vermont, Burlington, Vermont 05401 R. R. DEDOLPH Western Utilization Research and Development Division, United States Department of Agriculture, Albany, California 94710 ABSTRACT

position as that employed during growth or of the same composition excepting that the KCI level was increased from 10 to 30 mm. The 5-ml chamber containing the mounted section was removed from the aerated solution and placed on a microscope stage and perfused (10 ml/min flow rate) with the same concentration of nutrient solution employed during the holding period. Each membrane potential was measured after inserting a microelectrode of a tip diameter less than 1 pt into the upper or lower one-third of a cell by the techniques described elsewhere (4). In no case was the same cell used for both upper and lower measurements.

The electrical potentials across the cell membranes of the lower parts of Zea mays coleoptile cells are about 2 millivolts more negative than across the upper parts. This electrical polarization with respect to gravity occurs when coleoptiles are oriented with their apical ends either up or down and seems independent of the magnitude of the potential when the potential is modified by other treatments.

RESULTS

Sedimentation of starch grains occurs in the cells of many higher plants (8). Dedolph et al. (3) have proposed that this sedimentation could be followed by intracellularly polarized respiration rates due to the relatively larger concentrations of metabolically active particles in the lower parts of cells. We evaluated the above theory by measuring membrane electrical potentials in the upper and lower parts of plant cells. These measurements established the extent to which individual cells were electrically polarized with respect to gravity and were the basis for judging whether or not the electrical polarization was consistent with the theorized intracellular differences in respiration.

General Trends in Potential Differences. Transmembrane

potentials varied greatly from cell to cell both within and between tissues regardless of treatment. Since only one measurement could be made on each cell and seldom were more than four to five cells in a given tissue suitably located for measurement, it was apparent that a large number of measurements in each treatment would be required to establish rigorously the

validity of the small but quite consistent differences between the means of measurements made in upper and lower areas of the cell membrane (Table I). Therefore, experiments were continued until 40 measurements on 40 different cells in each of the discrete treatments had been obtained. The mean potentials thus obtained for each treatment when tested by appropriate analysis of variance indicated that the MATERIALS AND METHODS various treatment effects were almost wholly additive. That is, Coleoptile segments were excised from 5-day-old corn seed- the discrete effect of each treatment imposed was essentially lings (Zea inays cv. 64a X 22R, University of Wisconsin) constant over all of the other conditions (Table I). which had grown in the dark at about 25 C in sand moistened This constancy is verified by comparison of the mean values with a nutrient solution. Composition of the nutrient solution obtained by calculation assuming complete additivity of diswas KCI, 10 mM; NaH2PO4, 9.05 mM; Na2HPO,, 0.48 mM; crete treatment effects (calculated means) to observed means Ca(NO3)2, 10 mM; MgSO,, 2.5 mm. The pH was 5.3. To prepare (Table I). Of direct physiological bearing is the inference percoleoptiles for membrane potential measurements, the enclosed mitted by this demonstrated additivity. When membranes of leaf was removed, the tip 5 mm was cut off, and a remaining cells are toward the lower end with respect to gravity, their 1-cm segment was mounted on a holder with its apical end transmembrane potential tends to be more negative regardless either up or down. The lateral surfaces of parenchyma cells of location in tissues or orientation of tissues or nutrient salt were exposed by cutting a notch out of the upper end of each concentration. We infer from this that imposed treatments coleoptile. In those tissues with their apical ends up, the notch which greatly affect transmembrane potentials will not mateexposed cells 5 to 8 mm behind the morphological tip, whereas rially alter the gravity-induced intracellular difference in pothe exposed cells in the inverted coleoptiles were 12 to 15 mm tential. behind the morphological tip. Magnitude of Transmembrane Potential Differences AssociAfter mounting and notching, the coleoptile sections were ated with Treatment. Since the effect of combined treatments placed in Lucite chambers and held submerged for 2 to 5 hr was found to derive wholly from the addition of the independin 500 ml of aerated nutrient solution, either of the same com- ent effects of each of the treatments, determination of the 1019

1020

ETHERTON AND DEDOLPH

Table I. Effect of Locationi withini Cells, Ori enitation of Tissutes, anid KCI Conicenittrationis oni Membranie Potejitials Each observed mean is based on 40 measuriements. The sign of the potential is in reference to an assumed 0 p otential outside the cell wall. Correlation coefficient observed v'ersuts calculated = 0.999. Membrane Pcotentials KCI Concn

_ _ __ Apex down

Apex up

---

Upper

Lower

Upper

?tf.M

10

30

Lower

Observed Calculated

-91.4 -91 .8

-93.6 -93.9

Observed Calculated

-77.0 -76.9

-79.5 -79.0

-96.4

-97.8 -97.8

-95.7

-80.8

-82.9

80.

-80.8

-827

-82.9

Table II. Restults oj'ani Anialysis of Variancce o f the Data Used for Preparilng Table I Each number is the mean of 160 observation 'Mer3ibrane Potential Comparison Upper' Lower'

86.3 -88 2

-88 42

Apex up Apex down

- 85.4

10 mM KCI 30 mm KCl

-94.8 -94.8

-895.*89.3~

*79.93

1 Location in cell with respect to gravity. 2 Difference between means significant at th e S( level. Difference between means significant at th iel'level,

Plant

Physiol. Vol. 49,

1972

been extensively studied for higher plants in general, or for corn coleoptiles in particular. Research on oat coleoptiles shows that inhibitors of adenosine triphosphate synthesis such as dinitrophenol and cyanide make membrane potentials less negative (5, 7), indicating that membrane potentials are related to respiration. There are other possible interpretations for the current findings. The differences in membrane pctentials could reflect a change in the permeability of the cell membrane in the lower part of the cell, or it could result from changes in ion concentration gradients. These changes may or may not be related to differences in respiratory rates. The present data do not permit distinguishing between either of these alternatives or the respiratory hypothesis Our results suggest a connection between the gravity-related electrical polarization of a cell membrane (the cell geoelectric effect) and the gravity-related electrical polarization of an organ such as a coleoptile (the organ geoelectric effect). The organ geoelectric effect has been extensively studied (1, 6, 9). Briefly, it is a difference in electrical potential which can be measured between the upper and lower surfaces of a horizontally positioned plant organ such as a grass coleoptile or dicotyledonous stem. The lower side of a corn or oat coleoptile is 30 to 70 mv more positive than the upper side. This potential difference appears to depend upon the development of asymmetric auxin concentrations within the coleoptile and is not the cause of them (2, 6, 8), as had been previously postulated. The organ geoelectric effect may now be ascribed to electrically polarized cells. The sign of the cellular geoelectric potential is consistent with this hypothesis if one considers that electrodes external to cells are used for measuring the organ geoelectric effect, whereas the cellular geoelectric effect is

measured with intracellular microelectrodes. Since roughly 40 to 50 cells would be in series in a corn colecptile cross section, 9the magnitude of additive intracellular potentials agrees with that of the geoelectric potential. The demonstrated existence of a transmembrane geoelectric effect which is unaffected by other treatments which alter the membrane potential suggests future uses for this . pproach in studies of the effect of gravity on plants.

magnitude of response obtained is best undertaken by comparison of these main effect means (Table II). Based upon 160 measurements for each mean, the lower parts of the cells are 2.1 mv more negative than the upper parts. Membrane potentials of cells with their apex ends down were 3.9 mv more negative than those with their apex ends up. Cells in lOX nutrient solution containing 10 mm KCl had membrane potentials which were 14.9 mv more negative than those in lOX nutrient solution containing 30 mm KCI.

DISCUSSION The present data show that membrane potential differences between different parts of a cell are related to the cell's orientation in a gravity field. The cellular mechanisms which translate the gravity stimulus into this electrical effect are not clear, however. The findings are consistent with a gravity-induced increase in respiration in the lower parts of the cells which may be attributed to increased concentrations of starch grains. The above interpretation is a qualified one because the relationship between respiration and membrane potentials has not

.4cAktio'ldfmeqp, ts-We are giratefiil to Dr. S. A. Gordon for his stipport of this stuldv. The work was performed at Ar-gonniie Nationial Laboratory tinder tile auspices of tli-t-Inite(I States Atomic Energy Comumission andl funded, in part, t) Grant W- 12,792 0a) tfroi the Nationail Aeroniaitics and(1 Space Administration. LITERATURE CITED

1. BRAUNE,it. L. 1969. Effect of gravity oII ttie levelopiiiment of electr c potentials in plant tissuees. Ein(leav-ouir 28: 17-21. 2. D)EDOLPH, I{. t., J. J. BREEN, AND S. A. GOiRDON. 196.5. Geoelectric effect and geotrophiic curvature. Science 148: 1100-1101. 3. DEDOLPH, 1I. R., I). A. OEMICK, B. R. WILSON, AND) G. R. SMITH. 1967. The cauisal basis of gravity stimulus ntillification hv clinostat r-otation. Plaint PlSsisol. 42: 1373-1383. 4. ETHERTON, B. 1970. Effect of iil(lole-3-acetic acid on iiembrane potentials of oat coleoptile cells. Plant Piysiol. 43: 527-529. 5. ETHERTON, B. AND N. HIGINIBOTHAM. 1960. Transmenibrane potential m11eastuIeIiients of cells of ligiher plants as relate(d ti salt uptake. Science 131: 409-410. 6. GRAHNI, IL. 1964. Measurements of geoelectric ancl auixini induiticed( potentials inI coleoptiles Nvithaia efinedi vibrating electrloe technique. Phvsiol. Plant. 17: 236-262. 7. HIGINBOTHANM, N., J. S. GRANES. AN-D R. E. DAVIS, Jit. 1968. Evi(dence for an electrogenic ion transport pumi11p in highier plants. Planit Physiol. 43: 851. 8. AVILKINS, M. B. 1966. Geotropiism-1. Ainno. 1Rev. Planit Phyvsiol. 17: 379-408. 9. WXILKIN-S, M. B. AND A. E. R. WOODCOCK. 1965. Origini of the geoelectric effect in plants. Natuire 208: 990-992.