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This document from a 1976 journal explores the effects of inorganic salts on plant membrane permeability using beet root slices and rumex obtusifolius l. Leaf discs. The study reveals that cacl2 decreases betacyanin leakage and delays senescence, while (nh4)2so4 increases leakage and hastens senescence. The authors suggest that these effects may be due to alterations of interactions between water and macromolecules in the tissues, and discuss the possible relationship between these effects and the macromolecular stabilization or destabilization properties of inorganic salts.
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Plant Physiol. (1976) 58, 182-
Received for publication February 19, 1975 and in revised form February 24, 1976
ABSTRACT
Inorganic solutes are shown to alter the permeability of root and leaf tissues. Experiments with beet root tissues reveal that CaCI2 decreases leakage of betacyanin from the tissue, that (NH4)2SO4 increases leakage, and that each salt can relieve the effects of the other. A comparison of cations and anions shows a range of effects with the various solutes. Experiments with Rumex obtusifolius L. leaf discs reveal that whereas CaCI2 defers the development of senescence, (^) (NH4)2SO4 hastens senes- cence and increases the leakage of materials out of the leaf discs. The solute effect on Rumex obtusifolius L.^ is prevented^ by^ gibberellin.^ CaC can (^) relieve the (^) (NH4)2SO4 effect. The results are interpreted as indicat- ing that the inorganic solutes may serve to alter the^ permeability^ of membranes through^ alterations^ of^ interactions^ between water and mac- romolecules in^ the tissues; the^ interpretation^ is^ consistent^ with^ the evidence for opposite effects of Ca and NH4, the^ effective^ concentrations being about 1O-3^ M, and the reversibility of^ the^ effects^ of^ one^ solute by another of opposite stabilization-destabilization effect.
It is well known that inorganic salts can have large effects on the configuration of macromolecules; Hofmeister (2) described the range of effects of various salts on protein solubilities, and in
scribed the^ effects^ of^ inorganic salts^ as^ an^ alteration^ of^ macromo-
solutes on plant membranes. It is the intent of this paper to
relate to their effects as stabilization or destabilization agents.
into sections 1 cm^ in^ diameter and^1 to^2 mm^ thick. The^ sections
For the Rumex assay (Rumex obtusifolius L.), following the
ment (^) Station, Lafayette, Ind. 47907. (^2) Present address: Department of Horticulture, Washington State University, Pullman, Wash. 99163. (^3) Present address: Graduate College, University of Nebraska, Lin- coln, Neb. 68583.
house. Leaf discs (1 cm) were cut with a cork borer and^ random- ized, and 10 discs were floated in each Petri dish^ containing^10 ml of solution to be tested in the darkroom at 25 C for about 4 or 5 days. Chl was extracted in ethanol. Ten leaf discs were trans- ferred to 10 ml of ethanol in a closed vial and allowed to stand overnight, and absorbance was read at 665 nm. The absorbance of ambient solution was measured at 280 nm to assess the leakage from leaf discs. All experiments were repeated on at least five different occasions.
As a simple test of^ membrane permeability,^ we^ used^ slices of beet root suspended in water, and measured the^ extent^ of beta- cyanin leaking out into the ambient solution as an index of membrane leakiness. As a comparison of an extreme macromo- lecular destabilizer, CaCl2,^ and^ an^ extreme^ stabilizer, (NH4)2SO4, serial concentrations of^ these^ two salts^ were^ applied separately to beet root slices (Fig. 1); it can be seen that CaC produced a decrease in the amount of pigment leaking out, whereas (NH4)2SO4 caused an increase in the amount. If these opposite effects are, in fact, related to destabilization and stabilization effects, then it should be possible to relieve the effects of each salt with increasing amounts of the other. In order to test that possibility, a series of beet root sections was treated
whereas the (^) (NH4)2SO4 produced a marked increase in the amount of pigment leaking out of the root sections, the further addition of (^) CaCl2 at slightly above 10-3 M CaCl2 brought the leakage back down to the level of the sections in water alone. The converse experiment was^ carried out^ as shown^ in^ Figure^ 3; here, a group of sections was^ treated^ with^ CaCI2^ at^ 10-3^ M concentration, and the effects of further^ additions^ of^ (NH4)2SO
leakage of pigment by about half; the^ further addition^ of (NH4)2SO4 brought the amount of leakage back to^ the value of water controls. These two^ experiments^ indicate^ that^ the^ effects of each of these two^ salts^ may be^ erased^ by the simultaneous application of^ the other salt. As further evidence about the possibility that destabilization- stabilization effects were causing the leakage responses ob- served, we made a comparison of various cations and^ various
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Plant Physiol. Vol. 58, 1976
0
0
CD .4 w
2x1I0 2 2x162 m CONC. OF^ SA LT FIG. 1.^ Effects of^ (NH4)2S04 and^ CaC12^ on^ leakage^ of^ betacyanin from beet slices.
0 In a 0 w CD 4 4 -i
also caused an increase in leakage out of the leaf sections. Even in the presence of GA, an enhancement of leakage by (NH4)2SO4 was evident at the higher concentrations. If the enhancement of the rate of leaf senescence and of leakage by (NH4)2SO4 is related^ to^ the solute^ effecting the destabilization-stabilization state of^ the^ membranes, then^ one would expect that^ CaCl2^ additions^ might^ relieve^ the^ effects^ of^ the (NH4)2SO4. Experiments^ in^ which various^ concentrations^ of CaCl2 were added in the incubation medium are presented in Figure 5, from which it can be seen that even in the presence of the (NH4)2SO4, CaCI2 was able to defer the rate of senescence, as indicated by the maintenance^ of^ higher Chl^ contents^ in^ the leaf discs. The lower^ figure indicates^ that^ the^ leakage of^ materi- als absorbing at 280 nm^ was^ stimulated^ by (NH4)2SO0 as^ was seen previously in Figure 4, but the^ CaCI2 additions relieved^ the effects of the 3 x^ 10-3 M (NH4)2SO4 both^ with respect to^ Chl retention (Figure 5, above) and limiting the^ development of leakiness (Fig. 5, below).
0
0 0
(^04) w 4 w -i
1.2-
0.6-
0.2v
J II CONC. CO C FIG. 2. Promotion of betacyanin leakage from beet slices^ with 3^ mM (NH4)2S04 and the reversal of its effect with increasing concentrations of CaC12-
reduction of leakage was obtained with^ nitrate^ and^ carbonate. Collectively, these cation and anion^ experiments indicate^ that the leakiness effects are not^ specific to^ special cations^ or^ anions, but that (^) graded series of effects can be obtained with a wide array of anions and cations. Some further experiments were done with another tissue: the 1-cm discs of Rumex leaves. We had previously reported that the senescence of corn leaf discs was associated with a marked increase in leakiness, as measured by apparent free space and by permeability to tritiated water (8). It is known that the senes- cence of Rumex leaves is deferred by gibberellin (17), and that CaCI2 also markedly slowed the development of senescence (8) and abscission (9). In the present experiments, Rumex leaf discs were treated with various concentrations of (NH4)2SO4 to deter- mine whether this stabilizing salt would have effects opposite to those of CaCl2. Effects were measured in the presence and in the absence of GA. Chi content was used as a measure of senes- cence, and the^ relative^ absorbancy^ of the ambient^ solution^ at 280 nm was taken as an indication of the amount of leakage of organic materials from the leaf discs. The data in Figure 4 indicate that (NH4)2SO4 at concentrations of 10-3 M and above enhanced the rate of leaf senescence, as indicated by the greater loss of Chl, than in control pieces. While GA deferred senescence effectively, the addition of (NH4)2SO in the presence of the GA did not alter the rate of leaf senes- cence. The measurements of leakage out of the leaf discs indi- cate that the (NH4)2SO4 concentrations of 3 x 10-3 M^ or above
1O-4 - CONC. (NH4)2 S FIG. 3.^ Inhibition^ of^ betacyanin leakage from beet^ slices^ with^1 mm CaC12 and the reversal of its^ effect^ with^ increasing concentrations^ of (NH4)2S04.
Table I. Comparative Effects of Cations on Leakage of Betacyanin from Beet Slices Each cation supplied as the chloride at 3 x^ 10-3 M concentration.
Solute Betacyanin leakage
supplied (OD540)
H20 0.266 + 0.
Ammonium 1.300 +^ 0. Potassium 0.325 -^ 0. Lithium 0.200 - 0. Sodium 0.180 -^ 0. Magnesium 0.160 -^ 0. Calcium 0.115 -^ 0. Barium 0.111 + 0. Strontium 0.098 - 0. Manganese 0.030 - 0. Lanthanum 0.017 ±^ 0.
coci
I
0.so
that ammonium salts could increase the leakage of photosyn- thates out of cotton leaf cells, and Ca having the opposite effect. In some experiments with^ dynamic pumping^ systems,^ Satter^ et al. (13) have shown that the rhythmic movements^ of^ Albizzia pulvini are caused to exercise an opening action in the presence of calcium salts, a closing action in the presence of ammonium salts, and lesser or intermediate effects of other cations in^ the Hofmeister series. These experiments can be interpreted^ as indicating that inorganic solutes of the Hofmeister^ series^ can increase permeability of membranes or^ decrease the^ permeabil- ity, depending upon the stabilization or^ destabilization^ effects of the solutes. Through the experiments on^ interactions between solutes, our experiments have added strength to^ the^ concept^ of solute effects being through the^ destabilization-stabilization^ ef- fects. Our special interest in the solute alterations of membrane functions lies in the area of hormonal action; if hormones act through an attachment to a site of action on a membrane, then solute alterations of membrane characteristics might be expected to alter hormonal effectiveness.^ We^ have shown that^ each^ of the plant hormones is altered^ in^ effectiveness^ by^ calcium^ and^ ammo- nium salts (3, 11), and that^ the^ attachment^ of^ auxin^ to^ mem- brane pieces from^ corn^ coleoptiles can^ also be^ altered^ by^ these salts (10).
LITERATURE CITED
Exp. Pathol. Pharmakol. 24: 247-260.
Plant Physiol. Vol. 58, 1976