influence of decenylsuccinic acid water permeability plant ... · pdf filemal cells ofbulb...

Plant Physiol. (1972) 50, 608-615

Influence of Decenylsuccinic Acid on Water Permeability ofPlant Cells'

Received for publication July 30, 1971

0. Y. LEE, ED. J. STADELMANN, AND C. J. WEISERDepartment of Horticultural Science, University of Minnesota, St. Paul, Minnesota 55101

ABSTRACT

Decenylsuccinic acid altered permeability to water of epider-mal cells of bulb scales of Allium cepa and of the leaf midribof Rhoeo discolor. Water permeability, as determined bydeplasmolysis time measurements, was related to the dose ofundissociated decenylsuccinic acid (mX undissociated decenyl-succinic acid X minute). No relationship was found betweenpermeability and total dose of decenylsuccinic acid, or dose ofdissociated decenylsuccinic acid, suggesting that the undis-sociated molecule was the active factor in permeability changesand injury.At doses which did not damage cells (0.0008 to 0.6 [mm of

the undissociated molecule X minute]) decenylsuccinic aciddecreased water permeability. At higher doses (e.g., 4 to 8[mM X minute]) injury to cells was common and decenyl-succinic acid increased permeability. Doses above the 10 to20 (mM X minute) range were generally lethal. The plasmolysisform of uninjured cells was altered and protoplasmic swell-ing occasionally was observed. The dose-dependent reversalof water permeability changes (decreased to increased perme-ability) may reflect decenylsuccinic acid-induced changes inmembrane structure. Reported effects of decenylsuccinic acidon temperature dependence of permeability and frost resistancewere not verified.

Various external or internal factors are assumed to alterpermeability of cell membranes to water or solutes (cf. 20).Studies of entire plants or parts of plants may lead to erroneousconclusions about cell permeability because of other factorsinvolved and because cell viability usually is not tested. Withthe methods now available, changes in permeability can be in-vestigated directly at the cellular level (19). The difficulties inevaluating cell permeability changes with multicellular struc-tures is exemplified by the conflicting conclusions reached byworkers who have used detopped plants to study the influenceof decenylsuccinic acid in water permeability. DSA' is a waxysubstance which has a molecular weight of 256 and the formula

1 This research was supported by a grant from the Lois W. andMaud Hill Family Foundation and the Minnesota Agricultural Ex-periment Station. Minnesota Agricultural Experiment StationScientific Journal Series Paper No. 7716.

2 Abbreviations: DSA: decenylsuccinic acid; RCD: relativechange in deplasmolysis time.

CH3 -(CH2),,- CH = CH - CHs - CH(COOH) - CHa-COOH. Kuiper (9) reported that DSA increased the water per-meability of decapitated bean stumps as much as 8-fold. Usingsimilar techniques, Newman and Kramer (14) concluded thatincrease in permeability of roots treated with DSA was relatedto root injury. Application of lower, noninjurious concentra-tions of DSA led to reduced water permeability (14). Kuiper(10) also reported that DSA-treated bean plants survived frostsof -5 to -10 C; and that apple, pear, and peach blossomssprayed with DSA survived to -6 or -7 C. He attributed theincreased frost resistance to a purported DSA-induced increasein water permeability (10) which could reduce lethal intracellu-lar crystallization. Adverse effects of DSA on cold tolerance ofwinter wheat were recently reported (4).A number of other effects have been attributed to DSA

which could be related to permeability of cell membranes. Ithas been suggested that DSA reduces the temperature de-pendence of photosynthesis by increasing the water and CO2permeability of membranes at low temperatures (11); that DSAesters prevent stomatal opening and reduce transpiration (28;for failure of beneficial effects of DSA on internal waterbalance see 7 and 8); that DSA inhibits photophosphorylationand triphosphopyridine nucleotide activity (Siegenthaler andPacker, cited in ref. 11); that DSA acts as a metabolic inhibitor(14) and growth retardant (12); and that DSA alters the con-figuration of ATPase in membranes and protects this enzymefrom frost denaturation (12). DSA was also found to decreaseexudation from decapitated roots (J. W. O'Leary, personalcommunication) and to decrease the uptake of "P compoundsby roots (4).The objective of the present study was to examine the effect

of DSA on uniform plant cell material using deplasmolysistime measurements. The influence of DSA on temperature de-pendence of water permeability and on frost resistance was alsobriefly examined.

MATERIALS AND METHODS

DSA, supplied by Humphry-Wilkinson, Inc., North Haven,Connecticut was dissolved in water at 40 C. At concentrationsabove 5.0 mm, a precipitate formed at room temperature. At1.0 mm the DSA solution was colloidal and translucent andhad a low surface tension. At concentrations of 0.5 mm orlower DSA gave a clear solution with a surface tension closeto that of pure water (no foaming).When pH values of the 1.0 mm DSA solutions were adjusted

to neutrality with a few drops of NaOH or KOH, the solutionscleared and lost their colloidal appearance. The pK values ofDSA were estimated to be 4.5 and 5.7 by titration with arecording Corning Model 12 potentiometric pH meter. Withthese values the concentration, C, of undissociated DSA is

608

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Plant Physiol. Vol. 50, 1972 DECENYLSUCCINIC ACID AND WATER PERMEABILITY

calculated:

C= A * -cr1+ a, . Ca2

1 -aC2

(1)

where A = total DSA concentration of the solution; a1 and a!2are calculated from the relation pH = pK1 + log [a! (1 - a)] =pK, + log [a2/ (1-a,2)] a,. and a,2 are the degrees of dissociation ofthe undissociated molecule and monobasic ion, respectively.The influence of DSA on water permeability of cells of the

adaxial epidermis of onion (A Ilium cepa) bulb scales from threesources (variety Granex from Texas, Trapps Downing fromMinnesota, and market onions of unknown origin) was de-termined at room temperature by measuring the deplasmolysistime. The time required for complete deplasmolysis dependsmainly upon the permeability of the protoplasmic layer towater (the greater the deplasmolysis time, the lower the waterpermeability). Differences in the deplasmolysis time betweentreated and control tissues where all other external factors are

the same indicate changes in water permeability. Deplasmolysistime is a convenient means to obtain relative measures of waterpermeability.The difference in deplasmolysis time (treated-control) was

expressed as percentage of the deplasmolysis time of the con-

trol:

RCD

deplasmolysis time (treated) - deplasmolysis time (control)deplasmolysis time (control)

X 100

Negative values of RCD indicate higher water permeabilityof the treated sample with regard to the control and vice versa.

Similar studies were conducted on cells of the abaxial leafmidrib of a single clone of Rhoeo discolor. In a few Rhoeo ex-periments, the DSA effect on permeability was evaluated atlower than room temperatures (e.g., 10-15 C).

In a single study, six intact Rhoeo plants in a controlledenvironment chamber each growing in a 15-cm pot containingmixtures of 2 parts soil, 1 part sand, and 1 part peat moss(v/v) were treated daily with 1 mm DSA, pH 7, (200 ml of soilapplication per pot and foliar spray to run off) for 21 days.Six control plants randomly distributed in the same chamberwere treated similarly with distilled water. On the 16th through21 st days, all plants were subjected to progressively lower freez-ing temperatures for 3 hr during the dark period. Air tempera-ture was measured at plant height each night during test freez-ing and frost injury was rated visually on the following day.

Figure 1 presents a flow diagram which outlines the de-plasmolysis technique used in these studies. The adaxial epider-mis from the third bulb scale of Allium and the lower epidermisfrom the leaf midrib of Rhoeo were collected as previouslydescribed (3, 21). Paired adjacent sections were included ineach run; one a control and the other treated with DSA. Inas-much as the control and the treated section of epidermis couldnot be handled simultaneously with the same equipment, one ofthe epidermal sections, alternating between treatments in suc-

cessive experiments, was held in spring water' (from Glenwood,Minneapolis; total hardness as CaCO, 325 mg/l, Ca 2 78

mg/l. HCOr- 330 mg/l) until evaluation of the first section was

terminated.Pretreatment consisted of transferring epidermal sections to

3 Complete analysis available upon request.

* IN SOME EXPERIMENTS

FIG. 1. Flow diagram showing the individual steps of a typicalplasmolysis study.

spring water or a KCl-CaCl, solution, 20 mm each. These pre-treatments may counteract possible Ca deficiencies in theplasmalemma and tonoplast and hence yield more uniformvalues for the permeability constant and improve protoplastrounding during plasmolysis (cf. 13, 17 [p. 93], 23, 24, 26[p. 56]).The initial gradual plasmolysis (initial plasmolysis) in near

isotonic concentrations of mannitol (0.5-0.6 M for Allium;0.30-0.35 M for Rhoeo) is a tempering treatment which reducesosmotic shock. DSA treatments were administered duringinitial plasmolysis. Strength of treatment is calculated as dose(DSA concentration applied X time; the actual effectivestrength, however, may be modified by such factors as concen-

tration effects of diffusion leakage out of the membranes).After 15 min to 2 hr of initial plasmolysis, the tissue was

transferred in the same solution to a perfusion chamber (27)on a microscope stage. The chamber was sealed with a cover

slip and vaseline.Final plasmolysis was accomplished by passing mannitol

(0.7 M for A Ilium and 0.5 M for Rhoeo) through the perfusionchamber. After 0.5 to 1 hr, protoplasts had reached the final

Collection of epidermis(Allium or Rhoeo)

(1) Pretreatment of epidermis

il i'\CONTROL . TREATED

. Holding period/ (Springwater)

/(alternated between. treatment and control)

(2) Initial plasmolysis (2) Initial plasmolysis(DSA treatment)

(3) Final plasmolysis (3) Final plasmolysis

(4) Time-lapse photomicrography*

I l(5) Deplasmolysis (5) Deplasmolysis

I l(6) Test plasmolysis (6) Test plasmolysis

I e(7): Microscopic evaluation of deplasmolysis time from films*

609



LEE, STADELMANN, AND WEISER Plant Physiol.

7'

...

..............

.............

......

mm

mmw

1mm

. ..........-

L~~~~~~~~~~~~~~~~6

...............

J

FIG. 2. Frame of a time lapse microphotograph series of Alitim cepa epidermis about 30 sec after transfer from the plasmolyzing solution(0.8 m mannitol) into the deplasmolyzing solution (0.35 m mannitol).

degree of contraction (to about two-thirds or one-half of thecell volume) and osmotic equilibrium was established betweenthe vacuole and the external solution. In each experiment thecontrol and the treated sample were plasmolyzed for the samelength of time.

Deplasmolysis was induced by perfusing the sample with ahypotonic concentration of mannitol (usually 0.35 m for Al-lium and 0.1 m for Rhoeo). Deplasmolysis time was measuredas the time elapsed between the beginning of perfusion withthe hypotonic solution and deplasmolysis of 90% of the cells.The RCD was calculated from these values. Each determina-tion of deplasmolysis time was based on observation of ap-proximately 200 Alilium cells or 30 to 40 Rhoeo cells.

Analyses were made of the time course of deplasmolysisfrequency in several experiments by taking time-lapse photo-micrographs (Fig. 2) during deplasmolysis (cf. 18). In thisway the times for 50% and 90% deplasmolysis were de-termined exactly.

To test for cell viability, the cells were replasmolyzed follow-ing deplasmolysis by perfusion of plasmolyticum through thechamber. Injured cells which did not replasmolyze were ex-cluded from the evaluation of deplasmolysis time.

In the temperature dependence studies the perfusion solu-tions were cooled in a salt-ice water bath and solution tempera-tures were recorded by thermocouples in the outlet and inlettubes of the perfusion chamber. The given experimentaltemperatures were the means of these inlet and outlet tempera-tures.

RESULTS

Toxicity of DSA Solutions. The relationships between DSAconcentration, pH, duration of treatment and toxicity wereexamined in a series of 33 preliminary tests which are sum-marized in Table I. Controls which were run in each study arenot shown. The controls were exposed to the same concentra-

610 Vol. 50, 1972

.

.......

104I..% ".-

.:.:.




Table I. Viability of Allium anid Rhoeo Epidermal Cells as Relatedto DSA Conicentrationi, pH, anid Diaration of Treatment

DSA HConcnl PH2

Allium1.0 3.8 (u)

10 4.05 (u)0.01 5.0 (u)0.00115.6 (u)

Rhoeo1.0 3.8 (u)1.0 i4.2 (a)0.1 4.2 (u)1.0 5.2 (a)0.01 15.2 (u)1.0 l5.5 (a)0.00115.5 (u)1.0 6.0 (a)1.0 !6.2 (a)1.0 6.2 (a)

Treat-iConcn of Dose ofTrent- Undisso- Undisso-Tmet ciated ciatedTime DSA3 DSA4

1tf?n 7531 nim X Injin

30 0.83 2530 7.3 22090 10.002 0.18090 0.000041 0.00371

30 i0.8330 0.6660 0.06660 .0.1360 0.001367 0.0660 0.0000645 10. 0170 0.005125 0.005

25204.080.0840.0040.450.350.625

CellViability RCD5

% %70of control

00

100100

00

10020-6010060100100100100

.6

-6

6

-6

-51.17-6

+40.0+22+146

lDSA was applied in a 0.5 M mannitol solution.2 (a) = pH of DSA solution adjusted with NaOH or KOH;

(u) = pH of DSA solution unadjusted.I Calculated with equation 1.4Product of concentration of undissociated DSA (mM) and

the treatment time (min).5 Increase in deplasmolysis time in the cells of treated tissue

versats control (positive RCD values) indicates decrease in waterpermeability by treatment and vice versa.

6 Not measured.7Decrease in deplasmolysis time (increase in water permea-

bility) is related to cell damage (note lower cell viability).

(unadjusted) killed all cells. Freshly detached Rhoeo leaveswith cut bases immersed in 1.0 mm DSA at pH 3.8 sustainedgeneral injury. Control cells in distilled water at pH valuesabove 3.5 were never injured. Allium cells generally were moresensitive than those of Rhoeo to variation in DSA concentra-tion, treatment time, temperature, and pH.

These results are especially noteworthy since previous workwas conducted with 1 mm DSA at pH 3.65 (14) or at un-specified pH values (9, 10). Although species tolerance to DSAprobably differs, these findings support Newman and Kramer's(14) contention that injury to roots may account for the DSAeffects observed by Kuiper (9).

Effects of DSA on the Water Permeability of Cells. Rhoeoepidermal cells were partially injured and deplasmolyzed fasterwhen the treatment pH was below 5.2 to 5.5. An increase inpermeability without detectable cell damage was very rarelyobserved.

In order to evaluate the influence of DSA on uninjured cellsmost permeability studies (37 studies on Allium and 35 onRhoeo) were conducted at neutral pH over a wide range ofDSA doses. The results are summarized in Figures 3 and 4.The experimental material consisted primarily of dormantGranex onions which had been stored at 7 C, and fresh, de-tached leaves of Rhoeo, respectively. Deplasmolysis times forcontrol cells ranged from about 1 to 6 min for Allium and 15to 25 min for Rhoeo.

Relative changes in deplasmolysis time shown in Figures 3and 4 indicate that DSA treatments almost always decreasedpermeability to water of both Allium and Rhoeo cells. Rareexceptions (negative RCD values in Figs. 3 and 4) were in-variably associated with partially damaged tissues in whichonly some of the cells were replasmolyzable. A trend towarddecreasing permeability with increasing DSA dose was ap-

Dose of total DSA (mM x min)

tion of mannitol (0.5 M) adjusted with HCI to the pH measuredin the treatment, and there was 100% cell survival in all con-trols. Cell viability was determined by replasmolysis. The pHof DSA treatments was in some experiments adjusted withNaOH or KOH to pH values indicated.The data of Table I show that a threshold concentration or

dose (concentration X time) of the undissociated DSA existsfor survival of the cells. This is also illustrated by the smoothgraph obtained, when the dose of undissociated DSA is plottedversus survival (Fig. 6, part 1). Partial or complete injury alwaysoccurred in the concentration range 0.06 to 7.3 mm (4-200[mm X min]), while concentrations of 0.00006 to 0.06 mM un-dissociated DSA (0.0037-4.0 [mM X min]) did not causeinjury. Figures 3 and 4 indicate that the duration of treatmentaffects significantly the RCD values. It is therefore assumedthat neither concentration nor treatment time alone but ratherthe dose determines the effect on the cell.

Total DSA doses, disregarding dissociation, were not relatedto viability; e.g., in the various studies, total DSA doses over arange of 30 to 300 ( mM X min) killed all cells and total dosesover a range from 0.06 to 125 (mM X min) caused no apparentinjury.At the concentrations used, highly dissociated DSA was not

injurious to cells. This is illustrated by examining the survivalof Rhoeo cells treated with 1.0 mm DSA at pH values of 3.8,4.2, 5.2, 5.5, 6.0, and 6.2. At a pH adjusted to 6.2, there wasno injury even when treatment time was extended to 125 min,whereas 30 min of treatment at pH 4.2 (adjusted) or 3.8

120

100

80

c

v*Y

60

40

20

10 20 30 40 50 60 70 80

5 10 1 5 20 25 30 35 40

Dose of undissociated DSA (pjM x min)

FIG. 3. RCD of DSA-treated Allium epidermis (pH about 7;total DSA concentration applied: 1 mM). i: Extreme values forRCD: a = 336; b = 340. 0: RCD values from experiments with50 ,AM total DSA concentration at pH about 7 calculated as meanwith the following values:Time of treatment ............... 75 60 33 30 33

RCD ................. 69 50 58 50 46

,a

* 0I,-,

- 0

0

- 0~~~~

*

611

I lIb



LEE, STADELMANN, AND WEISER

Dose of total DSA ( mM x min)

c

vQ

r-

Dose of undissociated DSA (pM x min)

FIG. 4. RCD of DSA-treated Rhoeo epidermis (pH about 7; total DSA concentration applied: 1 m.t). The graph indicates the approx-mate relation between dose applied and RCD.

O RCD values from experiments with 50 AM total DSA concentra-tion. Calculated as mean with the following values:

Time of treatment (min) 33 30 67 30 30RCD. 11 0 100 90 100

parent in both Rhoeo and Alliumn, but was more pronounced inthe latter. The greatest decrease in permeability (RCD about+100 for Allium and +130 for Rhoeo, see Figs. 3 and 4) wasobserved at doses of 0.5 M undissociated DSA for 70 to 100min. With Rhoeo, where higher doses were also tested, theRCD decreased in two experiments where the dose was greaterthan 60 (,xM X min), probably indicating that permeabilityincreased with greater doses.

In order to obtain a quantitative evaluation of the timecourse of deplasmolysis, eight experiments were conducted inwhich time-lapse photomicrographs were taken of tissues dur-ing deplasmolysis at 5- to 10-sec intervals (see Fig. 2). De-plasmolysis frequencies of cells in four of these studies aregiven in Figure 5. Each curve is based on observations of about200 cells. Only cells which were replasmolyzable were counted.Similar cell counts have been previously described forplasmolysis frequency only (15).A and B of Figure 5 show the typical decrease in water

permeability in healthy Allium and Rhoeo cells induced byDSA at room temperature. The curve for control tissue usuallywas much steeper than for DSA-treated epidermis, indicatinghigher uniformity of the control cells. Figure SC shows thesimilar results which were obtained when the DSA treatmentwas administered at 0.5 C with subsequent perfusion at 12 to15 C. Figure SD illustrates the kind of anomalous resultswhich were obtained when the treated epidermis was injuredby a high dose of undissociated DSA. In this experiment only60% of the cells were replasmolyzable.

In contrast to Kuiper's findings (9), the data of the presentstudy indicate that DSA decreases rather than increases water

0: RCD values from experiments with 5 ,uM total DSA concentration.Calculated as mean with the following values:Time of treatment (min) 30 33 60 66 120 180RCD. 3 -5 4 7 -3 0

permeability of cells and that the DSA response does not ap-pear to alter the temperature dependence of permeabilityphenomena. These observations agree with Newman and Kra-mer's view (14) that the DSA-induced increase in waterpermeability described by Kuiper probably was an artifact.We did not observe any temperature-dependent effects of

DSA (see ref. 9) in additional studies in which the treatmentswere administered at 0 + 1.5 C, and the plasmolyzing anddeplasmolyzing solutions were cooled to between 10 C and15 C. As expected, the low temperatures reduced permeability(increased deplasmolysis time), but the reduction was es-sentially the same in both treated and control cells.

Cytomorphological Effects of DSA Treatments at NeutralpH. Doses of undissociated DSA (0.004 [mM X min]) causedextremely concave plasmolysis in Allium and Rhoeo and theappearance of numerous thick protoplasmic strands. Thesefeatures were observed much more frequently in treated cellsthan in control cells and less frequently at low doses than athigh doses. Low doses of DSA which did not affect deplasmoly-sis time generally did not alter plasmolysis forms. At dosesof 0.004 (mM X min) undissociated DSA, deplasmolysis wasoften incomplete, probably as a result of an increase in proto-plasmic viscosity. This indicates that DSA may have led toirreversible changes in the membranes and/or protoplasm.Low temperatures caused concave plasmolysis in both treatedand untreated cells, indicating increased protoplasmic viscosity.Swollen protoplasts were occasionally observed in Allii,m cellsat doses greater than about 2 ([cM X min) undissociated DSA.The swollen protoplasm usually persisted until the final stagesof deplasmolysis, disappearing when turgor pressure developed.

612 Plant Physiol. Vol. 50, 1972




A B

D 40 80 120 160 200 240

Deplasmolysis time in sec

-oVW= 80

t 6000a 40-

C0 20

0CLP-

7I -f I* 0-_

*-_ ~~~**

. I-0____ ---- L I1

.- ~~I I

.1

II

_ * I, ,l,,I III

- 11I

S

0001I

8 10 12 14 16 18 20 22

Deplasmolysis time in min24 26 28 30

clii l ll11 l

DO - * 0

---0 -*--- 0--80 _ * * - _

60 _*40 -*

* ~ ~ ~~ AII

30 34 38 42 46 50 54 58 62 66

Deplasmolysis time in mint FIG. 5. Time course of deplasmolysis frequency. *: Control; 0:treatedcoordinates for 50%!0andj90%7 deplasmolysis time.

110 IC

-5

u

44

0

%*60

0~

D

D0_ * ._~~*0

10

so

60 _ *11

_____ _____ __-Ii

ao-~~~~~~~

8 10 12 14 16 18 20 22

Deplasmolysis time in minepidermis. Concentration of DSA applied: 1 mM. Dotted lines

Treatment (dose)Diagram MIaterial Temperature pH Time

Total DSA |UndissociatedDSA

C min mm X minA Allium 22-24 7-8 50 50 0.025B RhoeO 22-24 7-8 60 60 0.030C Rhoeo 0.5 7-8 65 65 0.033D Rhoeo 22-24 4.4 60 50 34

Hernial protuberances of the protoplasm occurred frequentlyduring deplasmolysis, indicating hardening of external proto-plasm. The protuberances rarely led to a bursting of the proto-plast, and disappeared as deplasmolysis progressed (cf. 18, p.

54 and ff.). The osmotic ground values (19) of cells werenormally not affected by DSA treatment except at low pH asexosmosis occurred.

Effects of DSA on Frost Resistance. In a controlled freezingexperiment on intact Rhoeo plants the temperature in a con-trolled environment chamber was lowered on successive nightsto progressively lower freezing temperatures for 3 hr duringthe middle of the dark period. Control plants and plants thathad been treated with DSA for 21 days prior to freezing stresswere uninjured by exposure to -0.9 C and -1.4 C, but allwere severely injured at -4 C. DSA did not appear to inducea striking increase in frost resistance as reported by Kuiper(10).

DISCUSSION

The analysis of DSA influence at the cellular level indicatedthat its effects are more complex than previously suggested

(9). Many DSA treatments caused death of cells if the pH wasnot adjusted to neutrality. If the pH was adjusted to neutrality,or the dose was low enough to avoid killing the cells, DSA re-duced permeability of cells to water. At marginally injuriousconcentrations, DSA seemed to increase water permeability ofcells which survived the treatment. Dead cells, of course, arehighly permeable to water.

Toxicity of DSA was closely related to the pH of the DSAsolution. This relationship, and the DSA effects on per-meability, can be explained quite simply if it is assumed thatundissociated DSA molecules are the "effective" form that canpenetrate the plasmalemma and enter the mesoplasm, whileDSA ions either cannot penetrate or can enter only to sucha limited extent that no alteration occurs in the cell. This idea issupported by the data when survival, permeability, and cyto-morphological changes are plotted against the dose of undis-sociated DSA (Fig. 6). Plots of these data against the total ordissociated DSA dose do not reveal such clear-cut relation-ship. For example, there was little or no DSA toxicity at thehighest total DSA ion doses tested in these experiments. Pref-erential penetration of the undissociated molecule of a dis-sociable organic compound through the plasmalemma and

*

* 0._ * 0 _

* 0 -

*

- 101*0

;1 0 1-

100

80

60

-5lao0

0.#A 20a

0 O

-i2v

613

0

001 1(a

-

0

v

w

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2



614 LEE, STADELMANN, AND WEISER

Increase

2) /

I I I II - IL II11

Cellsdead

111111 l~~IIII .-~I II I I1II1I1

Decrease

3) ConcaveConvex

I1 I1111 11 11 111 li11 11 111 10.001~~~~~~~~~~~1

0.001 0.01 0.04 0.1 0.4 1 2 6 10

I, U

Dose of undissociated DSA (mMx min)

FIG. 6. Effect of DSA treatment on survival, permeability, andplasmolysis. 1: Survival of epidermal cells tested by plasmolysis.*: Rhoeo discolor, leaf, abaxial surface. *: Allium cepa, bulbscale, adaxial epidermis. *: Phaseolus vulgaris, root (tested bygrowth; calculated from Newman and Kramer's data; (14 p. 608).2: Assumed relation between doses and permeability changesderived from the results of these experiments (- - - no experimentsavailable in this dose range). 3: Dose limits for observation of con-cave and convex plasmolysis forms in Allium. 4: Dose range foroccasionally observed swollen protoplasts in Alliunm.

tonoplast is common in the case of vital stains (cf. 6), fusaricacid (1, 5 p. 342), and other acids and bases (2, p. 13 and ff., 16)and is closely related to their lipid solubility. The passive per-

meability of the plasmalemma to ions is very low (20) even forsmall ions. The toxicity of DSA to plant roots has been pre-viously discussed by Newman and Kramer (14) and Kozlowskiand Clausen (7, 8).Low doses of undissociated DSA generally caused a de-

crease in permeability of cells to water, whereas high doses in-creased permeability. This indicates that more than one process

may be involved in water permeability changes induced byDSA. Similar reversal of effects is known to occur in the per-

meability of cells treated with ionizing radiation (25, p. 490),fusaric acid (5, p. 352-353), metabolic inhibitors (cf. 14), andother substances. The reason for these reversals may lie in al-terations of the membrane structure; e.g., decrease in waterpermeability could be caused by changes in the ratio of themicellar to leaflet configuration in the lipid component of themembrane (cf. 20). Narrowing or decrease in the number ofassumed water channels around the globular membrane pro-

teins (cf. 22; G. Vanderkooi, 1971, personal communication)could also cause the lower water permeability. Increased per-

meability to water at high doses may be the manifestation ofchanges associated with partial injury of the membranes.The observation of cytomorphological changes induced by

DSA provides a basis for speculating about DSA effects at thesubcellular level. For example, the concave plasmolysis formsobserved in treated Allium cells may indicate that DSAstrengthens the binding of membrane proteins to the cell wall,thus impeding separation during plasmolysis. Hernial pro-

tuberances of the protoplasts of treated cells indicated thatthere may have been an alteration (hardening) of the external

Plant Physiol. Vol. 50, 1972

protoplasm. Concave plasmolysis indicates that DSA may havealso increased protoplasmic viscosity. The incomplete de-plasmolysis which was sometimes observed in DSA-treatedcells may also be attributable to increased protoplasmic vis-cosity. The swelling of protoplasm which was associated withDSA treatments further suggests that DSA may affect inter-molecular bonding in the ground plasm, perhaps by increasingthe water binding of macromolecules. This, however, is largelyconjectural since the molecular factors which are responsiblefor determining protoplasmic qualities such as wall attachmentor viscosity have not been resolved.

In the present study Allium and Rhoeo cells reacted similarlyto DSA treatment although Rhoeo cells were generally moreresistant than Allium, and cells from dormant Allium bulbswere more resistant to injury by DSA than cells from growingbulbs. Species differences have been previously observed byNewman and Kramer (14). The toxicity of DSA and our in-ability to demonstrate any DSA effect on temperature de-pendence of permeability or on frost resistance raises questionsabout conclusions reached in studies in which the pH of DSAtreatments was not controlled and where no observations weremade on cell viability following treatment.

Acknowledgments-The auithors wish to thank Drs. T. T. KozlIowski, Univer-sity of Wisconsin, P. J. Kramer, Dtuke University, and J. Lev-itt, Columbia Uni-versity for their suggestions and reviews of this paper.

LITERATURE CITED

1. BACHMAN-N, E. 1956. Der Einfluss von Fusarinsijure auf die Wasserperme-abilitiit von pflanzlichen Protoplasten. Phytopatholog. Z. 27: 255-288.

2. BRENNER, W. 1918. Studien tiber die Empfindlichkeit und Permeabilitatpflanzlicher Protoplasten fiir Siuren uncd Basen. Ofv-ersigt Finsk Vetensk.-Soc. Forhandl. 60 A (4) :1-124.

3. FITTING, H. 1915. Untersuchungen ilber die Aufnahme von Salzen in dielebende Zelle. Jahrb.Wiss. Botan. 56:1-64.

4. Green, D. G., W. S. FERGUSON, AND F. G. WARDER. 1970. Effects of dlecenvl-succinic aeid on 32p uptake and translocation by barley and winter wheat.Plant Physiol. 45: 1-3.

5. HEINRICH, G. 1962. Fusarinsaure in ihrer Wirkung auf die Wasserperme-abilitiit des Protoplasmas. Protoplasma 55: 320-356.

6. KINZEL, H. 1968. Vital staining of plant cells as a model of biologicaltransport and accumtulation phenomena. Abhandl. Detut. Aklad. Wiss.Berlin.1KI. Mledizin. 1968(4a) 205-215.

7. KOZLOwSsI, T. T. AND J. J. CLAUSEN. 1967. Effects of alkenylsuccinic acidsof moisture content of woody plants. Plant Physiol. 42: S-17.

8. KOZLOwN-SKI, T. T. AND J. J. CLAUSEN. 1970. Effect of decenylsucinic acidon neeclle moisture content and shoot growtlh of Pizius rcsiinosa. Can. J.Plant Sci. 50: 355-356.

9. KUIPER, P. J. C. 1964. Water transport across root cell membrane. Effect ofalkenylsuccinic acids. Science 143: 690-691.

10. KuipER, P. J. C. 1964. Inducing resistance to freezing and desiccation inplants by decenylsuecinic acids. Science 146: 544-546.

11. KUIPER, P. J. C. 1965. Temperature dependence of photosynthesis of beanplants as affected by decenylsuccinic acid. Plant Physiol. 40: 915-918.

12. KUIPER, P. J. C. 1967. Surface-active chemicals as regulators of plantgrowth, membrane permeability. ancl resistance to freezing. M\Ieded. Land-bouwhogesch. Wageningen 67-3:1-23.

13. LEE, 0. Y. 1967. The influence of decenylsuccinic acid on permiieabilityof plant cells to water and its possible effect on frost hardiness. Mastersthesis. University ofSMinnesota, St. Paul.

14. NEWMAN, E. I. AN-D P. J. KRA-MER. 1966. Effect of DSA on the permeabilityand growth of bean roots. Plant Physiol. 41: 606-609.

15. SCHaMIDT, H., DIWALD, K., AND 0. STOCKER. 1940. Plasmatischle Untersuchutngenan diirreempfindlichen tund diirreresistenten Sorten landwirtschaftlicherKtulturpflanzen. Planta 31: 559-596.

16. SINiON, E.W. AND S. H. BEEVER. 1951. The effect of pH on the biologicalactivities of weak acids and bases. I. The most usual relationship betweenpH and activity. New Phytol. 51:163-197.

17. STADELMANN, E. 1956. Plasmolyse undl Deplasmolyse. In: W. Ruhland, ed.,Encyclopedia of Plant Physiology. Vol. 2. Springer, Berlin. pp. 71-115.

18. STADELMANN, E. 1964. Zu Plasmolyse und Deplasmolyse von Alliam-Epider-men. Protoplasma 59:14-68.

19. STADELMAN-N, E. 1966. Ev-aluation of turgidity, plasmolysis, and deplasmolysis.In: D.M. Prescott, ed., Mlethods in Cell Physiology, Vol. 2. AcademicPress, New York. pp. 143-216.

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20. STADELMANN, E. 1969. Permeability of the plant cell. Annu. Rev. PlantPhysiol. 20: 58-606.

21. STADELMAXNN, E. AND J. WATrENDORFF. 1966. Plasmolysis and permeabilityof alpha-irradiated epidermal cells of Allium cepa L. Protoplasma 62:86-116.

22. VAmNDERKOOI, G. AND D. E. GREEN. 1971, New insights into biological mem-

brane structure. Bioscience 21: 409-415.23. VAN- STEVENINCK, R. F. M. 1965. The significance of calcium on the apparent

permeability of cell membranes and the effects of substitution with otherdivalent ions. Physiol. Plant. 18: 54-69.

24. VRErGDENHIL, D. 1957. On the influence of some environmental factors on

the osmotic behavior of isolated protoplasts of Allium cepa. Acta Bot.Neer. 6: 472-542.

25. W\ATTENDORFF, J. 1966. Dosisabhangige Xnderungen der Harnstoffpermeabi-litat unmittelbar nach alpha-Bestrahlung von Allium-Epidermiszellen. Bi-olog. Zbl. 85: 455-495.

26. WEIGL, J. 1967. Struktur und Funktion pflanzlicher Membranen. Fortschr.Botan. 29: 50-67.

27. WERTH, W. 1961. Vergleichende Untersuchungen uber die relative Permeabili-tiit des Protoplasmas fur Alkohol und Wasser. Protoplasma 53: 457-503.

28. ZELITCH, I. 1964. Reduction of transpiration of leaves through stomatal clo-sure induced by alkenylsuccinic acids. Science 143: 692-693.

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