PLANT PHYSIOLOGYVOLUME 34 JULY, 1959 NUMBER 4

STUDIES ON AUXIN-MEMBRANE PERMEABILITY RELATIONS INFRUIT AND LEAF TISSUES 1

JOSEPH A. SACHER2DEPARTMENT OF BIOLOGICAL SCIENCES, Los ANGELES STATE COLLEGE, Los ANGELES, CALIFORNIA

In a previous paper (22) I reported on the rela-tionship between auxin and membrane permeabilityin bean endocarp tissue and in abscission phenomenain Coleus. Segments of the pods of KentuckyWonder pole beans, prepared in the manner describedfor the Wehnelt bean test (4), remain rigid and plumpup to 17 days when stored in the dark at 25° C insolutions of 4 to 50 ppm naphthalene acetic acid(NAA) or indoleacetic acid (IAA). In contrast,control segments in distilled water become soft andflaccid in 3 days. A study of hand sections disclosesthat in auxin-treated segments the intercellular spacesof the endocarp are filled with air (a normal situa-tion), while in the control segments the spaces arefilled with liquid. This suggests that the increasedlongevity of bean segments in vitro results from aneffect of auxin on maintenance of membrane proper-ties. In the absence of added auxin movement ofsolutes and water into the intercellular spaces results.This water-logging of the spaces is followed by dis-solution of the middle lamella (cellular dissociation).

Permeability changes occur only in the endocarp(fig 1) of the bean segments, not in the exocarp.The different responses of adjacent tissues suggestthat permeability changes could be involved in ab-scission phenomena. Experiments with Coleus showthat 2 to 3 days after deblading, water-logging of theintercellular spaces and cellular dissociation occurin the abscission zone, followed by abscission. IAAapplied distally to debladed petioles prevents thesephenomena, supporting the hypothesis that auxin in-hibits abscission by maintaining normal membraneproperties.

This paper reports phenomena in fruit and leaftissues in vitro which contribute to an understandingof the role of auxin in delaying tissue senescence.

Received July 29, 1958.2 This work was supported by a Research Grant (RG73) from the Public Health Service, and was conducted

in the Division of Biology, California Instogy, Pasadena, California.litute of Technol-

METHODS AND EXPERIMENTAL RESULTSBEAN SEEDS AS A SOURCE OF AUXIN: It has been

reported that IAA occurs in young bean seeds (19).The following experiments were performed to ascer-tain if seeds function as a source of auxin in the ma-turing bean fruit. Bean pericarp segments were pre-pareAd with the seed removed and with the seed at-tached. The segments were kept on distilled water-moistened filter paper in Petri dishes at 25° C in thedark. Water-logging of intercellular spaces in theendocarp occurs in 2.5 to 3 days in deseeded segments,but not until 5 days in segments with the seed attached.The effect of the seed in these studies is comparableto the effect of IAA at a concentration of 4 ppm. Theresemblance between the effect of the attached seedand of applied auxin in preservation of membraneproperties suggests that in the bean fruit the seeds area source of auxin, which acts to retard the occurrenceof permeability changes. Observations on wholebeans during storage show that the endocarp under-goes permeability changes and cellular dissociation,occurring 1st in the tissue between the seeds, and withcontinued aging progressing to the region where theseed is attached.

EXUDATION IN ENDOCARP TISSUE: It is con-sistently observed that conspicuous droplets of liquidappear on the upper and lateral surfaces of water-treated segments (fig 5). This is manifest after 4to 5 days, or 1 to 2 days after water-logging occursin the endocarp. This phenomenon does not occurin auxin-treated segments. These observations sug-gest the following explanation. The alteration inmembrane properties in water-treated segments en-ables the leakage of solutes into the cell wall and inter-cellular space regions, which is accompanied by out-ward diffusion of water. When the intercellularregion becomes filled with liquid, the continued move-ment of water into the intercellular region results indisplacement of some of the intercellular contents asa surface exudate 3. Since cellular dissociation isevident during the period when exudation occurs, the

3 A subsequent publication will report on analyses ofthe exudate and pectin changes.

365

PLANT PHYSIOLOGY

{

q X .4.

.ffij.e ^ > . .;2gat , ,.!': .. :

... .:. 3'* ' :::eFIG. 1. Hand section of bean segment after 3 days

of water-treatment. The intercellular spaces in only theendocarp are water-logged. The exocarp shows air-filledintercellular spaces (they appear black, owing to reflectionphenomena). X 50.

FIG. 2. Hand section of a Rhoeo leaf slice after 28days of auxin-treatment to illustrate the abundance of in-tercellular air in the spongy chlorenchyma. Anthocyaninsshow in the abaxial epidermis (bottom). X 50-

FIG. 3. Hand section of a Rhoeo leaf slice after 5 days

of water-treatment. Intercellular spaces water-logged;anthocyanins have been lost by exosmosis. X 50.

FIG. 4. 1 mm thick Rhoeo leaf slices after 28 days

of auxin-treatment. The slices show no evidence of per-

meability changes as evidenced by their turgid, healthyappearance, absence of liquid-logging of air spaces, andretention of anthocyanin pigments. X 10.

(legradation of pectin in the middle lamella may be a

factor contributing to the osnmotic pressure of the in-tercellular mediunm.

PLAS.MOLYTIC STUDIES: The ability of cells to

unidergo plasmolysis and deplasmolysis has often beenusecl as a criterion of selective permeability of cellmembranes (6). In the following experiments theosmotic behavior of protoplasts of cells in whichchanges in pernmeability had occurred (water-treatedendocarp) is compared with cells of fresh en(locarpand auxin-treatedI endocarp in which there is no ap-

parent in(lication of alteration in menmbrane proper-

ties.Plasmolytic studies were made of hand sections of

the endocarp of fresh bean pods, of endocarp after3 to 7 days of water-treatment, an(d of endocarp after3 to 7 (lays of auxin-treatment. The plasmolvticagent used was 0.75 M\ sucrose; 0.1 M sucrose was

use(l for deplasmolysis. Under each of the 3 condi-tions plasmolysis and (leplasmolysis is observedthrough 2 cycles. Despite the evidlence for alterationin membrane properties in water-treated endocarp,the protoplasts retain characteristics of (lifferentialpermeability. It became obvious, however, that plas-mloly-sis occurs much more rapidly in water-treatedthan in auxin-treate(l tissue. The following pro-

cedure wras used to determine the rate at wlhich plas-molysis occurs in fresh, auxin- and wvater-treatedendocarp.

The time for plasmolysis was miieasuredI from thetime that 0.75 M\1 sucrose was addedl at the e(dge of thecoverslip, and drawn through using filter paper, untilthe protoplast had completely withdrawn from thecell wall. Deplasmolysis was recorded from the timeof adding 0.1 M sucrose until the protoplast resumedcontact with the cell wall. Observations on the rate

of plasmolysis were made on cells at the edges of thesections. This procedure minimizes the effect ofvariation in thickness of hand sections, which have tobe a few cell layers in thickness to ensure cells unin-jured by sectioning.

The more rapid rate of plasmolysis of water-treatedthan of fresh or auxin-treated endocarp is illustratedin table I. The plasmolytic studies were repeated on

numerous batches of beans over a period of 12 months.The lack of uniform response in auxin-treated seg-

ments among different experiments is probably at-

tributable largely to batch-wise variation in beansand their ability to respond to auxin. Regardless ofthe variability, it is clear that the time required for

366l

SACHER-AUXIN-MIEMBRANE PERMEABILITY IN TISSI'ES

TABLE IRATES OF PLASMOLYSIS IN FRESH, WATER- AND

AUXIN-TREATED ENDOCARP

TiSSUE TIME FOR TIME FORTISSUE PLASMOLYSIS* DEPLASMOLYSIS

Fresh endocarp 15-25 minutes 10-15 minutesWater-treated 15 seconds toendocarp 3 minutes 1-3 minutes(3-7 days)

Auxin-treatedendocarp 4-20 minutes 4-15 minutes(3-7 days)* Ranges of observations on rate of plasmolysis based

on 8 samples of fresh tissue; 18 of water-treated tissue;15 of NAA-treated tissue.

plasmolysis of water-treated tissue is significantly lessthan for fresh or auxin-treated tissue. The increasedrate of plasmolysis in water-treated tissue providesfurther evidence of a change in permeability.

An interesting observation associated with addi-tion of 0.75 M sucrose to sections of water-treatedendocarp is the instantaneous and marked increase inrefractivity of the protoplast, followed by plasmolysisin from 15 seconds to 3 minutes. The rapid increasein refractivity of the protoplast may be related to agreater permeability of the plasma membrane than ofthe tonoplast. MacRobbie and Dainty (15) reportthat in Nitellopsis flux through the plasma membraneis 20 to 100 times that through the tonoplast. Itseems that the increased refractivity depends upona change in cytoplasmic state, for it precedes the oc-currence of visible contraction of the protoplast(plasmolysis). WVhether the rapid loss of waterfrom the cytoplasm or rapid entry of the plasmoliticuminto the cytoplasm is the cause of the change in re-fractive index of the cytoplasm has not been deter-mined. In fresh tissue, increase in refractivity takesca. 20 minutes, attending retraction of the protoplastfrom the wall.

The effect of cellular dissociation on severing cvto-plasmic connections between cells does not appear tohave a significant effect on the rate of plasmolvsis,as plasmolysis of dissociated cells is initially crenulate(as in fresh tissue), suggesting attachment of the pro-toplast to the cell wall. Plasmolytic studies of endo-carp cells after 3 days and after 4 to 7 days of water-treatment reveal no consistent difference in the timerequired for plasmolysis, even though cellular dis-sociation is very conspicuous during the latter period.These observations suggest that the difference inmagnitude between rates of plasmolysis in water- andauxin-treated segments is not appreciably affected bycellular dissociation in the former.

The occurrence of water-logging of air spaces,conspicuous exudation, and the increase in rate ofplasmolysis in water-treated bean endocarp provideclear evidence of an alteration in membrane permea-bility, a process which is delayed by an exogenoussource of auxin. It is apparent, however, from theresponse to a plasmolytic medium that the membranes

still exhibit differential permeability. It may be in-ferred that the ability of cells to undergo plasnf6lysisand deplasmolysis may not give a full incli6at-ibn ofproperties of cell membranes unless the rate factoris considered.

c

FIG. 5. Dish on left shows bean endocarp segmentsafter 5 days of water-treatment. Note the droplets ofliquid exudate onl surface of segmeints. Dish oni rightshows segments after 5 days of auxin-treatmenit, with noexudation. X 3/8.

FIG. 6. HaInd sectioIn of a leaf of -Mesembryanthemum.Description in text. X 20.

FIG. 7. Han1d section1 of Rhoeo leaf (nmidrib region)depicting the colorless, compact adaxial tissue, centralspongy chlorenchyma (appearing black due to extensiveair-filled intercellular spaces), and the adaxial tissue zonecontaining anthocyanins. X 40.

-367

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4

PLANT PHYSIOLOGY

RESPIRATORY STUDIES: When bean segments areprepared without asepsis the water-treated segmentssuccumb to bacterial contamination after 5 days, or2 days after the completion of water-logging. Incontrast, auxin-treated segments do not become con-taminated until 12 to 20 days elapse (22), again fol-lowing the ultimate change in membrane properties.It seems that contamination results from the favorablesubstratum provided by the leakage of cellular sub-stances. Experiments performed with asepsis revealthat cells of water-treated endocarp appear normalcytologically over a 10-day period, notwithstandingthe dissociation of the endocarp into a loose mass ofcells. Thus it seemed attractive to conduct respira-tory studies on fresh and on water- and auxin-treatedendocarp to ascertain the effect of alterations in per-meability on rate of respiration.

Bean segments were prepared aseptically with exo-carp and mesocarp removed and kept in the dark at25° C in Petri dishes on filter paper moistened withdistilled water or a 50 ppm solution of NAA. Theremoval of the outer tissues provided segments ofhomogeneous endocarp for respiratory studies. Seg-ments were removed at 1-day intervals, quartered,blotted, weighed and placed into Warburg vessels.Samples were run in triplicate, each vessel containing300 to 600 mg of tissue.

It can be seen from a comparison of the curves infigure 8 that except for the 1st 3 days the Q 02 forthe auxin- and water-treated endocarp is similarthroughout the experiments (18). There occurs agradual decline in the rate of respiration until the6th day at which time the rate levels off. Similarresults were obtained in further experiments. RQdeterminations yield like results for both treatments.,ranging from ca. 1.11 at the beginning of the experi-ment to 1.06 on the 6th day. There is no effect onoxygen uptake of sucrose (0.04 M), glucose (0.02 M)or 0.02 M phosphate buffer at pH 7, or combinationsthereof, whether the materials are exposed to theadded substrates for a 2-hour period or overnight.In both water- and auxin-treated endocarp, however,there is an increase in the RQ (1.06 to 1.12) whensucrose is added (2). These results demonstratethat endocarp in which permeability changes and cellu-lar dissociation had occurred may exhibit a rate of

FIG. 8. Endogenous respiration of bean endocarp seg-

ments.I

respiration equal to that of tissue of similar agetreated with auxin to maintain membrane properties.As the following experiments show, however, water-treated segments undergo complete loss of protoplas-mic organization and respiratory activity over longerperiods of time, becoming disorganized at least 20clays prior to auxin-treated segments.

Auxin- and water-treated endocarp segments pre-frared as previously described were stored in the darkat 250 C for 5 days and then at 50 C in the dark for20 days. At the end of this period respiratory studieswere made 2 hours after removal of the materials fromcold storage. The water-treated segments show nooxygen uptake. Further, a study of hand sectionsreveals protoplasmic disorganization. In contrast,auxin-treated segments show an uptake of 80 ul0.,/g/hr, a rate comparable to the level exhibited bysegments during days 7 through 9 (fig 8). A similarQ O., (133 dul 0 /g/hr) is shown by auxin-treatedsegments after more prolonged storage, namely, for5 days at 250 C and 40 days at 50 C. No respiratorymleasurements were made on auxin-treated segmentsbeyond 45 days, but their appearance up to 60 dayssuggested that they had biological activity.

The conditions of the above experiment provide ameans of analyzing the effect of permeability changeson longevity of bean segments. The initial storageperiod at 250 C provides adequate time for the occur-rence of permeability changes and cellular dissociationin water-treated segments. The subsequent coldperiod a) reduces the possibility of contamination andb) lowers metabolic activity so that any effect ofpermeability changes on leakage of cellular materialsis evident before the depletion of metabolites limitsbiological organization. The auxin-treated segmentsexhibit respiratory activity 20+ days longer than dowater-treated segments. It seems plausible to regardpermeability changes in water-treated segments aspermitting excessive leakage of cellular materials,wlhich could bring about deficiencies of metabolitessufficient to cause cessation of biological activity.As a partial test of this hypothesis the following dryweight determinations of the diffusate from water-and auxin-treated segments were made. The termdiffusate refers to solutes which leak from the seg-ments into the external medium.

DIFFUSATE STUDIES: Bean endocarp segmentswere prepared aseptically and stored as describedabove (i.e., in the dark at 25° C for 5 days; thereafterin the dark at 50 C). In view of variations in theinitial size and water content of trimmed segments,the most reliable estimate of the relative amount ofleakage may be obtained from comparison of the ratioof the final dry weights of diffusate to segments foreach of the batches. To this end, 14 segments ofwater- and auxin-treated tissue respectively were re-moved at 7, 14 and 21 days for determination of dryweights of segments and diffusates. The diffusateswere washed from the Petri dishes with distilled water,filtered through Whatman no. 1 filter paper, anddiffusates and segments were dried in vacuum over

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369SACHER-AUXIN-'MEMBRANE PERMEABILITY IN TISSUES

sulfuric acid. The diffusates from auxin-treated seg-ments are clear, in contrast with the turbid-whitecharacter of diffusates from 14- and 21-day-old water-treated segments. The final dry weights of segmentsand diffusates are shown in table II.

As is shown in table I1 there is, over the 21-dayperiod, ca. 3 times more leakage from water-treatedsegments than from auxin-treated segments. Thesedata are consistent with the hypothesis that the earlierloss of respiratory activity in water-treated segmentsresults from excessive leakage, and that added auxin,through its effect in maintenance of membrane proper-ties, delays this process.

PERMEABILITY STUDIES ON LEAF TISSUES INVITRO: The evidence that auxin affects membranepermeability in bean endocarp tissue and in the abscis-sion zone in Coleus, prompted investigation of theeffect of auxin on leaf tissues. The leaves of Mesemiz-bryanthemumzmt sp. and Rhoeo discolor were used be-cause their thickness makes them adaptable to thestudy of fresh sections. It should be added thatRhoeo was also favored because of the anthocyaninpigments it contains.

Mesenmbryan thenium: Leaves were sterilized in10 % Clorox for 10 minutes and then cut transverselyinto segments 5 to 6 mm in length. The segmentswere transferred aseptically into Petri dishes on filterpaper moistened with sterile distilled water or a solu-tion of 4 ppm NAA, and stored in the dark at 250 C.The appearance of a fresh transection of the leaf isillustrated in figure 6. The central zone is occupiedby colorless mesophyll tissue having fairly prominentintercellular spaces. External to this is a layer ofpalisade chlorenchyma, limited outwardly by the epi-dermis.

In water-treated segments water-logging of inter-cellular spaces in the central zone occurs in 4 to 5days. Within 24 hours after water-logging the rigid-ity of this tissue is lost, and browning is apparent.

In contrast, treatment of the leaf segments with 4ppm NAA delays water-logging of the air spaces andbrowning until 9 days. This effect of auxin in main-tenance of membrane properties is best shown in seg-

ments of young leaves.

Rhoeo: Leaves were sterilized in 10 % Cloroxfor 10 minutes, after which a longitudinal segment ca.

12 mm in width containing the midrib was removedand cut transversely into slices 1 mm thick. Theslices were washed in sterile distilled water to re-move brei released from cells injured during section-ing. They were then placed aseptically into Petridishes containing distilled water or a 4 ppm NAAsolution and kept in the dark at 250 C.

Figure 7 illustrates a section of a Rhoeo leaf.The leaf consists of 3 horizontally arranged tissuezones. In the region of the adaxial surface there isa compact colorless tissue, 2 to 5 cell layers in depth,lacking intercellular spaces. The abaxial region con-sists of a compact tissue varying from 1 to severalcells in thickness, containing anthocyanin pigments.The central zone is occupied by photosynthetic tissuewith loose cellular arrangement and large intercellularspaces.

Within 6 days there are several obvious degenera-tive changes in water-treated Rhoeo slices. After 4days browning is evident in the colorless adaxialtissue region, and the intercellular spaces in the photo-synthetic tissue become water-logged (fig 3). Cellu-ular dissociation follows rapidly in the photosynthetictissue. By the 6th day the anthocyanins leak intothe medium. The identity of the pigments was dem-onstrated by chromatographic analysis. The dif-fusate was treated with 0.1 % HCl, concentrated bydistillation in vacuum and chromatographed on What-man no. 1 paper in a butanol: acetic acid: water(4:1 :5) solvent, along with a similarly concentratedcontrol consisting of pigments washed from freshleaf slices with 0.1 % HCl. The Rf values were thesame in all cases.

In contrast, auxin-treated leaf slices show no

browning, water-logging of the air spaces or loss ofanthocyanins for a period of 30 to 35 days. Figure 2depicts hand sections of Rhoeo leaf slices after 28days of auxin treatment. Figure 4 illustrates a groupof 1 mm thick leaf slices as they appear after beingmaintained on a solution of 4 ppm NAA for 28 days.

As in bean endocarp it seems clear that the addi-tion of auxin enables maintenance of normal mem-brane properties in Mesembryanthemum leaf segmentsand Rhoeo leaf slices. The 3 different changes, whichoccur in Rhoeo in the absence of added auxin, may

be attributed to permeability changes: a) The water-logging of the prominent air spaces in the photosyn-thetic tissue, followed by cellular dissociation, seems

LE II

ANALYSIS OF DIFFUSATE FROM NAA- AND WATER-TREATED ENDOCARP *

TREAT- 7 DAY 14 DAY 21 DAYMENT DRY \VT DRY WT PERCENTAGE** DRY WVT DRY WVT PERCENTAGE** DRY WT DRY WTT PERCENTAGE**

SEGM DIFFUSATE DRY WT SEGM DIFFUSATE DRY WT SEGM DIFFUSATE DRY WT

NAA 0.1271 0.0037 2.9 % 0.1150 0.0083 7.2 % 0.1272 0.0154 12.1 %H2O 0.110X 0.0095 8.6 % 0.1039 0.0187 18.0 % 0.0875 0.0312 356 %

* The figures are for batches of 14 bean segments, allweights in grams.

** Expressed as ratio of dry weight of diffusate to dryweight of segments.

PLANT PHYSIOLOGY

to parallel a similar effect observed in bean endocarpand in the abscission zone of Coleus. b) The leakageof anthocyanins resembles the loss of such pigmentsfrom beet tissue when its membranes are injured byheat or other agents (8). c) The browning reactionmay be attributable to permeability changes throughan effect on enzyme-substrate relationships, e.g.,polyphenols and polyphenolases (21).

It is of interest to note that the tissue containinga7thocyanins and the colorless tissue, both of whichare compact and devoid of intercellular spaces, do notexhibit cellular dissociation. Yet in each there oc-curs a phenomenon suggestive of changes in mem-brane permeability.

DISCUSSION

Experiments with segments of bean endocarp, andwith leaf slices of Rhoeo and Mesembryanthemum,provide clear evidence that auxin, long regarded as agrowth hormone, also effects the preservation of cer-tain nmembrane properties. To what extent auxinmay effect maintenance of membrane properties inroot andI stem tissues is being investigated. Theauxin-effects described in this paper are manifestedundler condlitions where little or no growth occurs.

The evidences of changes in permeability in theabsence of added auxin derived from studies on beanendocarp and certain leaf tissues are several, as fol-lows: 1) water-logging of intercellular spaces withina few days, 2) microbial contamination of tissues inabsence of asepsis, 3) a considerable decrease in thetime required for plasmolysis of bean endocarp, 4)conspicuous exudation at the surface of endocarp seg-ments, 5) the loss of anthocyanins from Rhoeo leafslices, 6) the occurrence of browning in Rhoeo andMesembryanthemum leaf tissues and 7) the leakageof ca. 36 % of the final (Iry weight in bean endocarpattended by a cessation of respiration. In the experi-mental materials these phenomena are inhibited byNAA for up to 30 days. Thus it is attractive to con-sider that auxin may act as a factor retarding sene-scence in leaf and fruit tissues in vivo, by effectingpreservation of membrane properties. Recent studies(20) demonstrate that kinetin increases longevity ofdetached Xanthium leaves by deterring proteolysis.The present investigation shows that kinetin has noeffect on the longevity of bean segments.

The water-logging of intercellular spaces is bestexplainedl by considering that a change in membranepermeability occurs, thereby permitting a movementof certain solutes, and hence of water into the inter-cellular spaces (to satisfy the diffusion pressuredeficit). It might be argued that water from themedium injects the air spaces in bean endocarp. This,however, is negated by the fact that this phenomenonoccurs in the abscission zone in Coleus (and inFuschia and Cestrum, unpublished) where the tissueis not exposed to an external source of water.

The water-logging of intercellular spaces and in-creased rate of plasmolysis in water-treated endocarpare evident preceding cellular dissociation. These

observations argue against an interpretation thatwater-logging is an effect of hydrolysis of pectins inthe middle lamella resulting in the formation of ahydrated pectin jelly in the air spaces. The fact thatthe Q 02 of water-logged tissue parallels that ofauxin-treated tissue for 9 days suggests no drasticdisorganization of the protoplasts, despite alterationsin permeability. Further, during the same interval,protoplasmic organization in water-logged tissue ap-pears normal. Uponl prolonged aging (20 days),however, the permeability changes accelerate proto-plasmic disorganization, as evidenced by the loss ofrespiratory activity in water-treated segments, attend-ed by marked leakage. Auxin effectively obviatesdisorganization for over 45 days. Thus it is con-cluded that auxin delays senescence by maintainingmembranes in their normal state.

The conspicuous browning in the colorless adaxialtissue in Rhoeo leaf slices attends leakage of antho-cyanins from the abaxial tissue zone and water-loggingof the intercellular spaces in the centrally situatedchlorenchyma. Thus it is interpreted as an effect ofalterations in membrane properties. A browning ef-fect analogous with this is the so-called "tea fer-mentation," attributed to cytoplasmic dlisorganizationwhich enables vacuolar polyphenols to come into con-tact witlh cytoplasmic polyphenolases (21). Brown-ing has been reported in maturing apples (10) andlattributed to "greater access of polyphenols to en-zymes." Also, it may be mentioned that the bruisingof apples without rupture of the skin brings on brown-ing. again an analogy with tea fermentation.

These reports together with the present observa-tions suggest that browning in aged or injured fruitand leaf tissues may be an effect of permeabilitychanges. An additional consequence of unnaturalpolypheniolase action in vivo may be the inhibition ofoxidative phosphorylation owing to the accumulationof oxidized polyphenols (13), thus acceleratingsenescence.

The demonstration of auxin-permeability relation-ships in bean and leaf tissues prompted studies ofcertain drupes and berries to see if maturation phe-nomena such as cellular dissociation, increase in sol-uble pectins, the climacteric rise in respiration (1),andl increased malate decarboxylation (9, 17) arepreceded by permeability changes. Although this in-vestigation is still in progress, preliminary observa-tions support this view as evidenced by the occurrenceof water-logging of air spaces in tissue segments invitro as well as in intact maturing fruits. Thus, thequestion is raised as to whether such phenomena areeffects of permeability changes. It is of interest topoint out that Blackman (1) and Kidd and West (11)suggested that the climacteric may be related to per-meability changes. Further investigation is neededon the role which permeability changes may play inripening fruits. The demonstrated water-logging ofair spaces in maturing fruit tissues, however, makesit attractive to consider changes in membrane per-meability as being the cause of phenonmena associatedwith the maturation of certain fruits.

370

SACHER-AUXIN-MEMBRANE PERMEABILITY IN TISSUES

Several investigators have shown that divalentions reduce leakage of cellular materials (12, 14).Laties (12) reports that exposure of tissue to a mono-

valent salt solution results in water-injection and lossof all aspects of semipermeability. These effects are

prevented if 10-4 M CaSO4 is in the bathing medium.The storage of bean endocarp in 5 X 10-4 CaSO4,however, has no effect compared to water, nor doesit prolong maintenance of membrane properties whenadded to an auxin solution. According to Helder (8)segments of plant tissue suspended in distilled waterundergo leakage of both organic and inorganic sub-stances. The storage of bean endocarp in 0.1 to0.3 AI sucrose or mannitol (loes not prevent permea-

bility changes. Thus, it is concluded that the auxin-membrane permeability relationship differs from theeffect of monovalent ions or (listilled water on per-

meability.The fact that water-treate(d endocarp plasmolyzes

more rapidly than fresh or auxin-treated endocarpdoes not establish that the permeability changes oc-

cur in both vacuolar and plasma membranes. Sinceit has been shown that the plasma membrane is 100times more permeable than the tonoplast (15), theincreased rate of plasmolysis could be accounted forby a change in permeability of the tonoplast only.The instantaneous increase in cytoplasmic refractivityof water-treated endocarp when exposed to the plas-moliticum, however, can best be explained by a markedchange in permeability of the plasma membrane, in-asmuch as the increase in refractivity precedes plas-molysis. Considering the apparent ubiquity of mem-brane structure the possibility should be consideredthat mitochondrial. as well as plasma and vacuolar,membranes are affected.

Although a scheme had been proposedI earlier(24) by which auxin (JAA) could affect membranepermeability directly, the possibility must also beconsidered that auxin effects maintenance of mem-

brane properties through some metabolic pathway.An interaction between auxin and iodoacetate affect-ing membrane permeability is shown by Christiansen(5). He reports that auxin-stimulated growth ofpea stem segments is blocked by iodoacetate, and isattendedl by a marked leakage of cellular substances.In the presence of iodoacetate or auxin alone there isno leakage. That this may differ from the permeabili-ty changes described in this paper is indicated byits reversibility when iodoacetate is removed. It isnot known if there w,as an accompanying water-

logging of the intercellular spaces.

Recent work by Galston on IAA oxidase gradientsin shoots (7) suggests that the occurrence of per-

meability changes in bean endocarp, and in abscissionzone tissue in Coleus, may be related to a higher rateof IAA destruction than in contiguous tissues. Cur-rent work reveals that IAA oxidase activity in thebrei of endocarp is 3 to 4 times higher than in thecontiguous exocarp. IAA oxidase determinations todate on abscission zone and adjacent tissues havebeen inconclusive.

SUMMARYStudies of fruit and leaf tissues in vitro demon-

strate that auxin acts to delay senescence. The ex-perimental work shows the dependence of these tissueson auxin for maintenance of membrane properties.'Whereas in water-treated tissues permeability changesoccur in ca. 5 days, the addition of auxin delays thisprocess for up to 30 days. In this paper evidencesof alterations in membrane permeability in the endo-carp in the absence of added auxin are 1) the rapidrate of plasmolysis in water-treated as compared withfresh and auxin-treated endocarp segments, 2) theconspicuous exudation which appears on the surfaceof water-treated endocarp segments, and 3) the con-siderably greater leakage of cellular contents fromwater-treated than from auxin-treated segments.

Respiratory studies of endocarp segments revealno significant difference under the 2 treatments overa 9-day period. Prolonged studies show that water-treated endocarp loses respiratory activity by 22days, while auxin-treated endocarp exhibits a moder-ate Q O., for 45 days or more. The earlier loss ofactivity in the former is believed attributable to theeffect of permeability changes on excessive leakageof cellular contents.

Striking effects are achieved in delaying senescenceof 1 mm thick Rhoeo leaf slices with 4 ppm NAA.Water-treated slices exhibit degenerative phenomenawithin 5 days, such as liquid-logging of intercellularspaces, cellular dissociation, browvning, and loss ofanthocyanins. These sequences are delayed for up to30 days by the addition of auxin only. Similar effectsare obtained with segments of leaves of AMesembryan-themum. The possible relationship between per-meability changes and phenomena attending sene-scence of certain fleshy fruits is discussed.

The author xvishes to gratefully acknowledge thehelpful discussions with Dr. G. Laties (luring thecourse of the investigation, and express appreciationto Drs. A. J. Haagen-Smit and G. Laties for review-ing the manuscript.

LITERATURE CITED1. BLACKMIAN, F. F. and PARIJA, P. Analytic studies

in plant respiration I. The respiration of a popu-lation of senescent ripening apples. Proc. Roy.Soc. (London) B 103: 412-445. 1928.

2. BONNER, B. A. and MACHLIs, L. Respiration ofthe mycelia and mitochondria of the filamentouswatermold, Allomyces macrogymsis. Plant Physiol.32: 291-301. 1957.

3. BONNER. J. Relations of respiration and growth inthe Avena coleoptile. Amer. Jour. Bot. 36: 429-436. 1949.

4. BON-NER. J. and ENGLISH, J., JR. A chemical andphysiological study of traumatin, a plant woundhormone. Plant Physiol. 13: 331-348. 1938.

5. CHRISTIANSEN, G. S. The metabolism of stem tis-sue during growth and its inhibition V. Natureand significance of the exudate. Arch. Biochem.29: 357-368. 1950.

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PLANT PHYSIOLOGY

6. CURRIER, H. B., ESAu, K. and CHEADLE, V. I. Plas-molytic studies of phloem. Amer. Jour. Bot. 42:68-81. 1955.

7. GALSTON, A. W. The effect of morphogeneticallyactive light on indoleacetic oxidase activity andgrowtlh. Photoperiodism and related phenomenain plants and animals. (Papers presented at asymposium held at Gatlinburg, Tennessee, August1957.) Amer. Assoc. Adv. Sci. (In press.)

8. HELDE.R, R. J. The loss of substances by cells andtissues (salt glands). Handb. d. Pflanzenphysiol.2: 468-488. 1956.

9. HULME, A. C. and NEAL, G. E. A new factor inthe respiration climacteric of apple fruits. Nature179: 1192-1193. 1957.

10. JAMES, W. 0. Plant Respiration. P. 198. Claren-don Press, Oxford 1953.

11. KIDD, F. and WEST, C. Physiology of fruit I.Changes in the respiratory activity of apples duringtheir senescence at different temperatures. Proc.Roy. Soc. (London) B 106: 93-109. 1930.

12. LATIES, G. The development and control of co-existing respiratory systems in slices of chicoryroots. Arch. Biochem. 79: 378-391. 1959.

13. LIEBERMANT, M. and BIALE, J. B. Oxidative phos-phorylation by sweet potato mitochondria and itsinhibition by polyphenols. Plant Physiol. 31: 420-424. 1956.

14. LUNDEGARDH, H. and STENLID, G. On the exudationof nucleotides and flavones from living roots. Ark.Bot. (Stockh.) Ser. A 31: 1-27. 1944.

15. MACROBBIE, E. A. C. and DAINTY, J. Ion transportin Nitellopsis obtutsa. Jour. Gen. Physiol. (Inpress.)

16. MARKS, J. D., BERNLOHR, R. and VARNER. J. E.Esterification of phosphate in ripening fruit. PlantPhysiol. 32: 259-262. 1957.

17. MARKS, J. D. and VARNER, J. E. The effect of bruis-ing injury on the metabolism of fruit. PlantPhysiol. 32 Suppl.: P. xiv. 1957.

18. MARRE, E. and FORT, G. Metabolic responses toauxin III. The effects of auxin on ATP level asrelated to the auxin induced respiration increase.Physiol. Plantarum 11: 36-47. 1958.

19. NITSCH, J. P. Methods for the investigation ofnatural auxins and growth inhibitors. In: Chem-istry and Mode of Action of Plant Growth Sub-stances, R. L. Wain and F. Wightman, eds. But-terworths Scientific Publications, London 1956.

20. RICHMIOND, A. E. and LANG, A. Effect of kinetinon protein content and survival of detached Xan-thium leaves. Science 125: 650-651. 1957.

21. ROBERTS, E. A. H. The chemistry of tea fermenta-tion. Adv. Enzymol. 2: 113-133. 1942.

22. SACHER, J. A. Relationship between auxin andmembrane-integrity in tissue senescence and abscis-sion. Science 125: 1199-1200. 1957.

24. VELDSTRA, H. Researches on plant growth sub-stances II. Contemplation on place and mechanismof the action of the growth suibstances. Enzymol-ogia 11: 137-163. 1944.

SOME METABOLIC RESPONSES OF ALFALFA SEEDLINGS TO FREEZING 1, 2C. R. SWANSON AND M. W. ADAMS

CROPS RESEARCH DIVISION, AGRICULTURAL RESEARCH SERvIcE, U.S.D.A. AND AGRONOMY DEPARTMENT,SOUTH DAKOTA STATE COLLEGE, BROOKINGS, SOUTH DAKOTA.

Species and varietal differences in cold hardinessor susceptibility have been studied for years and al-though many plant reactions to low temperatures havebeen reported, the physiological and biochemical basesfor recovery from low temperature stresses are stillnot clear. The extensive literature on methods oftesting for winter hardiness have been reviewed byDexter (2), and Levitt (7, 8) has reviewed numerouspapers perta ining to physiology of hardening andfrost hardiness.

Hansen and Nossal (4) have shown that in yeast,freezing not only increases permeability but alsomarkedly alters enzyme structure and cellular mor-phology. They showed that nucleotide coenzyme-enzyme bonds are destroyed by freezing, and they citeunpublished data indicating that in frozen cells oxi-dative phosphorylation is abolished. The latter hasalso been demonstrated in cell-free extracts of bac-terial particles (1). With succinate as a substrate,oxygen uptake of the particles was unaffected, yield-

1 Received September 3, 1958.2 Journal paper no. 406 of the South Dakota Agricul-

tural Experiment Station.

ing a very low ratio of phosphorus esterified to oxygenconsumed.

The hardened cell or tissue is better able to with-stand cold injury than is the unhardened, as shownby many past studies. Whether or not the physio-logical processes required for renewed growth afterfreezing are common to tissue which has been hard-ened and to unhardened tissue is not fully known.Some effects of different hardening conditions onrespiration and regrowth of alfalfa will be presentedelsewhere (11). The present paper reports the re-sults of investigations into the respiratory metabolismof alfalfa seedlings and mitochondria in relation tofreezing.

MATERIALS AND METHODS

Seeds of Teton, a hardy variety of alfalfa (Medi-cago niedia L.), were germinated in plastic boxes ongermination blotters in a dark germinator at 20° Cfor 4 to 5 days, at which time the radicle was 2 to 3cm long, and the cotyledons had not yet broken theseed coat. Seedlings of alfalfa were employed at thisstage of development in order to obtain a maximumamount of metabolically-active tissue. Relatively

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