PHYNIOt6LOGThe Effect of Reduced Water Potential on Soybean Mitochondria'
T. J. Flowers2 and J. B. HansonUniversity of Illinois, Departments of Agronomy and Botany, Urbana, Illinois 61801
Received January 14, 1969.
A bstract. The respiration of excised hypoeotyls and of isolated hypocotyl mitochondriafrom soybean [Glycine max (L.) Merr., var. Wayne] was determined in various concentrationsof sucrose and potassium chloride. Hypocotyl oxygen uptake deciined with increasing soluteconcentration; no specific effects of either solute were apparent. Mitochondrial state IIIrespiration was strongly inhibited as the solute concentrations were raised and there was inaddition a specific inhibitory effect of the salt. State IV respiration, however, was unaffectedby the presence of osmoticum. ADP/O ratios were also unaffected, except at high potassiumchloride concentrations (470 mil). The primary effect of solutes was thus to limit the rateof substrate oxidation.
Hydrostatic pressure did not reverse the decline in net phosphorylation accompanying reducedoxidation. It was inferred therefore that the inhibition was not due to lower water potentialper se! but rather to some other effect of water or solute ooncentration.
The effect of solutes on a mitochondrial enzyme, malate dehydrogenase, was also examined.Suerose inhibited malate oxidation by both the mitochondria and the isolated enzyme in parallel,while potassium chloride was more inhibitory on the isolated enzyme. It was concluded thatalthough the addition of solute lowers the water potential, the primary effects are exertedthrough specific effects of the solute on enzyme activity.
Although plant respiration is always retarded bythe net loss of relatively large amounts of water, theeffects of smaller changes in water content arevariable. There are two main responses, either therespiration is stimulated with small decreases inwater content or inhibition occurs at all water defi-cits (3, 7).
The reasons for these changes in respiratorymetabolism under conditions of stress have remainedlargely uinexplored. Kursanoov (15, see also 17)reasoning from the decrease in phosphorylated com-pounds reported by Zholkevich and Koretskava (24)and from the stimulated respiration that sometimesoccurs during drought, concluded that oxidation andphosphorylation become uncoupled. Flowers (11)found potato disks to show an increasing inhibitionof 14C-labeled acetate, glucose and succinate utiliza-tion, together with decreasing malonate sensitivity
1 This work was supported by a U.S.D.A. contract(Oil Seed Investigations) and a grant from the UnitedStates Atomic Energy Commission (AT/11-1/790).
2 Present address: University of Sussex, School ofBiological Sciences, Sussex, England.
and vesictulation of nmitochonidrial cristae as the waterpotential was lowered. The results were taken to beindicative of mitochondrial nmalftunction uinder waterstress.
Isolated mitochondria are affected by conditionsof lowered water potential, generally increasingsucrose concentration (see 4). Plant mitochondriamay show an increase in state IV respiration rateover a large range of solute concentrations (e.g. 0 toa'bout 800 mM). Alternatively stimulation may belimited to the range from 0 to about 400 mM, whenfurther increases in concentration either have noeffect on or decrease the rate of oxidation. Animalmitochondria show a more uniform response with anoptimal sucrose concentration of about 200 mM.State III respiration, however, is generally inhibitedin both plant and animal mitochondria as the soluteconcentration is raised (1 ;4; 12:21) although thedegree of inhibition may vary with the particularsubstrate being oxidized (12,22).
The results obtained on the effects of low waterpotential on phosphorylation are also somewhatvariable. Changes in concentration from about100 mm to 500 mM produced decreases in ADP/Oratio in 1 out of 2 of Stoner's t(22) experiments,
but had little effect in those of Johnson and Lardy(12) or Slater and Cleland (21). Cooper andLelhninger (5, 6), however, did report uncoupling bysucrose in digitonin fragments of rat liver mito-chondria.
The mitochondrial systemi wx-as an obvious choicefor the furtlher investigation of the effects of waterdeficit on plant respiration. However, since waterpotentials are most commonly lowered osmotically,evaluation of the data requires recognition of specificsolute effects and evaluation in distinction to waterpotential. Experinments wiJ]h these aims are reportedhere.
Materials and Methods
See(llings of soybean [Glycinle ;l-iax (L.) Merr.,var. \Vavne] were grown at 290 in the dark forapproximately 88 hr. Seeds were normally plantedin plastic trays containing about 450 g of vermiculiteand watered with 2.2 1 of 0.1 m\i calcium chloridesolution, 1.7 1 on planting and 0.5 1 after 40 hrgrowth. The surface of the vermiculite was lightlvdusted wvith "Spergon" bulb dust (Science Products,Chicago, Illinois) and the trays covered with alumi-num foil. Seedlings were raised at loNwer waterpotentials by raising the ratio of vermiculite to cal-ciunm chloride solution.
A modification of the procedure of Kenefick andHanson (13) was used to isolate mitochondria.About 100 g of excised hvpocotvls were washed withdistilled water (3 timiles With 500 nil at room tem-perature and 3 times with ice cold water) beforegrinding in an iced mortar with 250 tll of a coldisolationi mediumn consisting of sucrose (400 mIO);TES3 ( 50 ImlM, pH 7.8) cysteine (2 mil\0); EDTA(5 mn\) aind BSA (1 mg nilm1). The hoomogenatewas filtered through cheeseclotlh aind centriftuged at50)o at 0° for 10 min. After transfer of the stuper-natant to cleani tubes the mitoclhondria were sedi-menited at 1 5,400Q for 10 miin anId the pellets thenresuspenided in 50 il of cold 400 nIAmt sucrose. Fol-lowiig a fuirthier centrifugation at SOOg tlle suiper-natant waIs again transferred to cleall tuibes and10 mil of cold 600 milAi sucrose layered below eaclhsuspension. Finally after centrilfugation at 980ogfor 20 nmim the supernatant was discarded and thefinal sustpension micade inl 2 to 3 mil of 400 nmlM sucr-ose.
A Clark oxygen electrode was,;. uised in conljunictionwith a Gilsoni oxvgraph to measure oxygen uptake.Mitoclhondria (0.5-0.8 lg protein ) were susl)endedin 3 mil of a reaction miiixture conisisting- of BSA(1 nmg ml-1 )' ES (20 mn\, pH /7.4); malate(10 IlmM) pyrivate (10 niv) NAD (0.38 miM)
3 The following abbreviations are used in the text:TES for N-tris (hydroxymethyl) -methyl-2-aminoethane-sulphonic acid; BSA for bovine serum albumin; andTPP for thiamine pyrophosphate chloride.
TPP (03 mM); CoA (0.13 mM); magnesium chlo-ride (4 mM) and phosphate (3.8 mM) together withan osmoticum. Succinate (10 mntI) was used inplace of malate in a number of assays as indicatedin the text. The pH was adjusted with KOH. Theoxvgen content of reactionlmixtures was determinedusing protocatechluic 4-5-oxygenase with protocate-chuic acid as substrate; 1 mole of protocatechuic acidis equiivalent to 1 mole of 0., (cf. Dagley et al. 8).All measurements were made at room temperature(230 ± 1°). Acceptor control ratios (ACR) andADP/O ratios were determined after the additionof 200 to 350 nnioles of ADP, as described by, Esta-brook (10).
Oxygen uptake of hypocotvls was determined at250 bv standard \Varburg manometrv. Six 2-cmhypocotvl segments (approximately 0.4 g fresh wt)were placed in each flask and 0. measured over aperiod of 1 hr. Hypocotv\ls grown under normalconditions were used for experiments involvingosmoticallv induced water deficits and in these ex-periments the osmoticum was buffered with TES(50 mm, pH 7.5). The respiration rates of hvpo-cotvls from seedlings raised under drier conditionswere determined in dry flasks.
Both mitochondrial and hypocotyl protein wasestimated using the method of Lowry et al. (16)with BSA as standard. Dried hypocotyls were ex-tracted once with trichloroacetic acid (5 % w/v)before protein was dissolved overnight (at 400) inN-NaOH.
Phosphorvlation w as determined in the pressureexperimiients (see below) by estinmating the chaingein Pi in a mediunm consistin.g of BSA (1 mg ml-')TES (20 nMm, plH 7.4) ; pyruvate (10nimM) ; malate(10 nM); NAD (0.38 nt\mI); TPP (0.3 nimA); CoA(0.13 nim); MiIgCL, ( 1 3 ni- .); Pi (2.0 nis ); lhexo-kinase-Sigmila Tvpe III-(0.3 mg mil 1); glucose(50 11lsM) ADP ( 1.6 m-o); oslmioticutli and 0.3 or0.8 nig of nmitochonidr-ial protein. The inorganicphosphate was measured in supernatants containing5 % (w/v) cold trichloroacetic acid followring themethod of Penniall (20). Phos,phorvlation reactionswere carried out in 5 ml syringes an(Id where pressurewas alpl)lied this was achieved within 1 mini of addingthe mitochondria, by ap)plying weights to a triplebeam balance sutpporting the plunger of the syringeonl the pan. The reaction was not. allowed to exhaust0., in the solution, as determinieied previouisly with theoxygraph.
Malate dehvdrogenase (1 .1.1.3/7.) was preparedby sonicating (Bios,onik. Model BP1-BlackstoneCorporation, Jamestown, New York) freshly isolatedmitochoiidria in 400 mnal sucrose for approximately2 mmill at 00. The sonicated preparation was thencen.rifuged at 27,000g for 5 min and the supernatantused as enzyme, following a 250 fold dilution. En-zvmine activity \\was assayed at rooni temperatureaccording to the procedure of Ochoa (19). Changesin optical density at 340 mpt were recorded with aCarey model 14R spectrophotometer.
FLOWERS AND HANSON-WATER DEFICIT AND MITOCHONDRIAL ACTIVITY
Water potentials4 and osmotic potentials weremeasured with the thermocouple psychrometers ofBoyer and Knipling (2).
Results
Hypocotyl Respiration. The uptake of O2 byhypocotyl segments was retarded by lowering theirwater potential osmotically (Fig. 1). Tissue waterpotentials were estimated after correcting the solu-tion water potential for the amount of water lostfrom the tissue. There were no apparent differencesbetween the effects of potassium chloride and ofsucrose and from the combined results an inhibitionof 1.2 % per bar was calculated.
Although water deficits imposed on the seedlingsduring the 3.5 day growth period had a markedeffect on hvpocotyl length and weight (lowering thehypocotyl water potential from -2.2 bars to -4.0bars reduced the mean weight of a single hypocotylfrom 0.29 g to 0.09 g and the mean length from77 ± 4 mm to 24 + 2 mm) there was no significantreduction of the respiration rate. The constancy ofrespiration rate was correlated with a relatively
0~~~
0~~~ O
-35 -30 -25 -20 -15 -10 -5 0
SOLUTION WATER POTENTIAL (bars)
FIG. 1. The relationship between oxygen uptake andestimated hypocotyl water potential. Respiration rateswere determined in KCl (0) and sucrose ( 0) solutionsand expressed firstly on a mg protein basis and thenrelative to rates in water. The fitted linear regressionline is drawn. The estimated hypocotyl water potentialon harvesting was - 1.94 bars.
4 Water potential (T.) is defined by, =a- w/0
voV° where ,u' and V°.O are the chemical potential
and partial molal volume respectively of pure free waterat atmospheric pressure and a given temperature and p.t,is the chemical potential of water in the system underconsideration. The value of g. is a function of the num-ber of insoluble substances (matric potential, Pr), solublesubstances (osmotic potential, I'r) and pressure ('Vp)such that =-- Ir±+ T'r + 'Vp.
A
ADP
zTLZJ 54 n moles
() oxygen
ADP
T M E
1 min
FIG. 2. Polarographic determination of oxygen up-take by mitochondria in 230 mm sucrose. (A) malate+ pyruvate and (B) succinate + pyruvate as substratewas present prior to the addition of mitochondria (at M.)ADP (318 nmoles) was added as indicated.
unchanged water potential and/or water content.Mitochondria. The hypocotyls used for the iso-
lation of mitochondria had an average water potentialof -1.94 ± 0.06 bars. The osmotic component ofthe total potential was -5.24 ± 0.02 bars, givingan estimated turgor pressure of +3.3 bars. Theisolation was carried out under hypertonic conditionsat an osmotic potential of -13.8 bars and the mito-chondria remained at -11.0 bars in a 400 mm sucrosesolution at 00 until used. Storage did not normallyexceed 2 hr: acceptor control ratios measured in230 mm sucrose did not decrease in this time.
Addition of ADP to mitochondria oxidizingmalate+pyruvate caused a normal state IV tostate III transition (Fig. 2A). The return to stateIV respiration was followed after approximately 1min by a spontaneous increment in the respirationrate (see Fig. 2). This increment was apparent inall sucroSe concentrations used (up to 930 mM),although somewhat reduced in magnitude at thehigher concentrations. In KCI solutions the incre-ment was not apparent at concentrations greaterthan 230 mM. Since the nature of this stimulatedrespiration was not understood, only the resultsobtained with the initial state IV rate will be re-ported: acceptor control ratios and ADP/O ratioswere calculated from this initial state IV rate.
Succinate+pvruvate was oxidized at high rates,particularly during state IV respiration (Fig. 2B).Acceptor control ratios were consequently lower thanwith malate + pyruvate. There was no spontaneousincrement in the state IV rate with succinate. Addi-tion of KCl or sucrose to lower the water potentialdid not alter the relative rates of succinate and
malate oxidation (cf. 12 & 22): state III rateswere 1.3 times higher and state IV rates twice ashigh in succinate+pyruvate as in malate+pyruvateat all concentrations of solute. Only data obtainedwith malate + pyruvate are reported below.
Acceptorless (state IV) respiration was onlyslightly influenced as the concentration of osmoticumwas raised (Fig. 3): maximum rates occurred insolutions with an osmotic potential of about -10bars. Rates in KCl and in sucrose solutions weresimilar.
0
<~~~~///0
0
0-- -::o - °
240
200
c
160 <, s
Ha- L
120 cmz Ew(5-
80 >- '.x E0
0
It
-40 -30 -20 -10 0
SOLUTION OSMOTIC POTENTIAL (bars)
FIG. 3. Oxygen uptake by mitochondria at v-aricuswater potentials. Solid lines, state III rates in KCI(0) and sucrose (0) solutions: broken lines, state IV.Molarities of sucrose used were 0.23, 0.47, and 0.93and of KCI, 0.12, 0.27, and( 0.47.
State III respiration rates were strongly ihlhbitedas the water potential Nx'as decreased. Althouglh notshown, rates in the absence of added solute wereoccasionally lower than with 120 mM KCl or 230 mMsucrose. The inhibition of state III respiration wasmore marked than with intact tissue (cf. Fig. 1 );hypocotyl respiration was inhibited by 1.1 % per barwhile mitoclhondrial respiration was inhiibited by4.9 % per bar by sucrose. In addition there was apronounlced effect of salt over and above that ofsucrose (inhibition by KCI was 8.9 % per bar).Experiments with alterinative solutes indicated thatthe effects of glucose were similar to those of sucroseand NaCl to KCI.
Since state IV respiration was largely unaffectedby increased solute concentration, the marked declinein ACR (Fig. 4) occurring with decreased osmoticpotential was due to the decline in state III respira-tion.
Althotigh the ACR declined as the osmotic poten-tial was lowered this was not the case for ADP/Oratios. Neither sucrose nor KCI had any significanteffect upon the latter between 0 and about -115 bars
FIG. 4. Acceptor control ratios at various water po-tentials in KCI (0) and sucrose ( 0 ) solutions. Themean values from a number of experiments are plotted:there were no significant differences between ratios cal-culated from a first or a second addition of ADP.Standard errors Nere less than 1 %.
(Fig. 5). Below -15 bars, however, low-erinig thewater potential was accompanied by somiie decline inthe ADP/O ratio, particularly in KCI. It is clearthat the primary effect of a lowered water potentialoIn soYbean mitochondria Was not unlcouplilng of therespiration from phosphorylation. Evidently the in-hiibitory effects of increasing solute are only ob:erved
FLOWERS AND HANSON-WATER DEFICIT AND MITOCHONDRIAL ACTIVITY
Table I. Phosphate Esterificatiott at Variouis WaterPoteittials
Mitochondrial protein (0.3 mg) was incubated forapproximately 10 min in the phosphorylation mediumdescribed. 'fThe water potential was lowered by the ad-dition of KCI and then adjusted with hydrostatic pres-sure as indicated.
with high respiration rates. The limitations imposedon oxidation must be through effects on substratepenetration, electron transport or the coupled phos-phorylation mechanism.
At this point it became important to distinguishbetween effects of water potential and those due tosolute concentration. If the noted decline in respira-tion with increased solute were due to the loweredwater potential, it should be possible to reverse thedecline by raising the potential with pressure. Tech-nical difficulties prevented following respirationunder pressure. The procedure adopted for thisexperiment (determination of Pi) made use of thefact that total phosphorylation reflected respiration(since ADP/O ratios were not significantlychanged). A hexokinase trap was added to themedium and the reaction run in a hypodermic syringe
D_L
0*'50L 40.
CDE 40
w-ICE 300
0 200
10
I-
Ur0 0 2 0.4 0 6 0.8 1-0
MOLARITY OF ADDED SOLUTE
FIG. 6. Malate dehydrogenase activity as a functionof potassium chloride (0, ); sodium chloride (0,- - - -) and sucrose ( 0 ) concentration.
which could be loaded with weights to give a knownpressure. The experiment was terminated prior tothe exhaustion of oxygen in the solution as deter-mined by separate measurement with the oxygenelectrode.
At no time could an effect of pressure on netphosphorylation be detected (table I). Hence thereis no evidence that water potential per se affectsmitochondrial activity. The strong inhibition mustbe due to increasing solute concentration.
Malate Dehydrogenase. Further indication thatthe effect of solute was due to a specific effect onreaction rates was obtained by assays of malatedehydrogenase activity at various solute concentra-tions. The enzyme was niarkedly inhibited by bothsodium chloride and potassium chloride solutions(Fig. 6): rates in 470 mm solutions were 10 % ofthe rate in water. Sucrose also retarded the rate,but not as severely as the salt solutions: the ratein 930 mm sucrose was still 51 % of the control rate.A noticeable feature of the inhibition in sucrose wasthat the relative inhibition of malate dehydrogenaseactivity corresponded closely to the retardation of02 uptake (Fig. 7) by mitochondria oxidizing
ACTIVITYFIG. 7. The relationship between oxygen uptake and
malate dehydrogenase activity, in potassium chloride(0) and sucrose ( * ) solutions. Rates were deter-mined in each solute and expressed relative to the ratewith water. These relative values were then plotted asco-ordinates for equal concentrations of the given solute.
malate. This relationship was less obvious withpotassium chloride as the solute. At low concentra-tions of KCl '(120 mM) malate dehydrogenase activitywas greatly reduced although O2 uptake was hardlyaffected. With concentrations greater than 120 mMhowever, unit decrease of malate dehydrogenaseactivity was more nearly associated with unit declineof O2 uptake.
The explanation may lie in penetration of themitochondria by potassium chloride. In the absenceof substrate mitochondria swelled spontaneously whentransferred from sucrose to KCI solutions, presum-
ably due to the penetration of salt. With substratepresent. however, only a small change in opticaldensitv (520 m,u) was apparent. It was envisagedthat at low salt concentrations, little or no KCl pene-trated, so that there Nvas little inhibition of dehvdro-genase activity. As concentrations rose, however,greater salt penetration caused greater retardationof enzyme activity. The displacement of the KCIcurve from the 450 angle would have its explanationin the ability of the mitochondria to exclude most ofthe salt from the internal enzyme. The internalconcentration of salt would be less than the external.If the sucrose effect were in some way due to achange in water content of the mitochondria, thenit is probable that the effect of the salt was due toboth osnmotic and specific effects. The en,zyme activesite (or at least the site of action of potassiumchloride) would have to lie within the inner mem-brane. the presumed site of KCl exclusion, in thepresence of substrate.
Discussion
Increasing solute concentrations clearly depressedboth hypocotyl and mitochondrial state III respira-tion, being several fold more effective on the latter.The addition of solute has several effects which maybe responsible for this result: declining water poten-tial, osmotic withdrawal of water and/or increasingreaction of the solute.
Lowering the water potential should depress anyreaction in which water is a reactarrt. Dixon andWebb (9) attribute the inhibitory effects of highsucrose concentration in the experiments of Nelsonand Schubert (18) with fl-fructofuranosidase tolowvered water activity. However, in our experi-ments, hydrostatic pressure-an important factor inthe water activity in turgid plant cells-did not alternet plhosplhorylation in the presence or absence ofosmoticum (table I). Thus we have no evidencethat water potential as such plays any part in thesolute inhibition of respiration. Equally convincingis the sucrose inhibition of malate dehydrogenase(Fig. 6) which catalyzes a reaction where water isnot a reactant, at least in the formal chemical sense.
The effect of changing water potential on osmoticsystems must also be considered. Since plant mito-chondria show extensive water removal and conden-sation of the matrix in 400 mM sucrose (23), osmoticwater loss might concentrate certain endogenoussolutes to the inhibitory level or introduce physicallimitations manifested in lowering of substrate diffu-sion rates as proposed by Johnson and Lardy (12).In our exp)eriments, however, malate oxidation bymitochondria and malate dehydrogenase activity fromsonicated nmitochondria showed parallel sucrose in-hibition '(Fig. 7). If there were any physicalbarriers they were not uinique to the osmoticallycontracted mitochondrion but to the enzyme itself.
This analysis suggests that the critical responseto increasin,g solute lies with one or more enzymes.
Either the solute acts directly on the enzyme or doesso indirectly by altering some aspect of enzymehydration. In the case of the inhibition of malatedehydrogenase (Fig. 7) the steady decline in activitywith increasing sucrose concentration might beattributed to gradual changes of water structuresurrounding the enzvme (cf. 14). This may resultfrom specific interactions between water and solute.However, potassium chloride clearly produces inaddition some drastic change in the properties of theenzyme.
The absence of specific effects of either solute onthe hypocotyl respiration (Fig. 1) probably reflectsthe ability of the plasmalemna to restrict the pene-tration of KCl as compared with the mitochondrialmembrane. The different sensitivities of tissue andmitochondria may have resulted from the unlikely-hood that, in vivo, all of the mitochondria are re-spiring at state III rates, where the solutes areinhibitory.
As we noted in the introduction, it has beenproposed that lowered water content (or potential)decreases phosphorylative efficiency. This general-ization is definitely not true of soybean niitochondria,except possiblv at high solute concentrations. Itmay prove relevant that hypocotyl respiration is notstimulated in this tissue by water deficit. Net phos-phorylation is of course reduced concomitant withthe fall in respiration.
Although the water potenitial concept is extremelyuseful in problems of water flux we cannot see theabsolute relevance to the effects of water deficit onenzyme activity. The concept has its value, how-ever, in expressing the extent of water withdrawalfrom osmotic systems where metabolic functions arebeing determined.
Acknowledgments
We acknowledge the help of Dr. D. B. Peters whoarranged financial support for T. J. Flowers throughthe U.S.D.A. contract grant, of Dr. J. S. Boyer, forthe use of his psychrometers and of Mrs. D. M. Maddenfor her technical assistance.
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