CHATTERJEE AND LEOPOLD-CHANGES IN ABSCISSION WITH AGING
16. VAN STEVENINCK, R. F. M. 1959. Abscission accel-erators in Lupins. Nature 183: 1246-48.
17. VICKERY, H. B., G. W. PUCHER, A. J. WAKEMAN,AND C. S. LEAVENWORTH. 1937. Chemical investi-gations of the tobacco plant. VI. Chemical changesin light and darkness. Conn. Agr. Expt. Sta. Bull.399: 757-828.
18. WETMORE, R. H. AND W. P. JACOBS. 1953. Studies
on abscission: the inhibiting effect of auxin. Am.J. Botany 40: 272-76.
19. YEMM, E. W. 1956. The metabolism of senescentleaves. In: CIBA Colloquia on Aging. G. E. W.Wolstenholme ed. 2: 207-14.
20. YAGER, R. E. AND R. M. MUIR. 1958. Amino acidfactor in control of abscission. Science 127: 82-83.
Calcium Accumulation by Maize Mitochondria 1. 2T. K. Hodges3 and J. B. Hanson
Department of Agronomy, University of Illinois
A number of laboratories have reported that ani-mal mitochondria can actively accumulate various in-organic ions (8). Energy can be supplied by eithersubstrate oxidation or ATP. The antibiotic, oligo-mycin, inhibits ATP-driven Ca uptake, but not sub-strate-driven uptake (6,18). On the basis of thesefindings, plus the observation that ADP also inhibitssubstrate-driven ion accumulation, Brierley et al. (5)suggest that a high energy intermediate of oxidativephosphorylation is a common energy source for ATPformation and for ion transport.
Inasmuch as these studies may be relevant to theproblem of the connection between respiratory energyand ion transport in plants, we have conducted similarstudies on the accumulation of inorganic ions by plantmitochondria. Earlier attempts to demonstrate anenergy dependent accumulation of ions by plant mito-chondria had only limited success (17, 21). In gen-eral, our results are similar to those obtained withanimal mitochondria, and support the view that a highenergy intermediate of oxidative phosphorylation par-ticipates in Ca, Mg and phosphate uptake. However,a few differences do exist. The uptake of phosphate,Mg and Ca45 is dependent upon the presence of Ca.Substrate-driven Ca and phosphate uptake does notrequire exogenous Mg. In addition, we have beenunable to find Ca plus Mg: phosphate uptake ratiossuggestive of hydroxyapatite formation (5, 6, 18, 22).Preliminary communications on phases of this workhave appeared elsewhere ( 11, 13, 25).
1 Received July 20, 1964.2This research was supported by grants from the
Atomic Energy Commission (AT 11-1-790) and the Na-tional Science Foundation (G-16082).
3 Present address: Department of Horticulture, Uni-versity of Illinois.
Materials and MethodsIsolation of Mitochondria. Corn seeds (Zea
mays L., WF9 X M14) were germinated in the darkat 28° on paper towels saturated with 10-4 M CaC2.After 3 and one-half days, the shoots were excised,chilled, and ground in an ice-cold mortar with 0.25 Msucrose, 0.05 M KH2PO4 and 0.005M EDTA, ad-justed to pH 7.5 with Tris (hydroxymethyl) amino-methane. The slurry was strained through cheese-cloth. Mitochondria were isolated in a refrigeratedcentrifuge as the fraction sedimenting between 2000 Xg for 5 minutes and 12,000 X g for 10 minutes. Themitochondria were twice washed, first in the grindingmedium, next in 0.25 M sucrose and were finally sus-pended in 0.25M sucrose. The mitochondria werequite active, giving QO2(N) values of about 1500 andP/O values of about 2.5 when oxidizing a mixture ofpyruvate and succinate in the absence of inhibitors oruncouplers. Procedures for determining oxidativephosphorylation have been described (12).
Procedures for Measuring Ion Uptake. The ex-periments were carried out at 280 (except for experi-ments where temperature was varied) in centrifugetubes in a shaking waterbath. Unless otherwise noted,the reaction period was 10 minutes. Total volume ofmitochondria plus additives was 2.5 ml. Except forthe sucrose concentration of 0.25 M, buffering withTris to pH 7.5 and 4-8 X 104 cpm Ca45 per tube thereaction mixtures varied, and are given with the ex-perimental data. When pyruvate + succinate servedas substrate, 0.1 Smole coenzyme A, 0.4 pmole thia-mine pyrophosphate and 0.6 Mmole NAD per tubewere used. About 0.1 mg mitochondrial N was usedper tube, except for experiments where total Mg andP were determined; here about 0.7 mg N per tubewas used.
At the end of the experimental period, 5 nil of ice-cold 0.5 t sucrose was layered beneath the reactionmixture, the tubes were chilled for a few minutes incrushed ice, and the mitochondria centrifugethrough the sucrose in a centrifuge set at -4°.(Washing the mitochondria in sucrose by suspensionan l resedinmentation produced variable results, appar-ently from leaching of i-ons from the imiitochondria.The pellet was suspended in water for assay.
Analytical Methods. Calcium uptake was mea-sured Nwith Ca 45. Aliquots of the aqueous suspensionwere platedl on planchets, driecl, and counted with agas flow counter. For the determinationn of calcium,magnesium and phosphate (table VII and fig 7) theentire mitochondrial pellet was wet-ashed with H.1SO4andI H.O.,, the acid solution was diluted with water,boiled for 10 minutes to hydrolyze pyrophosphates,neutralized with KOH, an(l made to 10 nml with water.Aliquots were used for determination of inorganicphosphate by the method of Fiske and Subbarow(10); Mg by the procedure described by Brierley, etal. (5); nitrogen by nesslerization ; Ca by determnina-tion. of Ca45. Oxygen uptake was followed with thevibrating platinum electrode (Gilson Oxygraph).
ResultsGcieral Characteristics of Ca Uptake. The essen-
tial features of Ca accumulation as describedd for ani-mal mitochondria are readily found with corn mito-chondria. Table I gives a summary experiment show-ing succinate- and ATP-supported uptake, andI the in-hibition of both systems by 2,4-dinitroplhenol (DNP).In these early experiments, ADP inhibited succinate-(Iriven uptake (see later for exceptions). The cDim-bination of succinate and ATP produces miore Ca up-take than either separately, in agreement x-ith Vasing-ton and Miurphy (27) and Brierley et al. (6). but theeffects are not always additive. The level of Ca up-tako is lower than that reported with animal mito-chondria because lower concentrations of Ca wereused in these experiments. It was verified that mas-sive accumulations of Ca could be obtained (as highas 12.8 moless Ca/nmg N/10 min with 2.5 x 10-3 3r
Table I. Effect of Various .4dditiVes oni Ca UptakeReaction mixture contained 625 jimoles sucrose, 25
moless MgCl2, 0.25 molee CaCl2 + Ca45, 5 moless Pi,and where indicated 8 moless succinate, 6.5 moless ADP,6.5 utmoles ATP and 1.25 moless DNP. See text forconditions common to all experiments.
Ca), but there \vas no obvious advantage in loing so,and it would not be possible to make parallel studies inwhich oxidative phosphorylation was measured be-cause of the uncoupling action of Ca.
Figures 1 and 2 show that both succinate- andATP-supported Ca accumulation are temperature de-pendent, vith aIn optimum similar to that for growthof the corn seedling (28°-30°). The optimum foranimal mitochondria has been reported to be 360-40°(3, 27).
Time course stu(lies, such as those of figures 1 and2, did not always show continuous uptake for 10 nmln-utes. \Vith the optimum pH of 7.5, uptake vas occa-sionally very rapid for 5 minutes, followed by a grad-ual loss of the ion (fig 3). At pH 6.5 the rate of Cauptake xvas lower, but continued at a decreasing rattyfor 20 minutes with no subsequent loss of the ion.Exploration of this phenomenon showed that the rateof succinate oxi(lation, as determined with the GilsonOxygraph, often declined after 5 minutes at pH 7.5(fig 4). At pH 6.5 this did not occur, although therate of oxidation \Vas about 30 % less than the initialrate at 1)H 7.5.
The depressionn of succinate oxidation with timeat pH 7.5 is considered to be due to oxalacetate accu-nmulation (1,30). \Ve verified with the Warburg ap-paratus that the low rate of succinate oxidation couldbe greatly augmented by addition of pyruvate (29)or ATP (1, 26,,30). The oxidation rates of succinateor malate were nearly doubled by the addition of pyru-vate, which alone wvas not oxidized at significant rates.Table IT shows that these combinations of substrate
Table 11. Iffect of 1triotss SuIIsttrates 011 Ca UptakeReaction mixture for experiment 1 containedl 625
,umoles sucrose, 6.25 unmoles _MgCl2, 17 unmoles Pi, 0.25/iLmole CaCl2 plus Ca45, and 40 jumoles of the various sub-strates excel)t 5 unmioles of malate and succinate were us'dfor sparker quantities. Cofactors were present in alltreatments. Mitoch-ondria were preincubated for 5 min-utes with thle substrates prior to addition of other ingredi-ents. Final reaction time was 5 minutes. Reaction mix-ture for experiment 2 was the same except 12.5 molessMgCI2, 25.5 Mnmoles Pi, and where indicated 6.5 ptmolesATP and 5 /Amoles oxalacetate were used. Cofactorsxvere omitted.
ExperimentNo. Substrate
I NoneCitrateMalatea-KetoglutarateSuccinatePyruvatePyruvate + AfalatePyruvate + Succinate
Ca uptakemjumoles/mg N
8431
13418629698468487
2 OxalacetateSuccinateSuccinate + OxalacetateSuccinate + Oxalacetate + ATPSuccinate + ATP
HODGES AND HANSON-CALCIUM ACCUMULATION BY MAIZE MIITOCHONDRIA
acids were also more effective in Ca accumulation,and that ATP would partially alleviate the oxalace-tate inhibition of succinate-supported accumulation.The failure to obtain Ca uptake with citrate is attrib-uted to the chelation of the Ca ion (27).
Effect of Mg and Phosphate on Ca Uptake. Phos-
phate proved essential for substrate-supported Ca up-
zE 1000S
E 800
E
600
m400[u0
0C0
z
E
3.
u0
2 4 6 8 10 0 2 4 6TIME, min. Mg CONC., mM
take (table III). In other experiments it was foundthat arsenate, sulfate or chloride were ineffective.The addition of phosphate lowered the nonmetabolicbinding of Ca by the mitochondria (no substratetreatment), probably by lowering the effective Caconcentration.
Although not absolutely essential, phosphate
z
E
5..
0.
E
0L
z0
10 0 0.2 0.4 0.6 0.8 1.0Co CONC., mM
FIG. 1. Effect of temperature on substrate supported Ca uptake. Reaction mixture contained 625 moless sucrose,6.25 moles MgCI2, 17 /moles Pi, 0.25 mole CaCl2 plus Ca43, 40 Mmoles pyruvate, 5 /Amoles succinate and cofactors. Seetext for conditions common to all experiments.
FIG. 2. Effect of temperature on ATP supported Ca up ake. Reaction mixture contained 625 /Lmoles sucrose, 25moles MgCl2, 17 junoles Pi, 0.25 /mole CaCl2 plus Ca45 and 5 MAmoles ATP.
FIG. 3. Effect of reaction pH on succinate supported Ca uptake. Reaction mixture contained 625 moless sucrose,6.25 /Amoles MgCl2, 17 Amoles Pi, 0.25 Amole CaCl2 plus Ca'5, and 20 /Amoles succinate. Final pH's were-aj6usted& toA66Sand 7.5 with Tris.
FIG. 4. Relationship between Ca uptake and oxygen consumption with succinate as substrate at pH 7.5. Reactionmixture contained 625 moles sucrose, 6.25 moless MgCI2, 25.5 /Amoles Pi, 0.25 Amole CaCl2 plus Ca45, and 40 /Amolessuccinate. Reaction in Oxygraph vessel initiated by adding 0.1 ml mitochondria to above reactants in a volume of 2 mlat 25°. Oxygen consumption is replotted from the trace oblained with the Gilson Oxygraph.
FIG. 5. Effect of Mg and ATP on Ca uptake. Reaction mixture contained 625 moles sucrose, 4.5 gmotes Pi, and0.25 molee CaC12 plus Ca45.
FIG. 6. Effect of Ca concentrations on Ca, Mg and pho3phate uptake. Reaction mixture contained 625 moles su-
crose, 10 Lmoles MgCl2, 17 /Amoles Pi, 40 moless pyruvate, 5 moless succinate, cofactors, and Ca plus Ca45 as indicated.
Table III. Ca Uptake as a Fzinctionz ofPhosphate Concentration
Reaction mixture contained 625 moles sucrose, 0.5Mmole CaC12 plus Ca45, and cofactors. Substrate, whereindicated, was 40 moless pyruvate and 5 moless succinate.Reaction time was 6 minutes.
Phosphate Ca uptake (m/Lmoles/mg N)(,umoles/tube) + substrate - substrate net
0
8.517.034.0
840239027202710
843758620542
Table IV. Effect of Phosphate and ADP on ATPSupported Ca Uptake
Reaction mixture contained 625 moless sucrose, 25Moles MgCl2, 0.25 mole CaCI2 plus Ca45, and whereindicated 5 Amoles ADP, ATP, and 17 moless Pi.
Ca uptakeAdditives m~tmoles/mg N
Blank 79ATP 140ATP + Pi 497ATP + Pi + ADP 167
A very definite requirement for additional MIg inthe ATP-supported Ca uptake system is illustrated infigure 5. The total picture is complicated, however,by a competition between Mg and Ca for binding sites,and by what appears to be a lowering of effective Caconcentration by ATP. In the absence of energysource (ATP), the addition of MIg decreases the levelof nonmetabolic Ca binding. When ATP, but notMg, is added there is a sharp reduction in bound Ca,probably due to the formation of a Ca-ATP complex(7, 27). With the addition of Mg there is an increasein Ca uptake, suggestive of metabolic uptake. Toverify this we determined the effect of temperature onATP-stimulated Ca uptake in the presence and ab-sence of Mg (table VI). A significant temperature
Table VI. Effect of Temperature and Mg on ATPSupported Ca Uptake
greatly enhanced ATP-driven Ca accumulation (tableIV). The addition of ADP reversed the phosphateeffect.
Added Mg was of no value in substrate-supportedCa accumulation, and actually lowered both nonme-
tabolic binding and metabolic uptake (table V). TheMg probably "diluted" the Ca, competing with Ca forexchange and uptake sites. It is possible, of course,that the endogenous Mg (about 1 ,umole/mg mito-chondrial N) would be adequate for a Mg-requiringreaction. However, the mitochondria were isolatedand washed with EDTA, which should have loweredthe in vivo concentration, and one would expect addi-tional Mg to promote activity if Mg were actuallyneeded.
Table V. Effect of Mg on Substrate SupportedCa Uptake
Reaction mixture contained 625 moles sucrose, 17,Amoles phosphate, 1 jumole CaCl2 + Ca45 cofactors, andwhere indicated 40 moles pyruvate + 5 Atmoles succinateand 6.25 /Amoles MgCl2. Incubation was for 5 minutes.The endogenous Mg content of the mitochondria was 945mtmoles/mg N (0.154,umole/tube).
Ca uptakeSubstrate MgCl2 (mjumoles)
797+ 3420
+ 654+ + 3040
230504
dependence is only found with AIg. The small in-crease in Ca uptake at ice temperatures with Mg couldbe due to either the low rate of metabolic uptake(fig 2), or to some alleviation of the Ca binding toATP.
The Essentiality of Ca for Ca45, Mg and Phos-phate Uptake. Efforts to find Mg plus phosphate ac-cumulation similar to that reported for animal mito-chondria (3, 4, 5, 23) were completely unsuccessful.Only in the presence of Ca can Mg and phosphate beaccumulated (table VII). Without Ca some phos-phate is lost when the mitochondria are incubatedwith substrate, although the amounts are rathervariable.
The most striking observation was that the mito-
Table VII. Effect of Ca on Substrate Supported Mgand Phosphate Uptake
Reaction mixture contained 625 Amoles sucrose, 10moless MgC12, 17 1imoles Pi, cofactors and where indi-cated 0.5 Lmole CaCl2, 40 moless pyruvate and 5 jumolessuccinate as substrate.
chondria seem to require Ca to effectively accumulateCa45 (table VIII). Carrier-free Ca45 is not activelyaccumulated. The Ca45, however, is rapidly takenup if carrier Ca is added.
Effect of Inhibitors on Ca Uptake. As mentionedpreviously, the uncoupler 2,4-dinitrophenol inhibitsboth substrate- and ATP-driven Ca accumulation(table I). Chloramphenicol was also found to in-hibit both substrate- and ATP-supported Ca uptake(table IX). However as reported elsewhere (25),the sites of action of these 2 inhibitors appear to bequite different.
Arsenate in equimolar concentrations with phos-phate inhibited substrate-supported Ca uptake by 15to 20 %.
As with animal mitochondria (4, 6, 18) oligomy-cin blocks ATP-driven Ca uptake but does not inhibit
Table IX. Effect of Chloramphenicol on Substrate-and A TP-Supported Ca Uptake
Reaction mixture for experiment 1 contained 625uAmoles sucrose, 6.25 /moles MgC12, 17 /Amoles Pi, 0.25uAmole CaCl2 plus Ca45, cofactors and substrate (40MAmoles pyruvate plus 5 /Amoles succinate). Reaction mix-ture for experiment 2 was the same except 12.5 AmolesATP were substituted for the pyruvate and succinate.Incubation time was 5 minutes. Chloramphenicol wasadded in ethanol with equivalent amounts of ethanol inall tubes.
Chloramphenicol Ca uptakeEnergy source mg/tube m~umoles/mg N
Substrate None 1270"y 1 1210"y 2 713Pt 4 284
None None 104ATP None 426
Pi 2 181"9 4 87
Table X. Effect of Oligomycin on Substrate- andATP-Supported Ca Uptake
Reaction mixture contained 625 Moles sucrose, 12.5moles MgCl2, 0.25 Amole CaCl2 + Ca45, 17 Amoles Pi,cofactors, and where indicated 40 Mmoles pyruvate, 5Amoles succinate, and 6.5 Amoles ATP. Oligomycin wasdissolved in ethanol and all other treatments had anequivalent amount of ethanol.
Ca uptakeTreatment mumoles/mg N
Blank 183Pyruvate + Succinate 1195Pyruvate + Succinate + 2,gg/ml Oligomycin 1004+ ATP 1129+ ATP + 2,og/ml Oligomycin 269
the substrate-driven system (table X). Oligomycin(2 pg/ml) practically eliminates oxidative phosphory-lation of corn mitochondria (C. D. Stoner, unpub-lished data), and presumably functions by blockingone of the terminal steps of the phosphorylation se-quence (3, 15). For this reason, oligomycin shouldbe effective in reversing the ADP inhibition of Cauptake, as shown previously for phosphate uptake (5).That is, oligomycin by inhibiting ATP formation ata terminal step, should allow greater diversion ofenergy to ion accumulation. Such actually occurs,as shown in table XI.We did not always find ADP to inhibit Ca uptake
with pyruvate + succinate as substrate. Examplesare given in table XII where there were actually smallpromotions with 2.5 mm Mg and a large promotionwith 10 mm. This latter result is similar to that re-ported by Vasington and Murphy (26). However,the inclusion of a hexokinase trap, which would con-stantly regenerate the ADP, always produced an in-
Table XI. Effect of ADP and Oligomycin on SubstrateSupported Ca Uptake
Reaction mixture contained 625 /Amoles sucrose, 200moless glucose, 6.25 timoles MgCl2, 17 moless Pi, 0.25Smole CaCl2 plus Ca45, cofactors, and where indicated,substrate (40 /Amoles pyruvate plus 5 moless succinate),12.5 /moles ADP, 5 .g oligomycin and 20 K.M. units hexo-kinase. Oligomycin was suspended in ethanol and allother treatments had an equivalent amount of ethanol.Reaction time was 5 minutes.
Table XII. Effect of ADP oIL Substratc Supported Ca UptakeReaction mixtures for experiments 1 and 2 contained 625 gumoles sucrose, 6.25 jumoles MgCI2, 17 timoles Pi, 0.25
~Lmole CaCl2 plus Ca45, cofactors, and where indicated 40 Mmoles pyruvate, 5 unmoles succinate, 5 moles ADP, 100/Lmoles glucose and 20 K.M. units of hexokinase. Conditions were the same for experiment 3 except 25 moles Mgwere used.
hibition of Ca uptake (table XII). \We thereforeconcluded that the promotive effects of ADP alonewere due to ATP formed, and might be related to theATP plus substrate promotion found earlier (tables Iand II). No attempt was mlade to determine the re-
lationship between high Mg concentration and theADP promotion.
Tho Stoichiomnetry of Ca, Mg and Phosphate Ac-cuminulation. Figure 6 shows the changes in ion con-tent found by acid digestion of the mitoclhondria after10 minutes incubation with varying levels of Ca. Theminus Ca treatment was used to establish controllevels of ion since there is no active accumulationin the absence of Ca (tables VII and VIII), but thenonmetabolic binding of ions is compensated for.Some experiments were performed with cold per-chloric acid or trichloracetic acid extraction of themitochondria, but the extraction of inorganic phos-phate proved highly variable. This variability mayhave resulted from esterification of phosphate into var-
ious organic fractions. It was believed that the import-ant statistic was the total phosphate accumulated, re-
gardless of disposition after entry, and change in totalion content was thus determined.
At the lower levels of Ca, the divalent ion: phos-phate ratio is about unity. In 7 such experiments theion uptake in the presence of 0.2 mm Ca was 245+14mptmoles Ca, 309 47 mnimoles Mg, and 647 + 43mnmoles phosphate per mg N. As the concentrationof Ca is raised to 0.8 mm the ratio declines (fig 6)as a result of the drop in Mg uptake. Calcium andMg seem to compete for uptake, with Ca showing thegreater affinity for the accumulation mechanism.
Discussion
The initial task of demonstrating substrate and
ATP-powered Ca accumulation by plant mitochondriawas largely a matter of following descriptions in the
literature. Corn mitochondria clearly possess thesame basic system as animal mitochondria for using
high-energy intermediates in Ca, Mg and phosphateaccumulation. If one recalls, however, that plantsare the primary accumulators of the very diffusenutritive ions in the soil environment, certain adaptivespecializations of membrane transport might be ex-pected.
The Ca requirement for Cars, AM and phosphate
accumulation (tables VII and VIII) is an example ofthis. Animal mitochondria can accumulate Mi-g and
Pi in the absence of added Ca (3, 5) although Leb-ninger et al. (18) note that Ca is more effective thanMg in supporting P uptake. Corn mitochondria re-
iluire Ca, and perhaps it would be surprising if the)didl not. Calcium is the dominant cation on the ex-
change complex of most soils, and calcium is widelyknown to have some role in membrane integrity andlregulation of ion accumulation. Calcium deficientplants showv disintegration of membrane structure(19) and it has been shown that root tissues depletedof Ca by EDTA are ineffective in phosphate accum-
ulation (9). The fundamental question raised by theexperiments reported here is whether Ca has a struc-tural role in the membrane which prevents phosphatefrom leaking out as fast as it is accumulated, whetherit is a cofactor for a phosphate-transporting enzyme,
or if it simply immobilizes phosphate which has beenaccumulated. We (1o not have as yet sufficient evi-dence to distinguish between these alternatives.
Of equal interest is the failure of the corn mito-chondria to accumulate (livalent cations and phosphatein ratios typical of inorganic salt formation (5, 6, 18,22). This may be (lue in part to the technical pro-
cedures followed; i.e., use of low Ca concentrations,and analysis for total rather than acid-extractable in-organic phosphate. Under the condition of our ex-
periments, however, the stoichiometry of uptake isabout 1 phosphate molecule accompanied by 1 divalent
HODGES AIND HANSON-CALCIUM ACCUMULATION BY MA\IZE MITOCHONDRIA
ion (fig 7). (We have not yet studied the stoichi-ometry of uptake at high concentrations of Ca, norhave we investigated the uptake of other cations inthe medium, such as Tris.) Because no evidencecan be found for a Mg requirement in substrate-powered accumulation, perhaps only the Ca shouldbe considered. Here the ratio becomes approximately2 phosphate molecules accompanied by 1 Ca. Toolittle is known of the transport mechanism to attemptto attribute any biochemical significance to eitherratio.
There are additional observations which are puz-zling. One of these is the promotion of ATP-powered Ca accumulation by phosphate and the ADPreversal of this promotion (table IV). Another isthe action of DNP in depressing ATP-powered Cauptake. To explain these results we resort to thescheme given in figure 7. The scheme is adaptedfrom hypotheses and comments from several labora-tories [e.g., see the volume edited by Chance (8)], butmost directly from the comments of Boyer (2).
Calcium and phosphate uptake are assumed to beat the expense of the phosphorylated high-energyintermediate, X I ~ P. Slater et al. (24) considerthat the nonphosphorylated intermediate providesenergy for ion accumulation and other energy-linkedprocesses, with phosphorylation of the intermediateproviding a separate pathway to ATP formation.If this is true there must be an alternative phosphory-lated pathway from X I to Ca plus P accumulationin corn mitochondria, for Ca uptake is dependent uponphosphate (table III); we have not been able to findCa uptake where arsenate, sulfate or chloride weresubstituted. The participation of phosphorylated sub-stances in ion transport by a variety of animal sys-tems has been reported (14). For the present wewill assume a phosphorylated intermediate is involvedand for purposes of simplicity a single phosphorylatedintermediate is shown in figure 7.
The action of ADP in inhibiting Ca uptake (andADP is always inhibitory in the presence of a hexo-kinase trap, table XI and XII) is indicated to bethrough competition for the phosphorylated inter-mediate (5). The action of oligomycin on Ca uptakeby corn mitochondria (tables X and XI) is the sameas that reported for animal mitochondria, and figure7 is drawn in agreement with Brierley's basic scheme(3). According to figure 7, then, energy from sub-strate oxidation can be utilized either to form ATPor to power salt uptake.
When ATP is the energy source for Ca uptake,-the reversible formation of phosphorylated inter--mediate will lead in turn to the reversible formationof nonphosphorylated intermediate plus a stoichio-metric yield of inorganic phosphate. Now if addi-tional phosphate is added to the system (table IV)-mass action will tend to maintain a higher level ofphosphorylated intermediate, and thus higher rates ofCa uptake. A further addition of ADP (table IV),however, would for the same reason tend to reduce
SUBSTRATE 02
eI
x I pX~fl
ATP 'p
oligomycin
%X IMP
chloramphenicolFIG. 7. A scheme depicting how a high energy, phos-
phorylated intermediate may participate in Ca, Mg andPi uptake and the apparent sites of action of various in-hibitors.
the level of phosphorylated intermediate, loweringCa uptake.
When both ATP and substrate are provided thereis an enhanced Ca uptake (table I). To explain thisresult one must assume that there is some endogenous"leakage," when only 1 source of energy is provided,of 1 or both high energy intermediates, and that sup-plying energy from 2 sources tends to maintain ahigher level of X I - P.
The scheme also allows for the action of DNP indepressing ATP-powered Ca uptake. The nonphos-phorylated intermediate is assumed to be hydrolyzed(24, 28), thus setting up an irreversible degradationof ATP to ADP, Pi and the noncoupled intermediate(X I); in short, a DNP-stimulated ATPase. Cornmitochondria have such an ATPase (25). The re-duction in phosphorylated intermediate will be re-flected in lower Ca uptake (table I). This interpre-tation would similarly explain the DNP inhibitionof substrate driven Ca uptake (table I).
Chloramphenicol blocks both substrate and ATPpowered Ca uptake by apparently blocking the actionof the phosphorylated high energy intermediate (25).
Relationship to Ion Transport in Cells. Whetherion accumulation by isolated mitochondria is a phe-nomenon similar to ion transport across other mem-branes is uncertain. However, an attractive featureof the scheme outlined in figure 7 is that ion trans-port is not directly dependent upon substrate oxida-tion; a comparable DNP sensitive system operatingin other cell membranes could be powered by ATPcoming from mitochondria. In current work we findan ATP-stimulated binding of Ca by a microsome frac-tion which is indifferent to the presence of oxidizablesubstrate. However, the microsome fraction is muchless effective than the mitochondria (20-50 % increasein Ca binding due to ATP vs. 200-400 % with mito-
chondria), and the result might be attributed to con-
taminating mitochondrial fragments which for some
reason will not oxidize pyruvate + succinate.Peachey (20) has shown that divalent cations accum-
ulate in granules inside the mitochondria under invivo as well as in vitro conditions. Jackson et al. (16)have also linked phosphate accumulation by roots tooxidative phosphorylation by mitochondria. Morework is needed before a definite conclusion can bemade concerning the similarity of ion transport bymitochondria and ion absorption by entire roots.
Summary
The accumulation of Ca, Mg and phosphate byetiolated corn shoot mitochondria can be supported bysubstrate oxidation or by adenosine triphosphate.This accumulation is temperature dependent and issensitive to pH. Substrate-supported uptake is de-pressed by the presence of adenosine diphosphate.
Substrate-supported Ca uptake requires phosphatebut not exogenous Mg. Calcium is necessary forcarrier-free Ca45, Mg or phosphate uptake. The ratioof divalent cation: phosphate accumulated is approxi-mately 1 at low Ca concentrations.
The adenosine triphosphate-driven Ca uptake re-
quires exogenous Mg and is stimulated by inorganicphosphate; the stimulation is reversed by the additionof adenosine diphosphate.
Oligomycin inhibits adenosine triphosphate-sup-ported Ca uptake but has no effect on substrate-sup-ported Ca uptake. Dinitrophenol and chloramphen-icol inhibit with either energy source.
The data suggest that a phosphorylated high-energy intermediate of oxidative phosphorylation par-ticipates in the accumulation of divalent ions andphosphate by corn mitochondria.
Acknowledgments
The authors would like to express their appreciationto Mr. Hector Mirande for excellent technical assistanceand to Dr. George Webster for the gift of oligomycin.
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Photomorphogenic Responses of Dodder Seedlings 1
H. C. Lane and M. J. Kasperbauer2Crops Research Division, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland
In 2 previous reports the photocontrol of hookopening and twining of dodder seedlings was attrib-uted to action of phytochrome (8, 18). An addi-tional photoreaction has since been found to affectcontrol over these responses. This paper furnishesa description of both controlling photoreactions, andthe relationship between them.
Dodder seedlings respond to a single cycle of ex-posures to light. When the seedlings are grown indarkness, they form a hook much like the hypocotylhook of a bean plant. A single saturating red irra-diance causes the hook to open but the rate of openingis greatly accelerated under continuous white light.Twining depends on a prolonged exposure to blue orfar-red radiant energy. Its display is favored bya final brief irradiance with far-red. The seedlingsundergo circumnutational movements without a term-inal exposure to far-red, but they do not inclinesharply and twine.
Materials and Methods
All of the experiments were carried out with seed-lings of Cuscuta indecora Chois. Fresh mature seedswere used. They were scarified for 1 hour with con-centrated sulfuric acid. A measured number of seedswas planted in glass bowls (10 cm in diameter and3.8 cm deep) on 2 layers of moist Whatman No. 3filter paper. Five ml of water was added per bowl.After planting, each bowl was covered with the topof a petri dish. The seedlings were grown in dark-ness for 72 hours before the light treatments weregiven. All steps were carried out at 25°. Pre-liminary preparations and inspections were made in
1 Received June 24, 1964.2 Present address: Tobacco Physiology Investigations,
dim green light. Complete descriptions of the rou-tines and methods for each kind of experiment aregiven in the next section.
Standard Light Sources. Standard cool-whitefluorescent or incandescent lamps were used to pro-vide white light. Heat produced by the incandescentlamps was removed with a water filter. Far-red wasobtained by filtering the radiation from three 150-winternal-reflector incandescent lamps through 5 cm oftap water and either a sheet of black plastic (FRF 700filter, Westlake Plastics Company, West Lenni Road,Lenni Mills, Pennsylvania) or 2 layers each of redand dark blue cellophane. Red light was obtainedby filtering radiation from cool-white fluorescentlamps through 2 layers of red cellophane. The in-tensity of red and far-red radiation was adjusted toapproximately 450 /tw cm-2 over the wavelength bandsof 600 to 700 and 700 to 770 mg, respectively.
Three other lamps were used which, in additionto the standard red and far-red sources, providedsources that emit increasing amounts of far-red tored radiant energy. The 5 sources are listed belowin order of increasing far-red. The ratio of radiantenergy in the 700 to 770 mpu band, divided by theenergy in the 600 to 700 mgt band, appears in paren-theses: standard red source (0.147), standard incan-descent lamp (0.94), incandescent lamp with ruby-redbulb (2.0), incandescent lamp with dark-red bulb[GE-BCJ (5.3)], and the far-red source [plastic filter(64.0)].
Monochromatic Light Sources. An interference-filter monochromator, such as described by Withrow(17), and the Beltsville spectrograph were used todetermine action spectra. The interference filtersused are listed in table I. Copper sulfate solution(100 g/liter) was used in the aqueous filter at allwavelengths below 550 mIA. Water was used for alllonger wavelengths. Colored glass cut-off filters
