Plant Physiol. (1975) 56, 113-120

The Effect of ATP on the Photoconversion ofProtochlorophyllide in Isolated Etioplasts ofZea mays'

Received for publication September 17, 1974 and in revised form March 18, 1975

PETER HORTON2 AND RACHEL M. LEECHDepartment of Biology, University of York, Heslington, York, YOI 5DD, England

ABSTRACT

The transformation of protochlorophyllide (PChle) into chlo-rophyllide (Chle) has been studied in isolated etioplasts fromZea mays. ATP (1.5 mM) prevented the transformation of pho-toconvertible PChle 650 to PChle 630 in aged etioplasts. Curveanalysis indicated that the ATP effect on photoconvertibilitycould be entirely accounted for by changes in the proportions ofPChle 630 and PChle 650 and examination of the isolated pig-ments revealed that only unphytylated PChle could be activatedfor photoconversion by ATP. In etioplasts aged for 5 hours,ATP also stimulated photoconversion of PChle 630 into Chle670. The process was temperature-sensitive and involved PChle650 and Chle 680 as intermediates. AMP alone had no effect,but inhibited ATP retardation of PChle loss. ADP had a similarbut lesser effect than ATP. The ADP response, but not the ATPresponse, was considerably enhanced in the presence of an ATP-generating system (phosphoenolpyruvate/pyruvate kinase).UTP, GTP, and CTP gave 40 to 50% of the ATP response withintact etioplasts. In envelope-free etioplasts, ATP gave thegreatest response but the other nucleotides were now 80% aseffective as ATP. After primary photoconversion, ATP stimu-lated resynthesis of PChle 650. It is proposed that ATP bothgives the holochrome the ability to bind to the PChle moleculeand enables additional association of the pigment-protein com-plex to form PChle 650.

The photoconversion of PChl' into Chle is the first detect-able effect of illumination on dark-grown plants. The abilityto carry out the photoconversion varies with plant age (11,15, 34), and the extent of the conversion is sensitive to

1This research was supported by Science Research Council Grant No.B/SR/8692 to R. M. L. and a studentship to P. H.

2Present address: Department of Biological Sciences, Purdue Univer-sity, West Lafayette, Ind. 47907.

3 Abbreviations: PChle: protochlorophyllide; PChl: protochlorophyll;Chle: chlorophyllide; PEP/PK: phosphoenolpyruvate plus pyruvatekinase; PChl/e and Chl/e refer to mixtures of phytylated and unphytyl-ated pigments; PLB: prolamellar body. For identification purposes severalspectral forms of protochlorophyll and chlorophyll are referred to as fol-lows: PChl/e 630, absorption maximum 628 to 630 nm; PChle 650, maxi-mum at 650 nm; Chl/e 670, maximum at 669 to 674 nm; Chle 680, maxi-mum at 680 to 685 nm. The ranges of maxima refer to previously pub-lished values.

various physical treatments of the leaves (3, 4, 5, 10). Thechanges in the degree of photoconvertibility are due tochanges in the proportions of photoconvertible (mainly PChle650) and nonphotoconvertible protochlorophyllide (PChl/e630) (30). The relationship between these forms and themechanisms responsible for maintaining their relative propor-tions is still largely unknown. The nonconvertible form appearsto consist of partly phytylated pigment, and its inability totransfer absorbed photons to other PChle forms suggests thatit may be located at a different position in the etioplast (14,34). However, there is evidence that nonconvertible PChl canserve as a source for the rapid resynthesis of PChle 650immediately after photoconversion (8). This situation is clearlyseen when excess PChle 630 is accumulated by ALA treatment(7, 28-31). Gassman (6) has also shown interconversion ofPChle 650 and PChle 630 by H,S treatment of whole leaves.Recently using isolated intact etioplasts, we observed a slowtransformation of PChle 650 into PChle 630 during darkincubation (13). Addition of ATP prevented this transforma-tion and in addition stimulated photoconversion of PChle 650in aged etioplasts (13). In this paper, the results of a detailedanalysis of the effects of ATP on the protochlorophyll andprotochlorophyllide species of isolated maize etioplasts isgiven, and evidence for the direct conversion of PChle 630to PChle 650 in a cell-free system is presented.

MATERIALS AND METHODS

Etioplast Isolation and Incubation. Etioplasts were isolatedfrom dark-grown maize seedlings as described previously (18).One-ml samples of etioplasts were incubated in darkness at20 C under continuous slow rotation (13, 24) in a mediumcontaining 0.5 M sucrose, 0.4% BSA (Cohn Fraction V, KochLight), 2.5% Ficoll, 5 mm cysteine, 1 mm MgCl,, 1 mm EDTA,5 mm KHCO,, 0.5 mM sodium acetate, and 1 mM L-glutamicacid in 67 mm Na2HPO,-KH,PO, buffer adjusted to pH 7.5.When indicated ATP, UTP, GTP, ADP, and AMP (all Sigma)were added as sodium salts. Pyruvate kinase in glycerol wasobtained from Boehringer.

Determination of Photoconvertibility. Etioplast samples (1ml) were diluted with 2 ml of glycerol, and each absorptionspectrum was recorded at room temperature using a ShimadzuMPS 50L multipurpose spectrophotometer. The PChl/e con-centration was between 1.2 and 2.4 /-LM. The sample was thenilluminated for 40 sec using a 150-w tungsten filament lampfiltered through 17 cm of H20 and giving an intensity ofapproximately 4.4 X 10' erg cm-' sec' at the cuvette surface;this irradiance caused complete photoconversion of PChle.The spectrum of the sample was again recorded. The degree

113

HORTON AND LEECH

of photoconvertibility of an etioplast sample was measured byexpressing the Chle peak height at 680 nm as a percentage ofthat obtained by illuminating freshly isolated etioplasts. Acorrection for scatter was made by drawing a line to passthrough the absorbance values at increments of 10 nm from680 to 730 nm. All peak heights were measured from this line.Similar approximations have been made by previous workers(21, 22). The use of Chle 680 absorbance as a reliable in-dication of photoconvertibility was shown by comparisonwith pigment determinations on 80% acetone extracts. Etio-plast absorption spectra were also analyzed quantitatively bycurve analysis using a Dupont curve analyzer.

Determination of Phytylated and Unphytylated PChle. Phy-tylated and unphytylated chlorophyllous pigments were sepa-rated by partition between petroleum ether and aqueous ace-tone by the method of Treffry (33). Petroleum ether (1.8 ml)was added to 5 ml of an 80% acetone etioplast extract, andthe two layers were separated. In common with other workers,PChl and PChle concentrations were determined using thespecific absorption coefficients in 80% acetone described byAnderson and Boardman (2). These appear to be the onlyextinction coefficients available. The use of the value in 80%acetone for PChl (instead of in petroleum ether) apparentlyalters the extinction coefficient measured by only 5%. Thepurity of each phase was tested by TLC on Silica Gel H(Merck) using benzene-ethyl acetate-ethanol (8:2:2, v/v) asdescribed by Rebeiz and Castelfranco (23).

Determination of Adenine Nucleotides. ATP levels (duringexperiments when ATP was added initially) were determinedby the enzymic method described by Lamprecht and Traut-schold (16). ADP and AMP were determined as described byAdam (1). For the determination of the very low endogenousATP levels in the etioplast samples, the automated fluorometricanalytical procedure of Leese and Bronk (17) was employed.All cofactors and enzymes for these determinations wereobtained from Boehringer Corporation.

Wavelength (nm)

FIG. 1. Absorption spectra of etioplasts incubated for 5 hr in darknesswith 1.5 mm ATP compared to the spectra of a 5-hr sample incubatedwithout ATP and to the spectra of freshly isolated etioplasts. Spectra wererecorded after dilution with glycerol before ( ) and after ( ...) photo-conversion.

Table I. Effect of EDTA oni Degree of PhotoconzvertibilityEnvelope-free etioplasts were incubated for 5 hr under rotation

in darkness with and without ATP. The incubation medium con-tained tris-HCl buffer, pH 7.5, instead of phosphate, and MgCl2and EDTA only where indicated. Etioplasts were isolated in asimilar Mg-free medium. Results are the average of duplicate in-cubations. Photoconvertibility was determined as described inthe text.

PhotoconvertibilityTreatment

No additions1.5 mM ATP2 mM EDTA1.5 mm ATP + 2 mN EDTA

No MIg2+

,cl25484858

2 mm AMg2+

276732

RESULTS

Photoconvertibility with and without ATP. Figure 1 givesthe spectra obtained during a typical experiment, showingabout 75% retention of photoconvertibility in the etioplastsuspension incubated with ATP. Exactly the same results wereobtained with intact and envelope-free etioplasts (13). In addi-tion, it was found that washed etioplasts showed 80% photo-convertibility after 5 hr of incubation (not shown in Fig. 1).(Washed etioplasts are prepared by centrifuging the initialetioplast fraction through a layer of 0.6 M sucrose as describedpreviously [18], and the fractions so obtained contain approxi-mately 90% envelope-bound etioplasts.)

Identification of ATP as Primary Effector. Several additionalcontrol experiments were undertaken to determine whetherthe effects on photoconvertibility caused by adding ATP weredue to ATP per se. Since ATP has a high affinity for divalentcations, it is possible that the ATP effect could be accountedfor as a reduction in cation concentration. However, it wasfound that the metal chelator EDTA had a stimulatory effecton photoconvertibility (Table I) and that this effect waspartially additive to the ATP response. Addition of Mg2` fur-ther differentiated between the responses to ATP and to EDTAsince the addition of 2 mm MgCl2 enhanced the ATP effectbut inhibited the EDTA effect. The Mg2" requirement for fullpreservation of photoconvertibility by ATP is not a classicalone, since excess MgCl, (>5 mM) inhibits the response toATP (data not shown). From the results shown in Table I, itseems unlikely that the ATP effect can be explained in termsof a simple chelation.The question of whether the products of ATP hydrolysis

were active was next investigated under the standard condi-tions described under "Materials and Methods." It was foundthat changes in Pi concentration (0-100 mM) or addition of1.5 mm sodium PPi had no effect on photoconvertibility.Adenine nucleotide levels during a 5-hr incubation period werealso measured after the initial addition of 1.25 mm ATP (Fig.2). The AMP level rose from 0 at zero time to 0.6 mm at 5 hr,while the ADP level rose during the 1st hr, and afterwardsdecayed slowly to 0.15 mM. The level of ATP fell quickly to0.3 mm after 2 hr and to 0.1 mm by 5 hr. These kinetics areconsistent with a reaction sequence by which ATP is firstdegraded to ADP which is then broken down to AMP and notwith a simple myokinase reaction. Indeed if an adenylatekinase reaction were totally responsible for the ATP break-down, the "myokinase" ratio should remain constant; in

114 Plant Physiol. Vol. 56y 1975

ATP AND PROTOCHLOROPHYLLIDE

-E 10ATP

0

Qc0-5 - \............ADP,,v-

'--AMP4

0 1 2 3

Time (hr)

FIG. 2. Adenine nucleotide levels determined durincubation following initial addition of 1.25 mm ATP. Aland AMP (0) were determined using aliquots of duplisamples.

Table II. Photoconvertibilities of EnzvelopeEnivelope-free Etioplasts

Photoconvertibilities of etioplasts incubated inhr were determined as described in the text. Thiments 1, 2, 3, and 4 to 6 are means of 4, 6, 5, an

respectively.

Photoconvertibility

Etioplasts Treatment

-ATP +ATP(1.5 mm)

mM .~~~~~~~~0

1. Envelope-bound Control 30 53+ ADP (1.5) 42

2. Envelope-free Control 51 74+ ADP (1.5) 68

3. Envelope-free Control 39 55+ ADP (0.5) 49

4. Envelope-free Control 47 68+ ADP (0.5) 59 63

5. Envelope-bound Control 10 41+ AMP (1.5) 10 23

6. Envelope-free Control 47 68+ AMP (1.5) 60

fact, it changes from 1 at zero time to 3 after 5 hr incuba-tion.The extensive degradation of ATP suggested the possibility

that AMP or ADP rather than ATP may be the active agentsin the preservation of photoconvertibility in etioplast suspen-sions. Initial addition of ADP was found to cause a markedpreservation of photoconvertibility, but the response wasalways significantly below that obtained with ATP (Table II,experiments 1-4). An optimum ADP concentration between1 and 2 mm was found, i.e. similar to the optimum previouslyfound for ATP (13). The ATP and ADP effects were not addi-tive (Table II, experiment 4). Addition of AMP had no effecton the level of photoconvertibility and indeed caused inhibi-tion of the ATP effect (Table II, experiment 5), although thisinhibition was not as reproducible when envelope-free etio-plasts were used (Table II, experiment 6). The above observa-tions cannot be reconciled with an explanation that the effectof the added ATP is only due to its breakdown to ADP or

AMP.The greater ability of ATP, as opposed to ADP to preserve

photoconvertibility was clearly demonstrated when an ATP-regenerating system (phosphoenolpyruvate plus pyruvatekinase) was incorporated into the incubation medium (TableIII). PEP/PK markedly increased the preservation of photo-convertibility by suboptimal concentrations of ATP and byADP to a level equal to or even greater than the photo-

......... l convertibility found at optimal ATP levels (1.5 mM). PEP/PK induces a small increase in photoconvertibility in theabsence of added ATP or ADP, possibly due to regeneration ofendogenous ATP.

Determination of Specfficity of ATP Effect in Comparisonwith Effects of Other Nucleotide Triphosphates. The degree of

4 5 specificity with respect to the purine and pyrimidine residueswas next examined. GTP, CTP, and UTP were all capable

ig a 5-hr dark in-to some extent of preserving photoconvertibility (Table IV).

cate()mi etioplast Table III. Effect of ATP-regeneratinzg System PEP/PK on

Photoconivertibility of PChle inz Isolated Maize EtioplastsThe photoconvertibilities of envelope-free etioplast samples

-bound anid were determined after 5 hr of dark incubation as described in thetext. ATP and ADP were added at the final concentrations shown.

darkness for 5 Phosphoenolpyruvate (sodium salt) was 5 mM; 20 l (2 mg/ml ine data for exper- glycerol) of pyruvate kinase (EC 2.7.1 .40) were added just prior to

Id 2 incubations, incubation (PK). Each value is a mean of triplicate (+PEP/PK)or duplicate (-PEP/PK) incubations. Experiments 1 and 2 are

Photoconvertibility

Treatment-PEP/PK +PEP/PK by PEP/PK

1. No addition 38 49 11+ ATP (0.1 mM) 51 61 10+ ATP (0.5 mM) 52 65 13+ ATP(.S mM) 57 59 2+ ADP (0.S mM) 48 60 12Control (+PK)' 38

2. No addition 47 57 10+ ATP (0.1 mM) 58 68 10+ ATP (1.S mM) 68 65 3+ ADP (0.5 mM) 59 70 11Control (+PK)' 49

1 The absence of any effect due to 4 mM PEP alone was shown ina separate experiment presented earlier (13).

Table IV. Effect of Differentt Nucleotide Triphosphates onPhotoconivertibility Determinledfor Initact (Enzvelope-boutnd)

anzd Enivelope-free EtioplasisPhotoconvertibilities after 5 hr of dark incubation were deter-

mined as described in the text. Nucleotide concentrations (1.5mM) are final concentrations. Experiments 1, 2, and 3 are separateexperiments. Each value is an average of triplicate incubations.

PhotoconvertibilityNucleotide Triphosphate

Intact Envelope-free

mm %1. None 9 492. GTP (1.5) 25 653. UTP (1.5) 19 594. CTP (1.5) 28 675. ATP (1.5) 51 72

115Plant Physiol. Vol. 56, 1975

Plant Physiol. Vol. 56, 1975

With intact, envelope-bound etioplasts only about 40 to 50%of the level seen in the presence of ATP is observed. How-ever, with envelope-free preparations these values increaseto about 80 to 90%. In both cases, the order of specificitycould be tentatively described as ATP>CTP>GTP>UTP.

Since of all the adenine nucleosides and the nucleotide tri-phosphates examined, ATP had the largest and most con-sistent effect on photoconvertibility in intact isolated etioplasts,the effect of ATP on pigment interconversion in isolatedetioplasts was examined in greater detail.Measurement of Endogenous ATP Levels. The endogenous

ATP level was compared for fresh etioplasts suspended in themedium at zero time and after incubation for 5 hr. The ATPconcentration fell from 4.2 nmoles/ml total volume at zerotime to 2.5 nmoles/ml after 5 hr. This result suggests thatthe effect of exogenously added ATP is observed because theaddition is counteracting the effect of a depletion of endogenousATP which occurs during incubation.

63(10637 650 a

N

/\

el-

I

I'o, \'\

b

c

%/0totalPChI/e

n

% Photoconversion

_ 100

80

60

40

20

630637 650 A B

B8C

GLLRfl~0630 637 650 A B

6C

.40

20

630 637 650 A B

FIG. 3. Analysis of etioplast spectra into component curves. Absorp-tion spectra (-) were recorded in glycerol before and after 5 hr darkincubation. The spectra were analyzed into 3 component gaussian bands(- -) with maxima at 630, 637, and 650 nm and widths at half-maximaof 18 to 19 nm, 14 to 15 nm, and 12 to 13 nm, respectively. At the right-hand side are shown the areas of each PChle component as a percentageof the total PCnle. Tne p rcentages of photoconvertibilities were deter-mined from the curve analysis (A) and by the usual Chle 680 method (B).a: Freshly isolated etioplasts; b: 5 hr incubation + 1.5 mNi ATP; c: 5 hrincubation -ATP.

Curve Analysis of PChl/e Spectra. The spectra in Figure 1suggest that ATP preserves PChle 650 at the expense ofPChl/e absorbing near 630 nm. At least three different formsof PChl/e exist in etiolated leaves (14) with 77 K maxima at628, 637, and 648 nm. Therefore, to obtain a clearer definitionof the ATP effect, PChl/e absorption spectra were resolvedinto component curves. The aim was to obtain fit to all types ofPChl/e spectra by altering the proportions of two (or more)components whose peak positions and half-widths remainconstant. Gaussian bands with maxima at 630, 637, and 650nm were found to give perfect fit to spectra of freshly iso-lated etioplasts and of etioplasts incubated in vitro with andwithout ATP, by merely altering the peak heights of the 630and 650 nm bands (Fig. 3). The spectra could not be fitted byusing just two components with maxima 630 (±2 nm)or 650 (+2 nm). The proportional areas of each componentare also represented (on the right-hand side) in Figure 3.Clearly, the change from 0 to 5 hr in the absence of ATP canbe accounted for merely by changing the proportions of thetwo components PChl/e 630 and PChle 650 to obtain good fit.Similarly, the effect of ATP on the system can also be simulatedby altering the proportions of PChle 650 and PChl/e 630.The fact that PChle absorption bands in solution are notperfect Gaussian bands means that some caution must beapplied in using this method of determining the proportions ofdifferent spectral forms of PChl/ e. For this reason thevalidity of the simulations was checked by experimentationas follows:

1. At 0 hr the results of curve analysis indicated that 72%of the total PChl/e would be photoconvertible. This agreeswell with pigment analysis of acetone extracts which showedthat 69% of the total PChl/e was converted into Chl/e onillumination.

2. The percentage of the original (0 hr) PChl/e still photo-convertible after 5 hr can be determined by curve analysisand also directly by measuring the amount of formation ofChle 680 on illumination. Again, there is good agreement be-tween the two types of measurement. In the presence of 1.5mM ATP, curve analysis and Chle 680 measurement both in-dicate that 47% of the total PChl/e is photoconvertible,whereas in the absence of ATP, values of 37 and 29% respec-tively, are obtained. For easier comparison with previousexperiments, the values are shown in Figure 3, a and b, ex-pressed as percentage of photoconvertibilities rather than asthe percentage of the total PChl/e convertible. The similarityof the simulated and measured values in each case is clear.ATP-induced Photoconversion of PChle 630. It was pre-

viously reported that ATP stimulated photoconversion ofPChle 630 into Chle in 5-hr-old etioplasts (13). This trans-formation is a function of the total length of the light plusdark phases, rather than the total amount of irradiationreceived; thus treatment of etioplasts with the same totalillumination but given continuously without intervening darkperiods, induces very little PChl/e 630 conversion, suggestingthat dark reactions limit the accumulation of Chle. Thissuggestion is supported by the observation that the rate ofPChle 630 photoconversion is temperature-sensitive (Fig. 4).At 3 C, no PChl/e 630 conversion occurred and while in-creasing the incubation temperature to 10 C allowed a certainamount of extra Chl/e formation, not until the temperaturehad been increased to 20 C did PChl/e 630 conversion proceedat a significant rate.

Examination of absorption spectra recorded during PChletransformation supports the suggestion that transient spectralintermediates exist. The final absorption maximum of theChle (in this experiment 90% of that obtained by illuminating

116 HORTON AND LEECH

0

0

0

D

D

0

D

ATP AND PROTOCHLOROPHYLLIDE

0-06

0-04

0-02

0 4 8 12 16 20 24

No. of light and dark treatments

FIG. 4. PChle 630 photoconversion at 3, 10 and 20 C. Etioplasts,incubated in darkness for 5 hr were cooled to 3 C and exposed to a seriesof light + dark treatments as described in Table VI. After four treatments,the temperature was raised to 10 C and after 8 treatments to 20 C. Theabsorption spectra were determined following each flash and AA670measured.

Wavelength (nm)

FIG. 5. Absorption and difference spectra observed during ATP-induced PChle 630 photoconversion. Experimental details were as de-scribed under Table VI. a: Absorption spectra after 5 hr of dark incuba-tion: after primary photoconversion (- ----), after two light treatments

(- -) and after saturation ( ) i.e. eight light treatments; b: spectrumof a freshly isolated sample after photoconversion (not in glycerol);c: difference spectrum (1 min after illumination minus 4 min after illumi-nation) recorded after four light treatments; d: difference spectrum (afterillumination minus before illumination) recorded for fourth light treat-ment at 20 C (-) and 3 C (--).

freshly isolated etioplasts) is at 670 nm (Fig. 5, a and b). Aslight shift in the Chl/e absorption maximum from 672 nm

(1 min) to 670 nm (4 min) was observed when the spectra wererecorded after light treatments. The absorbance changes in-volved are small but a difference spectrum (1 min minus 4 min)did suggest that changes in a Chl/e component absorbing near680 nm were involved (Fig. 4c). In addition a slight decreasein absorbance at 630 nm accompanied by an increase at650 nm was observed during the postillumination dark period.The difference spectrum (after illumination minus beforeillumination) recorded at 20 C shows a wide Chl/e bandwith a maximum between 675 and 680 nm (Fig. 4d). Negativepeaks at 630 and 650 nm were also obtained. A similar differ-ence spectrum obtained after lowering the temperature to 3C before light treatment shows a clear Chl/e maximum at682 nm. No absorbance changes near 630 or 650 nm were

detectable. It is to be stressed that throughout, the absorptionchanges involved are small (10' to 102 optical density units)as well as rapid.The ability of other nucleotides to stimulate photoconver-

sion of PChl/e 630 are shown in Table V and indicate thatthe ATP effects on photoconvertibility and PChl/e 630 photo-conversion are different manifestations of the same basic

Table V. Effect of Different Nucleotides onz Rate and Amounti ofPChl/e 630 Photoconversionz

Etioplast samples were incubated in darkness for 5 hr withoutany added nucleotide and photoconvertibilities recorded (A =

37%, B = 19(%, C = 22%). One-ml samples were diluted with andwithout nucleotide (1.5 mm final concentrations) and given theusual 40-sec illumination to convert PChle 650 to Chl before expo-

sure to a series of light + dark treatments (5 sec photoconversionillumination plus 6 min darkness) continued until no further in-crease in absorbance at 670 nm was recorded. The absorptionspectra were recorded after each treatment and A67, plotted againsttreatment number. Rates of absorbance increase were measuredand expressed as AA/light treatment (in O.D. units). Total in-creases above that obtained by initial photoconversion were alsomeasured (AA in O.D. units). Experiments A, B, and C are sepa-

rate experiments. In experiment C the increase in A670 was biphasicfor CTP and GTP; the rates given are for the second, more rapidphase which began after nine treatments. For the first nine treat-ments rates approximately equal to those for no additions, were

recorded for CTP and GTP. For photoconversion of PChl/e630 samples were not diluted with glycerol.

Addition ~~Rate of Chli Total in- T

Addition formation crease incii NT'

103 X AA/light 103 X AA

treatment

A. None 5.0 22 14ADP 10.0 70 11ATP 14.0 68 8ADP + ATP 9.0 67 16

B. None 0.4 6 16AMP 0 0 1ATP 8.0 69 14ATP + AMP 0 0 1

C. None 0.8 12 16ATP 5.6 64 16CTP 1.9 42 29GTP 1.3 30 29UTP 0.8 19 29

1 NT: number of treatments needed for completion of conver-

sion.

30C 10 C 200C

a I

117Plant Physiol. Vol. 56, 1975

Plant Physiol. Vol. 56, 1975

FIG. 6. Reappearance of PChle 650 in the presence and absence of 1.5mM ATP. Etioplasts were incubated unstirred in the spectrophotometercuvette after primary photoconversion, and their spectra were recorded.a, b: Difference spectra, 15 min minus zero time (- -) and 90 min minuszero time ( ) for a sample plus ATP (a) and a sample minus ATP (b);c: difterence spectra (+ATP minus -ATP) at 15 min (- -) and 90 min(); d: difference spectra from measurements after 1 hr of incubationbefore and after a second illumination plus ATP ( ) and minus ATP(- -); e: time course for the absorbance increase at 650 nm followingprimary photoconversion +ATP (A) and -ATP (0).

effect. The same saturating Chl/e level was obtained withADP and ATP, although more light treatments were requiredwith ADP because the rate of Chl/e formation was less thanwith ATP. AMP was found to inhibit completely the actionof ATP as well as to prevent the low control conversion.Nucleotide triphosphates showed the same order of activity as

seen in the photoconvertibility experiments in Table IV, i.e.ATP>CTP>GTP>UTP. The rates of Chl/e formation in-duced by CTP, GTP, and UTP were all very slow and 29 lighttreatments were needed before a saturating Chl/e level was

reached.It can be seen from Table V that the varying rates of

photoconversion of PChl/e 630 in the presence of ATP may

be correlated with the degree of photoconvertibility at the timeat which light treatments commence. Thus in experiment Athe photoconvertibility was nearly 40% and high rates ofChl/e formation (0.014/treatment) resulted. In contrast, thelow rates in B and C (average 0.007/treatment) are accom-

panied by low photoconvertibility, at approximately 20%.

Effect of ATP on Resynthesis of PChle 650. In whole leavesafter primary photoconversion, reappearance of PChle 650occurs by conversion from PChle 630. A similar phenomenonwas observed in etioplast suspensions. Addition of ATP isable to stimulate the PChle 650 reappearance (Fig. 6). In thepresence of ATP (1.5 mM), difference spectra (e.g. 15 minminus 0 hr, 90 min minus 0 hr) show the emergence of a650 nm peak accompanied by a trough at 630 nm (Fig. 6a).In contrast, the same difference spectra recorded in the absenceof ATP show an absorbance increase due to the appearanceof Chl/e 670 and a smaller trough at about 634 nm (Fig. 6b).The possibility that the increase at 670 nm was obscuring thepresence of a large 650 nm band is discounted from theanalysis of difference spectra (+ATP minus -ATP) shownin Figure 6c; a peak at 650 nm (PChle 650) and a trough at630 nm (PChl/e 630) are clearly revealed. A time course forthe changes in absorbance at 650 nm recorded in the presenceand in the absence of ATP shows that an increase in the levelof PChle 650 induced by ATP can be seen within 15 min (Fig.6e). A maximum difference was observed at 3.5 hr, afterwhich decay of the newly produced PChle 650 becomes ap-parent.

Confirmation that the absorbance changes at 650 nm aredue to PChle 650 is shown by the effect of a second lighttreatment (20 sec of photoconversion illumination) given after90 min of dark incubation (Fig. 6d). In the presence of ATP,a large increase in absorbance at 682 nm accompanied by adecrease at 650 nm is induced by illumination (difference spec-trum: before illumination minus after illumination). WithoutATP no spectral change results, showing that no PChle 650is present. Measurements of the increase in Chl caused byfurther illumination show that the presence of ATP caninduce between 10 and 20% resynthesis of PChle 650 (ex-pressed relative to the original Chle 680 level) compared tonegligible amounts found in the absence of ATP.

Investigation of Phytylation of PChl/e. In both PChle 630photoconversion and PChle 650 reappearance experiments,it is clear that ATP is able to induce photoconversion onlyof part of the PChl/e pool. In Figure Sa, the final level of non-convertible PChle is approximately the same as in freshetioplasts (Fig. Sb). The data in Table VI suggest that ATP isable to induce photoconversion only of that part of thePChle pool which is unphytylated. At 0 hr the nonconvertiblePChl/e consists mainly of PChl, the level of which is unalteredby illumination. After 5 hr the proportion of phytylatedPChl is unchanged and again is not transformed to Chl onbrief illumination. The increase in nonconvertible PChl/e be-tween 0 and 5 hr is totally due to an increase in unphytylatedPChle. The ratios at 0 hr and 5 hr were consistently estimatedas 3.9 and 3.8, respectively. The amount of PChle at 0 hrnot transformed into Chle on brief illumination is just enoughto account for the 10 to 20% reappearance of PChle 650described above.

DISCUSSION

When etioplasts are incubated in a medium without addedATP, photoconvertible PChle becomes degraded. Since addedATP can partially prevent the decay and the etioplasts possessthe ability to hydrolyze ATP, the reason for this degradationseems to lie in a decrease in local ATP concentration. Wesuggested previously (13) that the preservation of photocon-vertibility induced by ATP was explainable in terms of twoor more interconvertible PChle forms with maxima near 630nm and 650 nm, the formation of the 650 photoconvertibleform requiring ATP. The results of the present experimentsadd considerable support to this explanation.

118 HORTON AND LEECH

ATP AND PROTOCHLOROPHYLLIDE

Table VI. Conients and Convertibility of Phlytylated anid unz-phyzylated PChl/e in Etioplasts before anid after InicutbationzEtioplast samples were incubated for 5 hr in darkness as de-

scribed. Determinations were repeated at 0 hr and 5 hr beforeand after photoconversion of PChle.

PChl/e ConcTreatment

Ohr 5 hr

tmoles/mI

Before illuminationPChle 1.49 1.36PChl 0.38 0.34Total 1.87 1.70

After illumination IPChle 0.19 1.16PChl 0.32 0.35Total 0.51 1.51

In freshly isolated etioplasts following primary photocon-version, ATP induces the formation of photoconvertiblePChle 650. The difference spectra for this effect suggest thatthe new PChle 650 is derived from PChle 630 and providedirect demonstration of PChle 630 transformation into PChle650 under the influence of ATP. The demonstration that therate of ATP induced photoconversion of PChle 630 into Chle670 in aged etioplasts is limited by temperature-sensitive dark re-actions, suggests the existence of one or more intermediates andanalysis of difference spectra indicates that PChle 650 andChle 680 are two of these intermediates. Curve analyses ofPChl/e spectra show that all degrees of photoconvertibilityobserved can be accommodated by altering just two forms ofPChle, PChle 630 and PChle 650. While curve analysis alonecannot prove any particular association, these results addcredence to the suggestion that ATP inhibits the transforma-tion of PChle 650 to PChle 630.

Since ATP has not been shown to induce complete trans-formation of PChle 630 into PChle 650 in aged etioplasts, fac-tors other than ATP may be involved in this conversion. Thetransformation appears to take place in discrete steps, thepre-existing PChle 650 being continually removed in thelight, thus allowing further formation of PChle 650 fromPChle 630. Similarly ATP does not induce PChle 650 forma-tion if added to fresh etioplasts unless the initial pool ofPChle 650 is first transformed into Chle. One possible ex-planation for this observation is that only a limited number ofsites for PChle 650 formation exist and that this numberfalls even lower in aged etioplasts. Thus the rate of ATP-induced PChle 630 photoconversion (i.e. the yield per flash)would be expected to depend on the degree of photoconverti-bility at the time of ATP addition. This indeed seems to bethe case as seen from the results of three experiments shownin Table V. (This correlation cannot be extrapolated to 100%photoconvertibility in fresh etioplasts since the PChle levelis now limiting).

It is possible to propose mechanisms for the ATP effectfrom a comparison of the results of the present experimentswith isolated etioplasts and those experiments in which wholeleaf tissue or holochrome preparations have been used. Alimitation in the number of sites for PChle photoconversion inetioplasts in vivo has already been well documented (30-32),and these sites have been identified as the binding sites forPChle on the holochrome protein. The association betweenPChle and the holochrome protein is thought to be necessaryfor photoconversion into Chle (12, 14, 22, 25, 27) and is sensi-

tive to various chemical and physical treatments which bringabout conversion of PChle 650 into PChle 630 (3, 4, 6, 10).PChle-holochrome has an in vitro absorption maximum near640 nm (6, 12, 14, 25) and in the etioplast membranes exists asan aggregate of two or more of these PChle/protein com-plexes. This result allows porphyrin ring interaction, giving riseto the 650 nm absorption maximum (4, 26). Thus the forma-tion of PChle 650 requires that the holochrome is both able tobind PChle (PChle 630) and to assume the correct orientationor association within the membrane. Photoconversion itselfmay only require the former. Since ATP is necessary notonly for photoconversion of PChle 630 but also induces theformation of and stabilizes PChle 650, it seems that ATPcould be affecting both these holochrome properties. Onepossible scheme to show the proposed role of ATP is given inFigure 7 and is based on the proposals of Mathis and Sauer(19, 20) that PChle 650-holochrome is a dimer, of Dujardinand Sironval (4) that PChle 630 is feebly linked to theholochrome, and of Sundqvist (30, 31) and others (32) that theholochrome acts as a reusable "photoenzyme." The proposalthat ATP induces specific binding between a porphyrin and aprotein unit is not without precedent. Haddock and Shairer (9)have described an ATP-induced reconstitution of cytochromeb oxidoreduction in Escherichia coli homogenates.At the present time, there is no evidence to indicate whether

ATP is acting directly on the PChle-holochrome or whetherthe effect is mediated through another effect on the prolamellarbody membranes. The scheme in Figure 7 can accommodateeither of these two possibilities. Clearly, the determination ofthe mechanism of the ATP effect will be important in anyconsideration of the process of PChle photoconversion invivo.The determination of whether ATP has any regulatory

significance in the biosynthesis of Chl remains a matter forfurther experimentation. Although ATP is the best agent forpreserving photoconvertibility and for initiating PChle 630photoconversion, both ADP and other nucleoside triphos-phates also have significant effects. The experiments are com-plicated by the apparent differential permeability of theetioplast envelope to ATP and other triphosphates, so thatwhen envelope-free preparations are used, the apparent speci-ficity for ATP is decreased. An alternative explanation, thatthe processes of dilution and homogenization involved inpreparing envelope-free etioplasts remove factors which conferspecificity cannot be ruled out. It is unlikely that the molarconcentrations of UTP, CTP, and GTP approach the con-

Chle

EP mPJ +ATP, EPzi

PChle 630 PChIe 650FIG. 7. Diagram illustrating the possible effects of the addition of

ATP to the PChle/holochrome complex. The holochrome (H) is asso-ciated with PChle (P) with feeble links (.* *) when the maximum is 630nm. In the presence of ATP the complex is activated, the process beingenvisaged as a conformational change in the holochrome (perhaps theformation of a dimeric species) enabling pigment-pigment interaction(;zs) to give the 650 nm form. Illumination (hv) induces Chle formationand allows further PChle 650 synthesis in the presence ofATP.

Plant Physiol. Vol. 56, 1975 119

120 HORTON A

centration of ATP in the cell, so these molecules may be ex-pected to be relatively unimportant in any effects on photo-convertibility in vivo. The ability of ADP to give as much as90% of the ATP effect is a more serious objection to thesuggestion that ATP itself has a regulatory effect. While it ispossible that the ADP effect be, in part, due to its priorenzymic conversion to ATP, the similar concentration rangesfor the ADP and the ATP effects suggest that the site ofaction has a similar affinity for both ADP and ATP. Theinhibition of the ATP effect by AMP, which is particularlyevident in the PChle 630 photoconversion experiments, (seeTable V) suggests that the ATP + ADP:AMP ratio ratherthan the ATP:ADP ratio could be important in regulatingphotoconvertibility. Since PChle 630 and PChle 650 appearto be intermediates in Chl biosynthesis (7, 8, 28-31, 34) theseeffects of adenine nucleotides could certainly be relevant toevents occurring in whole plants. Similarly, the fact that ATPonly induces photoconversion of unphytylated PChle 630 intoChle is consistent with unphytylated PChle (and not phytylatedPChl) being a major intermediate in Chl biosynthesis. Indeed,since there is competition between phytylation and PChle 650formation for the newly synthesized PChle, the ATP effectdescribed here may be relevant in determining the degree ofPChl/e phytylation. Of interest, therefore, is the observationof Rebeiz and Castelfranco (23) that ATP stimulates the in-corporation of '4C-8-aminolevulinic acid into PChle, but notinto PChl in homogenates of etiolated cucumber cotyledons.

LITERATURE CITED

1. ADAM, H. 1963. Adenosine 5'-diphosphate and adenosine-5'-monophosphate.In: H. U. Bergmeyer, ed., Methods of Enzymatic Analysis. Academic Press,New York, pp. 573-577.

2. ANDERSON, J. M. AND N. K. BOARDMAN. 1964. Studies on the greening of dark-grown bean plants. II. Development of photochemical activity. Aust. J.Biol. Sci. 17: 93-101.

3. BUTLER, W. L. AND W. R. BRIGGS. 1966. The relation between structure andpigments during the first stages of proplastid greening. Biochim. Biophys.Acta 112: 45-53.

4. DUJARDIN--, E. AND C. SIRONVAL. 1970. The reduction of protochlorophyllideinto chlorophyllide. III. The phototransformability of the forms of the pro-tochlorophyllide lipoprotein complex found in darkness. Photosynthetica 4:129-138.

5. EGNEUS, H. AND C. SUNDQUIST. 1970. An action spectrum for the transforma-tion of ALA-protochlorophyllide to ALA-chlorophyllide in the wavelengthregion 605-675 nm. Photosynthetica 4: 81-83.

6. GASSMAN-, M. 1973. A reversible conversion of phototransformable protochloro-phyllideea8 to photoinactive protochlorophyllideeaa by hydrogen sulphide inetiolated bean leaves. Plant Physiol. 51: 139-145.

7. GASSIMAN, M. 1973. The conversion of photoinactive protochlorophyllideezs tophototransformable protochlorophyllide6e0 in etiolated bean leaves treatedwith 5-aminolevulinic acid. Plant Physiol. 52: 590594.

8. GRANICK, S. AND M. GASSMAN. 1970. Rapid regeneration of protochlorophyl-lideess. Plant Physiol. 45: 201-205.

9. HADDOCK, B. A. AND H. U. SCcAIRER. 1973. Electron transport chains ofEscherichia coli: reconstitution of respiration in a 6-aminolevulinic acid-requiring mutant. Eur. J. Biochem. 35: 34-45.

10. HENN-INGSEN, K. W. 1970. Macromolecular physiology of plastids. VI. Changesin membrane structure associated with shifts in the absorption maxima ofthe chlorophyllous pigments. J. Cell Sci. 7: 587-681.

11. HENNINGSEN, K. W. AND J. E. BOYNTON. 1969. Macromolecular physiology of

LIND LEECH Plant Physiol. Vol. 56, 1975

plastids. VII. The effect of a brief illumination on plastids of dark-grownbarley leaves. J. Cell Sci. 5: 757-793.

12. HENNINGSEN, K. W., S. W. TsORNE, AND N. K. BOARDMAN. 1974. Properties ofprotochlorophyllide and chlorophyllide holochromes from etiolated andgreening leaves. Plant Physiol. 53: 419425.

13. HORTON, P. AND R. M. LEECH. 1972. The effect of ATP on photoconversion ofprotochlorophyllide into chlorophyllide in isolated etioplasts. FEBS Lett.26: 277-280.

14. KAHN, A., N. K. BOARDMAN, AND S. W. THORNE. 1970. Energy transfer betweenprotochlorophyllide molecules: evidence for multiple chromophores in thephoto-active protochlorophyllide-protein complex in vivo and in vitro. J.Mol. Biol. 48: 85-101.

15. KLEIN, S. AND J. A. SCHIFF. 1972. The correlated appearance of prolamellarbodies, protochlorophyllide species, and the Shibata shift during develop-ment of bean etioplasts in the dark. Plant Physiol. 49: 619-626.

16. LAMPRECHT, W. AND I. TRAUTSCHOLD. 1963. Adenosine 5' -triphosphate: de-termination with hexokinase and glucose-6-phosphate dehydrogenase. In:H. U. Bergmeyer, ed., Methods in Enzymatic Analysis. Academic Press,New York. pp. 543.

17. LEESE, H. J. AND J. R. BRONK. 1972. Automated fluorometric analysis of mi-cromolecular quantities of ATP, glucose and lactic acid. Anal. Biochem. 45:211-221.

18. LEESE, B. M., R. M. LEECH, AND W. W. THOMSON. 1972. Isolation of plastidsfrom different regions of developing maize leaves. In: G. Forti, M. Avron,and A. Melandri, eds., Proceedings of the 2nd International Congress on

Photosynthesis Research, Vol. 2. Dr. W. Junk N. V. The Hague. pp. 1485-1494.

19. MIATHIS, P. AND K. SAUER. 1972. Circular dichroism studies on the structureand the photochemistry of protochlorophyllide and chlorophyllide holo-chrome. Biochim. Biophys. Acta 267: 498-510.

20. MATHIS, P. AND K. SAUER. 1973. Chlorophyll formation in greening bean leavesduring the early stages. Plant Physiol. 51: 115-119.

21. MURRAY, H. E. AND A. 0. KLEIN. 1971. Relationship between photoconvertibleand nonconvertible protochlorophyllides. Plant Physiol. 48: 383-388.

22. NIELSON. 0. F. AND A. KAHN-. 1973. Kinetics and quantum yield of photocon-version of protochlorophyllide to chlorophyllide. Biochim. Biophys. Acta292: 117-129.

23. REBEIZ, C. A. AND P. A. CASTELFRANCO. 1971. Protochlorophyll biosynthesisin a cell-free system from higher plants. Plant Physiol. 47: 24-32.

24. RIDLEY, S. M. AND R. MI. LEECH. 1969. Chloroplast survival in vitro. In: H.Metzner, ed., Progress in Photosynthesis Research, Vol. 1. Tubingen, Ger-many. pp. 229-244.

25. SCHOPFER, P. AND H. W. SIEGELMAN. 1968. Purification of protochlorophyllideholochrome. Plant Physiol. 43: 990-996.

26. SELISKAR, G. J. AND B. KE. 1968. Protochlorophyllide aggregation in solutionand associated spectral changes. Biochim. Biophys. Acta 153: 685-691.

27. SIRONVAL, C., M. R. MICHEL-WOLWERTZ, AND A. MADSEN. 1965. On the natureand possible functions of the 673 and 684 mA forms of in vivo chlorophyll.Biochim. Biophys. Acta 94: 344-354.

28. SUNDQUIST, C. 1969. Transformation of protochlorophyllide formed fromexogenous 3-aminolevulinic acid in continuous light and in flashlight. Phys-iol. Plant 22: 147-156.

29. SUNDQUIST, C. 1970. The conversion of protochlorophyllideesm to protochloro-phyllideso in leaves treated with 3-aminolevulinic acid. Physiol. Plant. 23:412424.

30. SUNDQUIST, C. 1973. The relationship between chlorophyllide accumulation,the amount of protochlorophyllideems and protochlorophyllide&ae in dark-

grown wheat leaves treated with 8-aminolevulinic acid. Physiol. Plant.28: 464-470.

31. SUNDQUIST, C. 1973. The influence of varying light intensities on the photo-transformation of protochlorophyllideess in dark-grown wheat leaves treatedwith 8-aminolevulinic acid. Physiol. Plant. 29: 434-439.

32. SUZER, S. AND K. SAUER. 1971. The sites of photoconversion of protochloro-phyllide to chlorophyllide in barley seedlings. Plant Physiol. 48: 60-63.

33. TREFFRY, T. 1970. Phytylation of chlorophyllide and prolamellar body trans-

formation in etiolated peas. Planta 91: 279-284.34. TIORNE, S. W. 1971. The greening of etiolated bean leaves. I. The initial pho-

toconversion process. Biochim. Biophys. Acta 226: 113-127.