Mar 18, 1975 - SUNDQUIST, C. 1969. Transformation of protochlorophyllide formed from exogenous 3-aminolevulinic acid in continuous light and in flashlight.
Plant Physiol. (1975) 56, 113-120
The Effect of ATP on the Photoconversion of Protochlorophyllide in Isolated Etioplasts of Zea mays' Received for publication September 17, 1974 and in revised form March 18, 1975
HORTON2 AND RACHEL M. LEECH Department of Biology, University of York, Heslington, York, YOI 5DD, England PETER
various physical treatments of the leaves (3, 4, 5, 10). The changes in the degree of photoconvertibility are due to changes in the proportions of photoconvertible (mainly PChle 650) and nonphotoconvertible protochlorophyllide (PChl/e 630) (30). The relationship between these forms and the mechanisms responsible for maintaining their relative proportions is still largely unknown. The nonconvertible form appears to consist of partly phytylated pigment, and its inability to
The transformation of protochlorophyllide (PChle) into chlorophyllide (Chle) has been studied in isolated etioplasts from Zea mays. ATP (1.5 mM) prevented the transformation of photoconvertible PChle 650 to PChle 630 in aged etioplasts. Curve analysis indicated that the ATP effect on photoconvertibility could be entirely accounted for by changes in the proportions of PChle 630 and PChle 650 and examination of the isolated pigments revealed that only unphytylated PChle could be activated transfer absorbed photons to other PChle forms suggests that for photoconversion by ATP. In etioplasts aged for 5 hours, it may be located at a different position in the etioplast (14, ATP also stimulated photoconversion of PChle 630 into Chle 34). However, there is evidence that nonconvertible PChl can 670. The process was temperature-sensitive and involved PChle serve as a source for the rapid resynthesis of PChle 650 650 and Chle 680 as intermediates. AMP alone had no effect, immediately after photoconversion (8). This situation is clearly but inhibited ATP retardation of PChle loss. ADP had a similar seen when excess PChle 630 is accumulated by ALA treatment but lesser effect than ATP. The ADP response, but not the ATP (7, 28-31). Gassman (6) has also shown interconversion of response, was considerably enhanced in the presence of an ATP- PChle 650 and PChle 630 by H,S treatment of whole leaves. generating system (phosphoenolpyruvate/pyruvate kinase). Recently using isolated intact etioplasts, we observed a slow UTP, GTP, and CTP gave 40 to 50% of the ATP response with transformation of PChle 650 into PChle 630 during dark intact etioplasts. In envelope-free etioplasts, ATP gave the incubation (13). Addition of ATP prevented this transformagreatest response but the other nucleotides were now 80% as tion and in addition stimulated photoconversion of PChle 650 effective as ATP. After primary photoconversion, ATP stimu- in aged etioplasts (13). In this paper, the results of a detailed lated resynthesis of PChle 650. It is proposed that ATP both analysis of the effects of ATP on the protochlorophyll and gives the holochrome the ability to bind to the PChle molecule protochlorophyllide species of isolated maize etioplasts is and enables additional association of the pigment-protein com- given, and evidence for the direct conversion of PChle 630 to PChle 650 in a cell-free system is presented. plex to form PChle 650.
MATERIALS AND METHODS Etioplast Isolation and Incubation. Etioplasts were isolated from dark-grown maize seedlings as described previously (18). One-ml samples of etioplasts were incubated in darkness at
The photoconversion of PChl' into Chle is the first detectable effect of illumination on dark-grown plants. The ability to carry out the photoconversion varies with plant age (11, 15, 34), and the extent of the conversion is sensitive to 1 This 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 University, West Lafayette, Ind. 47907. 3 Abbreviations: PChle: protochlorophyllide; PChl: protochlorophyll; Chle: chlorophyllide; PEP/PK: phosphoenolpyruvate plus pyruvate kinase; PChl/e and Chl/e refer to mixtures of phytylated and unphytylated pigments; PLB: prolamellar body. For identification purposes several spectral forms of protochlorophyll and chlorophyll are referred to as follows: PChl/e 630, absorption maximum 628 to 630 nm; PChle 650, maximum at 650 nm; Chl/e 670, maximum at 669 to 674 nm; Chle 680, maximum at 680 to 685 nm. The ranges of maxima refer to previously published values. 113
20 C under continuous slow rotation (13, 24) in a medium containing 0.5 M sucrose, 0.4% BSA (Cohn Fraction V, Koch Light), 2.5% Ficoll, 5 mm cysteine, 1 mm MgCl,, 1 mm EDTA, 5 mm KHCO,, 0.5 mM sodium acetate, and 1 mM L-glutamic acid 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 was obtained from Boehringer. Determination of Photoconvertibility. Etioplast samples (1 ml) were diluted with 2 ml of glycerol, and each absorption spectrum was recorded at room temperature using a Shimadzu MPS 50L multipurpose spectrophotometer. The PChl/e concentration was between 1.2 and 2.4 /-LM. The sample was then illuminated for 40 sec using a 150-w tungsten filament lamp filtered through 17 cm of H20 and giving an intensity of approximately 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
HORTON AND LEECH
of photoconvertibility of an etioplast sample was measured by expressing the Chle peak height at 680 nm as a percentage of that obtained by illuminating freshly isolated etioplasts. A correction for scatter was made by drawing a line to pass through the absorbance values at increments of 10 nm from 680 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 indication of photoconvertibility was shown by comparison with pigment determinations on 80% acetone extracts. Etioplast absorption spectra were also analyzed quantitatively by curve analysis using a Dupont curve analyzer. Determination of Phytylated and Unphytylated PChle. Phytylated and unphytylated chlorophyllous pigments were separated by partition between petroleum ether and aqueous acetone by the method of Treffry (33). Petroleum ether (1.8 ml) was added to 5 ml of an 80% acetone etioplast extract, and the two layers were separated. In common with other workers, PChl and PChle concentrations were determined using the specific absorption coefficients in 80% acetone described by Anderson and Boardman (2). These appear to be the only extinction coefficients available. The use of the value in 80% acetone for PChl (instead of in petroleum ether) apparently alters the extinction coefficient measured by only 5%. The purity of each phase was tested by TLC on Silica Gel H (Merck) using benzene-ethyl acetate-ethanol (8:2:2, v/v) as described by Rebeiz and Castelfranco (23). Determination of Adenine Nucleotides. ATP levels (during experiments when ATP was added initially) were determined by the enzymic method described by Lamprecht and Trautschold (16). ADP and AMP were determined as described by Adam (1). For the determination of the very low endogenous ATP levels in the etioplast samples, the automated fluorometric analytical procedure of Leese and Bronk (17) was employed. All cofactors and enzymes for these determinations were obtained from Boehringer Corporation.
Plant Physiol. Vol. 56y 1975
Table I. Effect of EDTA oni Degree of Photoconzvertibility Envelope-free etioplasts were incubated for 5 hr under rotation in darkness with and without ATP. The incubation medium contained tris-HCl buffer, pH 7.5, instead of phosphate, and MgCl2 and EDTA only where indicated. Etioplasts were isolated in a similar Mg-free medium. Results are the average of duplicate incubations. Photoconvertibility was determined as described in the text. Photoconvertibility Treatment 2 mm AMg2+
No additions 1.5 mM ATP 2 mM EDTA 1.5 mm ATP + 2 mN EDTA
25 48 48 58
27 67 32
RESULTS with and without ATP. Figure 1 gives Photoconvertibility the spectra obtained during a typical experiment, showing about 75% retention of photoconvertibility in the etioplast suspension incubated with ATP. Exactly the same results were obtained with intact and envelope-free etioplasts (13). In addition, it was found that washed etioplasts showed 80% photoconvertibility after 5 hr of incubation (not shown in Fig. 1). (Washed etioplasts are prepared by centrifuging the initial etioplast fraction through a layer of 0.6 M sucrose as described previously , and the fractions so obtained contain approximately 90% envelope-bound etioplasts.) Identification of ATP as Primary Effector. Several additional control experiments were undertaken to determine whether the effects on photoconvertibility caused by adding ATP were due to ATP per se. Since ATP has a high affinity for divalent cations, it is possible that the ATP effect could be accounted for as a reduction in cation concentration. However, it was found that the metal chelator EDTA had a stimulatory effect on photoconvertibility (Table I) and that this effect was partially additive to the ATP response. Addition of Mg2` further differentiated between the responses to ATP and to EDTA since the addition of 2 mm MgCl2 enhanced the ATP effect but inhibited the EDTA effect. The Mg2" requirement for full preservation of photoconvertibility by ATP is not a classical one, since excess MgCl, (>5 mM) inhibits the response to ATP (data not shown). From the results shown in Table I, it seems unlikely that the ATP effect can be explained in terms of a simple chelation. The question of whether the products of ATP hydrolysis were active was next investigated under the standard conditions described under "Materials and Methods." It was found that changes in Pi concentration (0-100 mM) or addition of 1.5 mm sodium PPi had no effect on photoconvertibility. Adenine nucleotide levels during a 5-hr incubation period were also 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 afterwards decayed slowly to 0.15 mM. The level of ATP fell quickly to Wavelength (nm) 0.3 mm after 2 hr and to 0.1 mm by 5 hr. These kinetics are consistent with a reaction sequence by which ATP is first FIG. 1. Absorption spectra of etioplasts incubated for 5 hr in darkness to ADP which is then broken down to AMP and not with 1.5 mm ATP compared to the spectra of a 5-hr sample incubated degraded without ATP and to the spectra of freshly isolated etioplasts. Spectra were with a simple myokinase reaction. Indeed if an adenylate recorded after dilution with glycerol before ( ) and after ( ...) photo- kinase reaction were totally responsible for the ATP breakdown, the "myokinase" ratio should remain constant; in conversion.
ATP AND PROTOCHLOROPHYLLIDE
Plant Physiol. Vol. 56, 1975
photoconvertibility -E 10
Time (hr) FIG. 2. Adenine nucleotide levels determined durin
cubation following initial addition of 1.25 mm ATP. Al and AMP (0) were determined using aliquots of dupli samples.
5-hr dark in-
was clearly demonstrated when an ATPregenerating system (phosphoenolpyruvate plus pyruvate kinase) was incorporated into the incubation medium (Table III). PEP/PK markedly increased the preservation of photoconvertibility by suboptimal concentrations of ATP and by ADP to a level equal to or even greater than the photoconvertibility found at optimal ATP levels (1.5 mM). PEP/ PK induces a small increase in photoconvertibility in the absence of added ATP or ADP, possibly due to regeneration of endogenous ATP. Determination of Specfficity of ATP Effect in Comparison with Effects of Other Nucleotide Triphosphates. The degree of specificity with respect to the purine and pyrimidine residues was next examined. GTP, CTP, and UTP were all capable to some extent of preserving photoconvertibility (Table IV).
Photoconivertibility of PChle inz Isolated Maize Etioplasts The photoconvertibilities of envelope-free etioplast samples
Table II. Photoconvertibilities of Enzvelope -bound anid Enivelope-free Etioplasts Photoconvertibilities of etioplasts incubated in darkness for 5 hr were determined as described in the text. Th e data for experiments 1, 2, 3, and 4 to 6 are means of 4, 6, 5, anId 2 incubations, respectively.
were determined after 5 hr of dark incubation as described in the text. ATP and ADP were added at the final concentrations shown.
Phosphoenolpyruvate (sodium salt) was 5 mM; 20 l (2 mg/ml in glycerol) of pyruvate kinase (EC 2.7.1 .40) were added just prior to incubation (PK). Each value is a mean of triplicate (+PEP/PK) or
duplicate (-PEP/PK) incubations. Experiments 1 and 2
3. Envelope-free 4. Envelope-free 5. Envelope-bound
Control + ADP Control + ADP Control + ADP Control + ADP Control + AMP Control + AMP
+ATP (1.5 mm)
38 51 52 57 48 38 47 58 68 59 49
49 61 65 59 60
11 10 13 2 12
57 68 65 70
10 10 3 11
-ATP 1. Envelope-bound
30 42 51 68 39 49 47 59 10 10 47
53 74 55
68 63 41 23 68 60
fact, it changes from 1 at zero time to 3 after 5 hr incubation. The extensive degradation of ATP suggested the possibility that AMP or ADP rather than ATP may be the active agents in the preservation of photoconvertibility in etioplast suspensions. Initial addition of ADP was found to cause a marked preservation of photoconvertibility, but the response was always significantly below that obtained with ATP (Table II, experiments 1-4). An optimum ADP concentration between 1 and 2 mm was found, i.e. similar to the optimum previously found for ATP (13). The ATP and ADP effects were not additive (Table II, experiment 4). Addition of AMP had no effect on the level of photoconvertibility and indeed caused inhibition of the ATP effect (Table II, experiment 5), although this inhibition was not as reproducible when envelope-free etioplasts were used (Table II, experiment 6). The above observations cannot be reconciled with an explanation that the effect of the added ATP is only due to its breakdown to ADP or AMP. The greater ability of ATP, as opposed to ADP to preserve
1. No addition + ATP (0.1 mM) + ATP (0.5 mM) + ATP (.S mM) + ADP (0.S mM) Control (+PK)' 2. No addition + ATP (0.1 mM) + ATP (1.S mM) + ADP (0.5 mM) Control (+PK)'
1 The absence of any effect due to 4 mM PEP alone was shown in a separate experiment presented earlier (13).
Table IV. Effect of Differentt Nucleotide Triphosphates on
Photoconivertibility Determinledfor Initact (Enzvelope-boutnd) anzd Enivelope-free Etioplasis Photoconvertibilities after 5 hr of dark incubation were determined as described in the text. Nucleotide concentrations (1.5 mM) are final concentrations. Experiments 1, 2, and 3 are separate experiments. Each value is an average of triplicate incubations. Photoconvertibility
Nucleotide Triphosphate Envelope-free
None GTP (1.5) UTP (1.5) CTP (1.5) 5. ATP (1.5)
1. 2. 3. 4.
9 25 19 28 51
49 65 59 67 72
HORTON AND LEECH
With intact, envelope-bound etioplasts only about 40 to 50% of the level seen in the presence of ATP is observed. However, with envelope-free preparations these values increase to about 80 to 90%. In both cases, the order of specificity could be tentatively described as ATP>CTP>GTP>UTP. Since of all the adenine nucleosides and the nucleotide triphosphates examined, ATP had the largest and most consistent effect on photoconvertibility in intact isolated etioplasts, the effect of ATP on pigment interconversion in isolated etioplasts was examined in greater detail. Measurement of Endogenous ATP Levels. The endogenous ATP level was compared for fresh etioplasts suspended in the medium at zero time and after incubation for 5 hr. The ATP concentration fell from 4.2 nmoles/ml total volume at zero time to 2.5 nmoles/ml after 5 hr. This result suggests that the effect of exogenously added ATP is observed because the addition is counteracting the effect of a depletion of endogenous ATP which occurs during incubation. 63(10637 650
%/0total % Photoconversion
n 630637 650
630 637 650
630 637 650
FIG. 3. Analysis of etioplast spectra into component curves. Absorption spectra (-) were recorded in glycerol before and after 5 hr dark
incubation. The spectra were analyzed into 3 component gaussian bands (- -) with maxima at 630, 637, and 650 nm and widths at half-maxima of 18 to 19 nm, 14 to 15 nm, and 12 to 13 nm, respectively. At the righthand side are shown the areas of each PChle component as a percentage of the total PCnle. Tne p rcentages of photoconvertibilities were determined 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 hr incubation -ATP.
Plant Physiol. Vol. 56, 1975
Curve Analysis of PChl/e Spectra. The spectra in Figure 1 suggest that ATP preserves PChle 650 at the expense of PChl/e absorbing near 630 nm. At least three different forms of PChl/e exist in etiolated leaves (14) with 77 K maxima at 628, 637, and 648 nm. Therefore, to obtain a clearer definition of the ATP effect, PChl/e absorption spectra were resolved into component curves. The aim was to obtain fit to all types of PChl/e spectra by altering the proportions of two (or more) components whose peak positions and half-widths remain constant. Gaussian bands with maxima at 630, 637, and 650 nm were found to give perfect fit to spectra of freshly isolated etioplasts and of etioplasts incubated in vitro with and without ATP, by merely altering the peak heights of the 630 and 650 nm bands (Fig. 3). The spectra could not be fitted by using just two components with maxima 630 (±2 nm) or 650 (+2 nm). The proportional areas of each component are also represented (on the right-hand side) in Figure 3. Clearly, the change from 0 to 5 hr in the absence of ATP can be accounted for merely by changing the proportions of the two components PChl/e 630 and PChle 650 to obtain good fit. Similarly, the effect of ATP on the system can also be simulated by altering the proportions of PChle 650 and PChl/e 630. The fact that PChle absorption bands in solution are not perfect Gaussian bands means that some caution must be applied in using this method of determining the proportions of different spectral forms of PChl/ e. For this reason the validity of the simulations was checked by experimentation as follows: 1. At 0 hr the results of curve analysis indicated that 72% of the total PChl/e would be photoconvertible. This agrees well with pigment analysis of acetone extracts which showed that 69% of the total PChl/e was converted into Chl/e on illumination. 2. The percentage of the original (0 hr) PChl/e still photoconvertible after 5 hr can be determined by curve analysis and also directly by measuring the amount of formation of Chle 680 on illumination. Again, there is good agreement between the two types of measurement. In the presence of 1.5 mM ATP, curve analysis and Chle 680 measurement both indicate that 47% of the total PChl/e is photoconvertible, whereas in the absence of ATP, values of 37 and 29% respectively, are obtained. For easier comparison with previous experiments, the values are shown in Figure 3, a and b, expressed as percentage of photoconvertibilities rather than as the percentage of the total PChl/e convertible. The similarity of the simulated and measured values in each case is clear. ATP-induced Photoconversion of PChle 630. It was previously reported that ATP stimulated photoconversion of PChle 630 into Chle in 5-hr-old etioplasts (13). This transformation is a function of the total length of the light plus dark phases, rather than the total amount of irradiation received; thus treatment of etioplasts with the same total illumination but given continuously without intervening dark periods, induces very little PChl/e 630 conversion, suggesting that dark reactions limit the accumulation of Chle. This suggestion is supported by the observation that the rate of PChle 630 photoconversion is temperature-sensitive (Fig. 4). At 3 C, no PChl/e 630 conversion occurred and while increasing the incubation temperature to 10 C allowed a certain amount of extra Chl/e formation, not until the temperature had been increased to 20 C did PChl/e 630 conversion proceed at a
Examination of absorption spectra recorded during PChle transformation supports the suggestion that transient spectral intermediates exist. The final absorption maximum of the Chle (in this experiment 90% of that obtained by illuminating
Plant Physiol. Vol.
ATP AND PROTOCHLOROPHYLLIDE
freshly isolated etioplasts) is at 670 nm (Fig. 5, a and b). A slight shift in the Chl/e absorption maximum from 672 nm (1 min) to 670 nm (4 min) was observed when the spectra were 0-06 recorded after light treatments. The absorbance changes involved are small but a difference spectrum (1 min minus 4 min) did suggest that changes in a Chl/e component absorbing near 680 nm were involved (Fig. 4c). In addition a slight decrease 0-04 in absorbance at 630 nm accompanied by an increase at 650 nm was observed during the postillumination dark period. The difference spectrum (after illumination minus before illumination) recorded at 20 C shows a wide Chl/e band 0-02 with a maximum between 675 and 680 nm (Fig. 4d). Negative peaks at 630 and 650 nm were also obtained. A similar difference spectrum obtained after lowering the temperature to 3 C before light treatment shows a clear Chl/e maximum at 24 682 nm. No absorbance changes near 630 or 650 nm were 20 16 12 8 0 4 detectable. It is to be stressed that throughout, the absorption No. of light and dark treatments changes involved are small (10' to 102 optical density units) FIG. 4. PChle 630 photoconversion at 3, 10 and 20 C. Etioplasts, as well as rapid. incubated in darkness for 5 hr were cooled to 3 C and exposed to a series The ability of other nucleotides to stimulate photoconverof light + dark treatments as described in Table VI. After four treatments, sion of PChl/e 630 are shown in Table V and indicate that the temperature was raised to 10 C and after 8 treatments to 20 C. The the ATP effects on photoconvertibility and PChl/e 630 photoabsorption spectra were determined following each flash and AA670 conversion are different manifestations of the same basic measured.
Table V. Effect of Different Nucleotides onz Rate and Amounti of PChl/e 630 Photoconversionz Etioplast samples were incubated in darkness for 5 hr without any added nucleotide and photoconvertibilities recorded (A 37%, B = 19(%, C = 22%). One-ml samples were diluted with and without nucleotide (1.5 mm final concentrations) and given the usual 40-sec illumination to convert PChle 650 to Chl before exposure to a series of light + dark treatments (5 sec photoconversion illumination plus 6 min darkness) continued until no further increase in absorbance at 670 nm was recorded. The absorption spectra were recorded after each treatment and A67, plotted against treatment number. Rates of absorbance increase were measured and expressed as AA/light treatment (in O.D. units). Total increases above that obtained by initial photoconversion were also measured (AA in O.D. units). Experiments A, B, and C are separate experiments. In experiment C the increase in A670 was biphasic for CTP and GTP; the rates given are for the second, more rapid phase which began after nine treatments. For the first nine treatments rates approximately equal to those for no additions, were recorded for CTP and GTP. For photoconversion of PChl/e 630 samples were not diluted with glycerol. =
of Chli formation
~~Rate 103 X
103 X AA
A. None ADP ATP ADP + ATP B. None AMP ATP ATP + AMP C. None ATP CTP GTP UTP
FIG. 5. Absorption and difference spectra observed during ATPinduced PChle 630 photoconversion. Experimental details were as described under Table VI. a: Absorption spectra after 5 hr of dark incubation: after primary photoconversion (- ----), after two light treatments (- -) and after saturation ( ) i.e. eight light treatments; b: spectrum of a freshly isolated sample after photoconversion (not in glycerol); c: difference spectrum (1 min after illumination minus 4 min after illumination) recorded after four light treatments; d: difference spectrum (after illumination minus before illumination) recorded for fourth light treatment at 20 C (-) and 3 C (--).
10.0 14.0 9.0
70 68 67 6 0 69 0 12 64 42 30 19
0.4 0 8.0 0 0.8
5.6 1.9 1.3 0.8
NT: number of treatments needed for completion of
16 16 1 14 1 16 16 29 29 29 conver-
HORTON AND LEECH
Plant Physiol. Vol. 56, 1975
Effect of ATP on Resynthesis of PChle 650. In whole leaves after primary photoconversion, reappearance of PChle 650 occurs by conversion from PChle 630. A similar phenomenon was observed in etioplast suspensions. Addition of ATP is able to stimulate the PChle 650 reappearance (Fig. 6). In the presence of ATP (1.5 mM), difference spectra (e.g. 15 min minus 0 hr, 90 min minus 0 hr) show the emergence of a 650 nm peak accompanied by a trough at 630 nm (Fig. 6a). In contrast, the same difference spectra recorded in the absence of ATP show an absorbance increase due to the appearance of Chl/e 670 and a smaller trough at about 634 nm (Fig. 6b). The possibility that the increase at 670 nm was obscuring the presence of a large 650 nm band is discounted from the
analysis of difference spectra (+ATP minus -ATP)
FIG. 6. Reappearance of PChle 650 in the presence and absence of 1.5 ATP. Etioplasts were incubated unstirred in the spectrophotometer cuvette after primary photoconversion, and their spectra were recorded. a, b: Difference spectra, 15 min minus zero time (- -) and 90 min minus ) for a sample plus ATP (a) and a sample minus ATP (b); zero time ( c: difterence spectra (+ATP minus -ATP) at 15 min (- -) and 90 min (); d: difference spectra from measurements after 1 hr of incubation ) and minus ATP before and after a second illumination plus ATP ( (- -); e: time course for the absorbance increase at 650 nm following primary photoconversion +ATP (A) and -ATP (0). mM
effect. The same saturating Chl/e level was obtained with ADP and ATP, although more light treatments were required with ADP because the rate of Chl/e formation was less than with ATP. AMP was found to inhibit completely the action of 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 induced by CTP, GTP, and UTP were all very slow and 29 light treatments 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 time at which light treatments commence. Thus in experiment A the photoconvertibility was nearly 40% and high rates of Chl/e formation (0.014/treatment) resulted. In contrast, the low rates in B and C (average 0.007/treatment) are accompanied by low photoconvertibility, at approximately 20%.
in Figure 6c; a peak at 650 nm (PChle 650) and a trough at 630 nm (PChl/e 630) are clearly revealed. A time course for the changes in absorbance at 650 nm recorded in the presence and in the absence of ATP shows that an increase in the level of PChle 650 induced by ATP can be seen within 15 min (Fig. 6e). A maximum difference was observed at 3.5 hr, after which decay of the newly produced PChle 650 becomes apparent. Confirmation that the absorbance changes at 650 nm are due to PChle 650 is shown by the effect of a second light treatment (20 sec of photoconversion illumination) given after 90 min of dark incubation (Fig. 6d). In the presence of ATP, a large increase in absorbance at 682 nm accompanied by a decrease at 650 nm is induced by illumination (difference spectrum: before illumination minus after illumination). Without ATP no spectral change results, showing that no PChle 650 is present. Measurements of the increase in Chl caused by further illumination show that the presence of ATP can induce between 10 and 20% resynthesis of PChle 650 (expressed relative to the original Chle 680 level) compared to negligible amounts found in the absence of ATP. Investigation of Phytylation of PChl/e. In both PChle 630 photoconversion and PChle 650 reappearance experiments, it is clear that ATP is able to induce photoconversion only of part of the PChl/e pool. In Figure Sa, the final level of nonconvertible PChle is approximately the same as in fresh etioplasts (Fig. Sb). The data in Table VI suggest that ATP is able to induce photoconversion only of that part of the PChle pool which is unphytylated. At 0 hr the nonconvertible PChl/e consists mainly of PChl, the level of which is unaltered by illumination. After 5 hr the proportion of phytylated PChl is unchanged and again is not transformed to Chl on brief illumination. The increase in nonconvertible PChl/e between 0 and 5 hr is totally due to an increase in unphytylated PChle. The ratios at 0 hr and 5 hr were consistently estimated as 3.9 and 3.8, respectively. The amount of PChle at 0 hr not transformed into Chle on brief illumination is just enough to account for the 10 to 20% reappearance of PChle 650 described above. DISCUSSION When etioplasts are incubated in a medium without added ATP, photoconvertible PChle becomes degraded. Since added ATP can partially prevent the decay and the etioplasts possess the ability to hydrolyze ATP, the reason for this degradation seems to lie in a decrease in local ATP concentration. We suggested previously (13) that the preservation of photoconvertibility induced by ATP was explainable in terms of two or more interconvertible PChle forms with maxima near 630 nm and 650 nm, the formation of the 650 photoconvertible form requiring ATP. The results of the present experiments add considerable support to this explanation.
Plant Physiol. Vol. 56, 1975
ATP AND PROTOCHLOROPHYLLIDE
Table VI. Conients and Convertibility of Phlytylated anid unzphyzylated PChl/e in Etioplasts before anid after Inicutbationz Etioplast samples were incubated for 5 hr in darkness as described. Determinations were repeated at 0 hr and 5 hr before and after photoconversion of PChle. PChl/e Conc Treatment Ohr
Before illumination PChle PChl Total After illumination PChle PChl Total
1.49 0.38 1.87
1.36 0.34 1.70
0.19 0.32 0.51
I 1.16 1.51
In freshly isolated etioplasts following primary photoconversion, ATP induces the formation of photoconvertible PChle 650. The difference spectra for this effect suggest that the new PChle 650 is derived from PChle 630 and provide direct demonstration of PChle 630 transformation into PChle 650 under the influence of ATP. The demonstration that the rate of ATP induced photoconversion of PChle 630 into Chle 670 in aged etioplasts is limited by temperature-sensitive dark reactions, suggests the existence of one or more intermediates and analysis of difference spectra indicates that PChle 650 and Chle 680 are two of these intermediates. Curve analyses of PChl/e spectra show that all degrees of photoconvertibility observed can be accommodated by altering just two forms of PChle, PChle 630 and PChle 650. While curve analysis alone cannot prove any particular association, these results add credence to the suggestion that ATP inhibits the transformation of PChle 650 to PChle 630. Since ATP has not been shown to induce complete transformation of PChle 630 into PChle 650 in aged etioplasts, factors other than ATP may be involved in this conversion. The transformation appears to take place in discrete steps, the pre-existing PChle 650 being continually removed in the light, thus allowing further formation of PChle 650 from PChle 630. Similarly ATP does not induce PChle 650 formation if added to fresh etioplasts unless the initial pool of PChle 650 is first transformed into Chle. One possible explanation for this observation is that only a limited number of sites for PChle 650 formation exist and that this number falls even lower in aged etioplasts. Thus the rate of ATPinduced PChle 630 photoconversion (i.e. the yield per flash) would be expected to depend on the degree of photoconvertibility at the time of ATP addition. This indeed seems to be the case as seen from the results of three experiments shown in Table V. (This correlation cannot be extrapolated to 100% photoconvertibility in fresh etioplasts since the PChle level is now limiting). It is possible to propose mechanisms for the ATP effect from a comparison of the results of the present experiments with isolated etioplasts and those experiments in which whole leaf tissue or holochrome preparations have been used. A limitation in the number of sites for PChle photoconversion in etioplasts in vivo has already been well documented (30-32), and these sites have been identified as the binding sites for PChle on the holochrome protein. The association between PChle and the holochrome protein is thought to be necessary for photoconversion into Chle (12, 14, 22, 25, 27) and is sensi-
tive to various chemical and physical treatments which bring about conversion of PChle 650 into PChle 630 (3, 4, 6, 10). PChle-holochrome has an in vitro absorption maximum near 640 nm (6, 12, 14, 25) and in the etioplast membranes exists as an aggregate of two or more of these PChle/protein complexes. This result allows porphyrin ring interaction, giving rise to the 650 nm absorption maximum (4, 26). Thus the formation of PChle 650 requires that the holochrome is both able to bind PChle (PChle 630) and to assume the correct orientation or association within the membrane. Photoconversion itself may only require the former. Since ATP is necessary not only for photoconversion of PChle 630 but also induces the formation of and stabilizes PChle 650, it seems that ATP could be affecting both these holochrome properties. One possible scheme to show the proposed role of ATP is given in Figure 7 and is based on the proposals of Mathis and Sauer (19, 20) that PChle 650-holochrome is a dimer, of Dujardin and Sironval (4) that PChle 630 is feebly linked to the holochrome, and of Sundqvist (30, 31) and others (32) that the holochrome acts as a reusable "photoenzyme." The proposal that ATP induces specific binding between a porphyrin and a protein unit is not without precedent. Haddock and Shairer (9) have described an ATP-induced reconstitution of cytochrome b 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 whether the effect is mediated through another effect on the prolamellar body membranes. The scheme in Figure 7 can accommodate either of these two possibilities. Clearly, the determination of the mechanism of the ATP effect will be important in any consideration of the process of PChle photoconversion in vivo.
The determination of whether ATP has any regulatory significance in the biosynthesis of Chl remains a matter for further experimentation. Although ATP is the best agent for preserving photoconvertibility and for initiating PChle 630 photoconversion, both ADP and other nucleoside triphosphates also have significant effects. The experiments are complicated by the apparent differential permeability of the etioplast envelope to ATP and other triphosphates, so that when envelope-free preparations are used, the apparent specificity for ATP is decreased. An alternative explanation, that the processes of dilution and homogenization involved in preparing envelope-free etioplasts remove factors which confer specificity cannot be ruled out. It is unlikely that the molar concentrations of UTP, CTP, and GTP approach the conChle
PChle 630 PChIe 650 FIG. 7. Diagram illustrating the possible effects of the addition of ATP to the PChle/holochrome complex. The holochrome (H) is associated with PChle (P) with feeble links (.* *) when the maximum is 630 nm. In the presence of ATP the complex is activated, the process being envisaged as a conformational change in the holochrome (perhaps the formation of a dimeric species) enabling pigment-pigment interaction (;zs) to give the 650 nm form. Illumination (hv) induces Chle formation and allows further PChle 650 synthesis in the presence of ATP.
HORTON ALIND LEECH
centration of ATP in the cell, so these molecules may be expected to be relatively unimportant in any effects on photoconvertibility in vivo. The ability of ADP to give as much as 90% of the ATP effect is a more serious objection to the suggestion that ATP itself has a regulatory effect. While it is possible that the ADP effect be, in part, due to its prior enzymic conversion to ATP, the similar concentration ranges for the ADP and the ATP effects suggest that the site of action has a similar affinity for both ADP and ATP. The inhibition of the ATP effect by AMP, which is particularly evident in the PChle 630 photoconversion experiments, (see Table V) suggests that the ATP + ADP:AMP ratio rather than the ATP:ADP ratio could be important in regulating photoconvertibility. Since PChle 630 and PChle 650 appear to be intermediates in Chl biosynthesis (7, 8, 28-31, 34) these effects of adenine nucleotides could certainly be relevant to events occurring in whole plants. Similarly, the fact that ATP only induces photoconversion of unphytylated PChle 630 into Chle is consistent with unphytylated PChle (and not phytylated PChl) being a major intermediate in Chl biosynthesis. Indeed, since there is competition between phytylation and PChle 650 formation for the newly synthesized PChle, the ATP effect described here may be relevant in determining the degree of PChl/e phytylation. Of interest, therefore, is the observation of Rebeiz and Castelfranco (23) that ATP stimulates the incorporation of '4C-8-aminolevulinic acid into PChle, but not into PChl in homogenates of etiolated cucumber cotyledons. LITERATURE CITED
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