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Studies on chloroplast degradation in vivo II effects of aging on hill activity of plastids from Cucurbita cotyledons
Lehrstuhl fur Biochemie cler Pflanze, Gottingen
Studies on Chloroplast Degradation in vivo II Effects of Aging on Hill Activity of Plastids from Cucurbita Cotyledons GOTZ
HARNISCHFEGER
With 11 figures Received July 4, 1973
Summary The rate of dichlorophenol-indophenol (DPIP) and ferricyanide reduction was investigated as a function of the age of the cotyledon from which the plastids were isolated. The pseudooscillatory data obtained could be transformed into normal growth curves by assuming an age dependent pH-shift for optimum activity. The possible reasons for this proton dependency of electron transport are discussed.
Introduction The correlation between structure and function of biological membranes is a topic of considerable interest. A better understanding of their specific interconnection provides a means to elucidate the mechanics of many biological systems, among them respiration and photosynthesis. The investigations described in this paper center around the interdependence of photochemical activity and membrane integrity in chloroplasts of higher plants. Changes in membrane structure due to different physiological conditions which in turn cause alterations in rate of electron transport were achieved by judicious use of the process of aging in vivo. The detailed specifics of the plant system used have been described previously (HARNISCHFEGER, 1973). In brief, chloroplasts were isolated at various stages of development from pumpkin cotyledons grown under controlled conditions. Through continual removal of the epicotyl, cotyledons were obtained which remained green throughout their period of growth and contained plastids which possessed a functionally intact, qualitatively unchanged pigment system. This feature facilitates the study of enzymatic alterations in electron transport attributable to membrane rearrangement and degradation during aging. This paper reports the correlation between age-induced structural changes and electron transport activity of pumpkin chloroplasts.
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
302
G. HARNISCHFEGER
Material and methods Pumpkin, Cucurbita maxima cv. «gelbe, genetzte Riesenmelone» was grown under the conditions described previously (HARNISCHFEGER, 1973). The epicotyl of the plants was continually removed resulting in cotyledons which expanded considerably beyond the size of those found in normal growing plants. Plastids with qualitatively unchanged pigment characteristics were isolated from leaves of various age in the usual manner. Chloroplast preparation always took place at the same time of day in order to minimize the influence of photoperiodic phenomena (BARTELS, 1971). The plastids were stored in 0.4 M sucrose-O.l M phosphate buffer pH 6.3-1 mg/ml BSA in ice under light exclusion. Chlorophyll content was determined according to ARNON (1949). The dichlorophenol-indophenol (DPIP) and ferricyanide (FeCN) Hill-reaction was carried out using the method of POSTIUS and JACOBI (1971). Light was provided by a 300 watt Philips floodlight lamp. The beam was condensed by a set of lenses, passed through a water and an IR filter and was modified through a red plexiglas filter (l > 600 nm) giving a saturating incident intensity of 1.2 X 105 ergs/cm2 X sec. The assay samples contained 2 ml 0.4 M sucrose, 0.5 ml 0.2 M buffer, chloroplasts equivalent to 30-40 fig chlorophyll and 0.1 ml of electron acceptor (2 X 10-3 M DPIP or 3 X 10-2 M FeCN). The direct readout together with standartization and triple measurement of individual points permits a confidence limit of under 5 %. The buffer systems developed by GOOD (1972) viz., MES for the pH-range 5.6-6.8, TES from 6.8-7.8 and TRICINE between 7.6 and 8.3 were used to determine the pH profiles. Overlapping points with these different buffers were measured and the pH of the assay solution was rechecked immediately after illumination on a Metrohm E-516 Titriskop pH-meter.
160 L..
.J::.
140
o
x
~120 0-
o
5100
E80 "0
~ 0-
~ 40
o
o
000 .........
0
"'0
o
/
o
'a
0
I
I
0,I o
I
o
:J..
0
/00
~
E 20
//00
0
/
60
a::
0/
o
:cu
o
/--;---,
o
I
I
10
20
30
40
50
60
70
80
days after seeding
Fig. 1: DPIP-Hill activity of isolated chloroplasts as a function of the age of the cotyledonary material used. The values were measured under saturating light conditions in a medium of pH 6.3.
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Chloroplast Degradation in vivo II
303
10 8 6
20
o
20
40
60
40
60
days after seeding
80
Fig. 2: DPIP-Hill activity as a function of cotyledonary age, plotted on the basis of unit cotyledon area. The data of Fig. 1, measured at pH 6.3, are shown in Fig. 2 a, while Fig. 2 b shows the values obtained at pH 7.5.
~
~ 120
6.0
7.0
pH Fig. 3: pH-profiles of DPIP-Hill activity obtained with plastids isolated from cotyledons of various age (indicated as days after sowing in brackets above each curve).
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
304
G.
HARNISCHFEGER
Results
Effects of aging on the DPIP-Hill reaction The course of the DPIP-Hill reaction, measured at a constant pH of 6.3 and under saturating light intensity was followed through the life span of the cotyledon. Fig. 1 depicts the values obtained and shows a wide scatter of points around the ideal line expected for changes in electron transport properties during development and death. Deviations of this nature are normally attributed to the inherent variability of the plant system. However, when the measured points are replotted on a basis of unit cotyledon area, the curve of Fig. 2 a is obtained, showing what appears to be periodic oscillations. This feature is also reflected, but to a lesser extent, in Fig. 2 b which depicts data measured at pH 7.5. The oscillations were observed not only in all
:r:
0.
o
20
40
60
days after sczczding
80
a.o
7.0 :r:
0.
6.0
o
20
40
60
00
days after seeding
Fig. 4: Position of pH optima of the DPIP-Hill reaction as a function of cotyledonary age. Fig. 4 a and b show two possible ways to connect them.
z.
Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Chloroplast Degradation in vivo II
305
five experimental series performed over a period of two years, but also showed up in all partial reactions tested (compare figs. 7 and 11). Since the yearly season influenced the duration of the total cotyledonary life span not allowing a comprehensive graphical presentation of all results, only data from one representative experimental series are shown in the following.
7.0 I
a.
6.0
10
20
40
60 80
days after seeding
Fig. 5: Logarithmic decline of the pH-optimum starting at the pH 7 value of Fig. 4 b. '-
.c ~
160
.? ~ 120 .Q .c u
~ 80
0: ~ 40 (j)
~
§ :1..
/°-0-0
0
/0 o
I
0 L-..O.--l.---L_..!-..----L...-_ 20 40 60 80
1: x
ru ~
N
ro 6 E u
0: 0: 0
(j)
4 2
~
0
E
:1..
0
0
f
0.-
o~o
/ 20
40
60
80
days after seeding
Fig. 6: DPIP-Hill aCtiVIty as a function of cotyledonary age. The values were measured taking into account the pH shift of the optimum starting at pH 7. They are plotted in Fig. 6 a on a chlorophyll basis and in Fig. 6 b on the basis of unit cotyledon area.
Z. PJlanzenphysiol. Bd. 71. S. 301-312. 1974.
306
G.
HARNISCHFEGER
The pattern of the curves is clearly somewhat difficult to interpret in a direct manner but a possible means of explanation is afforded by examination of the pH-profiles of the DPIPHill reaction obtained with plastids from cotyledons of various age (Fig. 3). One general feature emerges from comparison of these profiles, namely that the pH-optima shown do not always occur at the same position. Further analysis indicates that a straight line cannot easily be fitted when the position of the various observed optima is plotted as a function of cotyledonary age. Fig. 4 a and Fig. 4 b represent two sets of curves which can be used to arrange the points in order. By connecting the points in these ways one assumes a shift of the pH optima and can thereby explain the oscillations observed in Fig. 2 as merely a reflection of their migration through the constant pH values employed during measurement. Thus, Fig. 2 provides a means of distinguishing between the models of Fig~ 4 a and Fig. 4 b. Table 1 shows the time of maximal rate measured vs. the time it should be expected according to the models of Fig. 4 a or Fig. 4 b, both for pH 6.3 and pH 7.5. One can conclude from this comparison that the model of Fig. 4 b seems to be an adequate description of the supposed pH shift, a concept which is supported by its logarithmic decline and exemplified in Fig. 5. In order to obtain meaningful curves of Hill activity as a function of age one, therefore, has to use data taken along the pH shift lines. Only under these circumstances can one reduce the multifaceted effects to a single one, i.e. activity decline vs. time. Fig. 6 shows the changes in DPIP-Hill reaction obtained when the points were taken along the activity line of the optimum starting at pH 7. The data give the result expected from theoretical considerations.
Table 1: Comparison of measured and calculated time of occurrence of maximal activity of the DPIP-Hill reaction in chloroplasts isolated from aging pumpkin cotyledons. pH
6.3
time of occurrence of maximal activity (days after sowing) measured
17
calculated from Fig. 4 a
7.5
24 20
32
42
30
45
55 60
calculated from Fig. 4 b
17
24
32
55
measured
20
32
45
73
calculated from Fig. 4 a calculated from Fig. 4 b
25 21
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
57 32
45
70
74 74
70 70
77
Chloroplast Degradation in vivo II
307
Effects of aging on FeeN reduction The introduction of the concept of a pH-shift with age, supported by the abovementioned observations, works well also for the FeCN reduction by isolated plastids. Fig. 7 shows the changes in activity measured in this reaction at a constant pH of
.cx =::
.? Q.
240
§ 200
Lu
en
160
-0 ~
120
~
z
u
~ 80 1Il
~
o E 40 ::l..
o
10
20
30 40 50 60 days after seeding
70
80
Fig. 7: Rate of Ferricyanide reduction of pumpkin chloroplasts in relation to the age of the cotyledons used. The measurements were taken at a constant pH of 6.3.
160
0-0/ 800
o
o
,. 0 - - - 0
"-
/
0,
.,0, 0
(63)
'0_0
0 '0
\o
40
0L-JL.....-IL.--l---1---L---I.--L--.L..-..J...-.L.--I--..L..-..L-
uO
7.0
ao
pH Fig. 8: Variations in pH-profile of the FeCN-Hill reaction obtained with chloroplasts isolated from cotyledons of different age (indicated as days after sowing in the parentheses above the curves).
z.
Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
308
G.
HARNISCHFEGER
_H2°-+FeCN
~g
8.0
0
0
/
0
\~oljJI. o~ I.
7.0 :r:
0-
0
o
o~o
-:~----
6.0
0
~oo
0
40
20
0
0
60
80
days after seeding
8.0
H2O-FeCN
0 0
'~
o\~~
7.0 :r:
~
0-
0
o~
"
o~o o .... - ... ___
6.0
0
- - .... _-
o~o
__ 0
0
10
20
30
40
50
60
70
80
days after seeding
Fig. 9: Position of the pH-optima of the FeCN-Hill reaction as a function of cotyledonary age. Fig. 9 a and 9 b show two possible ways of connecting the data. Table 2: Comparison of measured and calculated time of occurrence of maximal activity of the FeCN reduction in chloroplasts isolated from aging pumpkin cotyledons.
pH 6.3
z.
time of occurrence of maximum activity (days after sowing) measured calculated from Fig. 9 a calculated from Fig. 9b
13
42
17 17
Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
72 60
41
70 71
75
78
Chloroplast Degradation in vivo II
309
6.3 as a function of age. Again, the pseudo-oscillatory phenomenon is observed. In analogy to Fig. 3, Fig. 8 shows that the pH optima occur at different positions in aging cotyledons. Fig.9 depicts curves similar to those of Fig. 4, two of the various possible ways to connect the scattered points. Again, one can decide that a decline of the pH-optima takes place, the model of Fig. 9 b, by using the observed time of maximal activity shown in Fig. 7. Table 2 shows the calculated vs. measured occurrence of these peaks. '.c ~ 160 .c
u
E120
~
z 80 u ~
40
til ~
0
E
:I-
0
'.c
8
N
x
E
~
iz
6 4
u
~ til
o
~
E
:l.
2 0
f~\
( /0
20
60
40
.
~
80
(\~
•
•
I
20 40 60 80 days after seeding
Fig. 10: Rate of FeCN-reduction of pumpkin chloroplasts in relation to cotyledonary age. The data were taken along the pH curve starting at the pH 7.4 value of Fig. 9 b. • DPIP o FeCN () NADP(HzO) + NADP(ASC)
6.0 2 log(days after seeding)
Fig. 11: Logarithmic decline with age of the pH optimum starting around pH 7.5 for various partial reactions of isolated pumpkin chloroplasts.
z.
Pflanzenphysiol. Bd. 71. S. 301-312. 1974.
310
G.
HARNISCHFEGER
The appearance of the data when analyzed along the line of the pH optimum starting at a pH of 6.5 and 7.5 is depicted in Fig. 10. In a similar way to the pattern shown in Fig. 6, a progressive increase in activity to a maximal rate followed by a decline can be observed reflecting the respective growth phases of greening, expansion and aging in vivo. Other reactions The other partial reactions of photosynthesis show related but somewhat more complicated phenomena due to additional cyclic processes. Fig. 11 illustrates that the described pH shift might be common. In their uncoupled, strictly electrontransporting form all the major reactions show a similar decline of their pH optima, the optimum starting around 7.5 is depicted as an example.
Discussion The data obtained for changes in Hill activity with age give simple and meaningful results only if the concept of a migration of the pH-optima towards more acidic positions is introduced into the analysis of these reactions. All the observations reported above support such an interpretation but the cause of this shift remains unknown. Several possible explanations may be put forward. Firstly, it is well known that chloroplasts break up during the preparation procedure resulting in a mixture of whole and broken plastids even under the most careful conditions. Everyone of these fractions might possess its own pH-optimum for maximal activity. JACOBI and LEHMANN (1969) have shown that th~ pH of maximal activity becomes more acidic with decrease in size of the plastid fragments. Thus, one can envision that a chloroplast preparation from older cotyledons contains a higher amount of fragments than a suspension obtained from young cotyledons, a reasonable assumption born out by their structural degradation seen in the electron microscope (HARNISCHFEGER, 1973). Since structural decay occurs gradually, the fraction of small particles in the preparation should increase slowly, their pH optimum becoming more and more dominant. Functionally one would obtain the observed pH shift. In order to test this possibility experimentally, a separation of the various whole and broken plastid types by the counter-current-distribution method of KARLSTAM and ALBERTSSON (1972) was performed with chloroplast preparations from both young and old cotyledons. Superimposable distributions were obtained which is at variance with the above argument. Moreover, the more subjective method of microscopic inspection for intact and broken plastids leads to a similar negative conclusion. A second possible explanation for the observed shift can be derived in analogy to the results of KAHN (1971) with Euglena. He showed with extensive use of various inhibitors that this organism contains not one but two different compartments in its thylakoids with counteracting proton pumps. Their performance in relation to each Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Chloroplast Degradation in ruivo II
311
other and to the surrounding fluid is dependent on the pH of the medium. It is conceivable that pumpkin thylakoids might also contain more than one compartment and that membrane rearrangement and decay upon aging leads to a slow but irreversible loss of one type of proton pump. The overall functional result would be, again, the observed pH shift. A third possibility to explain the pH shift can be derived from thermodynamic considerations. In order for a reaction to occur the emf is critical, and this holds true also for the redox steps of the electron transport chain. For the quinone-cytochrome step the Nernst equation reads as follows: E = Eo -
RT [Q] ~ In [QH 2 ]
-
RT [Fe++J - F - In [Fe+++J
+
RT 2.3F pH.
It follows that a change in the ratio of the redox components has to be counterbalanced by a shift in pH in order to achieve a stable emf value. Let e. g. [Qtotal] decline. Since [Qtotal] = [Q] + [QH2 ] and [QH2 ] can be considered constant during the reaction (non-equilibrium conditions because light forces the electrons toward cytochrome), this will result in a smaller Q/QH 2 ratio which has to be counteracted by a decrease in pH. A similar argument can be made for the cytochrome component. Observations supporting this notion have been published by LICHTENTHALER (1969). He showed that during plastid aging in vivo more and more plastoglobuli are formed from the decaying membranes. These lipid globules contained, besides fatty acids, carotenes and plastoquinone which is rendered inaccessible for electron transport in the thylakoid. Furthermore, the decline in cytochrome f content with age (HARNISCHFEGER, 1973) also points strongly in this direction and could well constitute a major component involved in the pH-shift. It may thus be concluded that the concept of a pH shift of the electron transport optima upon aging explains well the observed data and can be easily worked into the current knowledge of the photosynthetic apparatus. Its implications, however, for the evaluation of functional parameters in isolated plastids can only remain speculative. Acknowledgements I want to thank Ms. S. FORBACH for excellent technical assistance. These studies were made possible through a grant from the Deutsche Forschungsgemeinschaft.
Literature ARNON, D. 1.: Plant Phys. 24, 1 (1949). BARTELS, F.: Protoplasma 72,27 (1971). GOOD, N. E., and S. IZAwA: In: Methods in enzymology Vol. XXIVB, (A. San Pietro ed.) p. 53. Academic Press, New York (1972). HARNISCHFEGER, G.: ]. expo Bot" in press (1973).
z.
Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
312
G. HARNISCHFEGER
JACOBI, G., and H. LEHMANN: In: Progress in Photosynthetic Research Vol. 1 (H. Metzner ed.) p. 159. Tubingen (1969). KAHN, J. S.: Biochim. Biophys. Acta 245,144 (1971). KARLsTAM, B., and P. A. ALBERTSSON: Biochim Biophys. Acta 255, 539 (1972). LICHTENTHALER, H. K.: Protoplasma 68, 315 (1969). POSTIUS, S., and G. JACOBI: Planta 99, 222 (1971). GOTZ HARNISCHFEGER, Lehrstuhl fur Biochemie der Pflanze, D-34 Gottingen, Untere Karspule 2.
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Studies on Chloroplast Degradation in vivo II Effects of Aging on Hill Activity of Plastids from Cucurbita Cotyledons GOTZ
HARNISCHFEGER
With 11 figures Received July 4, 1973
Summary The rate of dichlorophenol-indophenol (DPIP) and ferricyanide reduction was investigated as a function of the age of the cotyledon from which the plastids were isolated. The pseudooscillatory data obtained could be transformed into normal growth curves by assuming an age dependent pH-shift for optimum activity. The possible reasons for this proton dependency of electron transport are discussed.
Introduction The correlation between structure and function of biological membranes is a topic of considerable interest. A better understanding of their specific interconnection provides a means to elucidate the mechanics of many biological systems, among them respiration and photosynthesis. The investigations described in this paper center around the interdependence of photochemical activity and membrane integrity in chloroplasts of higher plants. Changes in membrane structure due to different physiological conditions which in turn cause alterations in rate of electron transport were achieved by judicious use of the process of aging in vivo. The detailed specifics of the plant system used have been described previously (HARNISCHFEGER, 1973). In brief, chloroplasts were isolated at various stages of development from pumpkin cotyledons grown under controlled conditions. Through continual removal of the epicotyl, cotyledons were obtained which remained green throughout their period of growth and contained plastids which possessed a functionally intact, qualitatively unchanged pigment system. This feature facilitates the study of enzymatic alterations in electron transport attributable to membrane rearrangement and degradation during aging. This paper reports the correlation between age-induced structural changes and electron transport activity of pumpkin chloroplasts.
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
302
G. HARNISCHFEGER
Material and methods Pumpkin, Cucurbita maxima cv. «gelbe, genetzte Riesenmelone» was grown under the conditions described previously (HARNISCHFEGER, 1973). The epicotyl of the plants was continually removed resulting in cotyledons which expanded considerably beyond the size of those found in normal growing plants. Plastids with qualitatively unchanged pigment characteristics were isolated from leaves of various age in the usual manner. Chloroplast preparation always took place at the same time of day in order to minimize the influence of photoperiodic phenomena (BARTELS, 1971). The plastids were stored in 0.4 M sucrose-O.l M phosphate buffer pH 6.3-1 mg/ml BSA in ice under light exclusion. Chlorophyll content was determined according to ARNON (1949). The dichlorophenol-indophenol (DPIP) and ferricyanide (FeCN) Hill-reaction was carried out using the method of POSTIUS and JACOBI (1971). Light was provided by a 300 watt Philips floodlight lamp. The beam was condensed by a set of lenses, passed through a water and an IR filter and was modified through a red plexiglas filter (l > 600 nm) giving a saturating incident intensity of 1.2 X 105 ergs/cm2 X sec. The assay samples contained 2 ml 0.4 M sucrose, 0.5 ml 0.2 M buffer, chloroplasts equivalent to 30-40 fig chlorophyll and 0.1 ml of electron acceptor (2 X 10-3 M DPIP or 3 X 10-2 M FeCN). The direct readout together with standartization and triple measurement of individual points permits a confidence limit of under 5 %. The buffer systems developed by GOOD (1972) viz., MES for the pH-range 5.6-6.8, TES from 6.8-7.8 and TRICINE between 7.6 and 8.3 were used to determine the pH profiles. Overlapping points with these different buffers were measured and the pH of the assay solution was rechecked immediately after illumination on a Metrohm E-516 Titriskop pH-meter.
160 L..
.J::.
140
o
x
~120 0-
o
5100
E80 "0
~ 0-
~ 40
o
o
000 .........
0
"'0
o
/
o
'a
0
I
I
0,I o
I
o
:J..
0
/00
~
E 20
//00
0
/
60
a::
0/
o
:cu
o
/--;---,
o
I
I
10
20
30
40
50
60
70
80
days after seeding
Fig. 1: DPIP-Hill activity of isolated chloroplasts as a function of the age of the cotyledonary material used. The values were measured under saturating light conditions in a medium of pH 6.3.
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Chloroplast Degradation in vivo II
303
10 8 6
20
o
20
40
60
40
60
days after seeding
80
Fig. 2: DPIP-Hill activity as a function of cotyledonary age, plotted on the basis of unit cotyledon area. The data of Fig. 1, measured at pH 6.3, are shown in Fig. 2 a, while Fig. 2 b shows the values obtained at pH 7.5.
~
~ 120
6.0
7.0
pH Fig. 3: pH-profiles of DPIP-Hill activity obtained with plastids isolated from cotyledons of various age (indicated as days after sowing in brackets above each curve).
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
304
G.
HARNISCHFEGER
Results
Effects of aging on the DPIP-Hill reaction The course of the DPIP-Hill reaction, measured at a constant pH of 6.3 and under saturating light intensity was followed through the life span of the cotyledon. Fig. 1 depicts the values obtained and shows a wide scatter of points around the ideal line expected for changes in electron transport properties during development and death. Deviations of this nature are normally attributed to the inherent variability of the plant system. However, when the measured points are replotted on a basis of unit cotyledon area, the curve of Fig. 2 a is obtained, showing what appears to be periodic oscillations. This feature is also reflected, but to a lesser extent, in Fig. 2 b which depicts data measured at pH 7.5. The oscillations were observed not only in all
:r:
0.
o
20
40
60
days after sczczding
80
a.o
7.0 :r:
0.
6.0
o
20
40
60
00
days after seeding
Fig. 4: Position of pH optima of the DPIP-Hill reaction as a function of cotyledonary age. Fig. 4 a and b show two possible ways to connect them.
z.
Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Chloroplast Degradation in vivo II
305
five experimental series performed over a period of two years, but also showed up in all partial reactions tested (compare figs. 7 and 11). Since the yearly season influenced the duration of the total cotyledonary life span not allowing a comprehensive graphical presentation of all results, only data from one representative experimental series are shown in the following.
7.0 I
a.
6.0
10
20
40
60 80
days after seeding
Fig. 5: Logarithmic decline of the pH-optimum starting at the pH 7 value of Fig. 4 b. '-
.c ~
160
.? ~ 120 .Q .c u
~ 80
0: ~ 40 (j)
~
§ :1..
/°-0-0
0
/0 o
I
0 L-..O.--l.---L_..!-..----L...-_ 20 40 60 80
1: x
ru ~
N
ro 6 E u
0: 0: 0
(j)
4 2
~
0
E
:1..
0
0
f
0.-
o~o
/ 20
40
60
80
days after seeding
Fig. 6: DPIP-Hill aCtiVIty as a function of cotyledonary age. The values were measured taking into account the pH shift of the optimum starting at pH 7. They are plotted in Fig. 6 a on a chlorophyll basis and in Fig. 6 b on the basis of unit cotyledon area.
Z. PJlanzenphysiol. Bd. 71. S. 301-312. 1974.
306
G.
HARNISCHFEGER
The pattern of the curves is clearly somewhat difficult to interpret in a direct manner but a possible means of explanation is afforded by examination of the pH-profiles of the DPIPHill reaction obtained with plastids from cotyledons of various age (Fig. 3). One general feature emerges from comparison of these profiles, namely that the pH-optima shown do not always occur at the same position. Further analysis indicates that a straight line cannot easily be fitted when the position of the various observed optima is plotted as a function of cotyledonary age. Fig. 4 a and Fig. 4 b represent two sets of curves which can be used to arrange the points in order. By connecting the points in these ways one assumes a shift of the pH optima and can thereby explain the oscillations observed in Fig. 2 as merely a reflection of their migration through the constant pH values employed during measurement. Thus, Fig. 2 provides a means of distinguishing between the models of Fig~ 4 a and Fig. 4 b. Table 1 shows the time of maximal rate measured vs. the time it should be expected according to the models of Fig. 4 a or Fig. 4 b, both for pH 6.3 and pH 7.5. One can conclude from this comparison that the model of Fig. 4 b seems to be an adequate description of the supposed pH shift, a concept which is supported by its logarithmic decline and exemplified in Fig. 5. In order to obtain meaningful curves of Hill activity as a function of age one, therefore, has to use data taken along the pH shift lines. Only under these circumstances can one reduce the multifaceted effects to a single one, i.e. activity decline vs. time. Fig. 6 shows the changes in DPIP-Hill reaction obtained when the points were taken along the activity line of the optimum starting at pH 7. The data give the result expected from theoretical considerations.
Table 1: Comparison of measured and calculated time of occurrence of maximal activity of the DPIP-Hill reaction in chloroplasts isolated from aging pumpkin cotyledons. pH
6.3
time of occurrence of maximal activity (days after sowing) measured
17
calculated from Fig. 4 a
7.5
24 20
32
42
30
45
55 60
calculated from Fig. 4 b
17
24
32
55
measured
20
32
45
73
calculated from Fig. 4 a calculated from Fig. 4 b
25 21
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
57 32
45
70
74 74
70 70
77
Chloroplast Degradation in vivo II
307
Effects of aging on FeeN reduction The introduction of the concept of a pH-shift with age, supported by the abovementioned observations, works well also for the FeCN reduction by isolated plastids. Fig. 7 shows the changes in activity measured in this reaction at a constant pH of
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240
§ 200
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160
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120
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z
u
~ 80 1Il
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10
20
30 40 50 60 days after seeding
70
80
Fig. 7: Rate of Ferricyanide reduction of pumpkin chloroplasts in relation to the age of the cotyledons used. The measurements were taken at a constant pH of 6.3.
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0-0/ 800
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ao
pH Fig. 8: Variations in pH-profile of the FeCN-Hill reaction obtained with chloroplasts isolated from cotyledons of different age (indicated as days after sowing in the parentheses above the curves).
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Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
308
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HARNISCHFEGER
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10
20
30
40
50
60
70
80
days after seeding
Fig. 9: Position of the pH-optima of the FeCN-Hill reaction as a function of cotyledonary age. Fig. 9 a and 9 b show two possible ways of connecting the data. Table 2: Comparison of measured and calculated time of occurrence of maximal activity of the FeCN reduction in chloroplasts isolated from aging pumpkin cotyledons.
pH 6.3
z.
time of occurrence of maximum activity (days after sowing) measured calculated from Fig. 9 a calculated from Fig. 9b
13
42
17 17
Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
72 60
41
70 71
75
78
Chloroplast Degradation in vivo II
309
6.3 as a function of age. Again, the pseudo-oscillatory phenomenon is observed. In analogy to Fig. 3, Fig. 8 shows that the pH optima occur at different positions in aging cotyledons. Fig.9 depicts curves similar to those of Fig. 4, two of the various possible ways to connect the scattered points. Again, one can decide that a decline of the pH-optima takes place, the model of Fig. 9 b, by using the observed time of maximal activity shown in Fig. 7. Table 2 shows the calculated vs. measured occurrence of these peaks. '.c ~ 160 .c
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Fig. 10: Rate of FeCN-reduction of pumpkin chloroplasts in relation to cotyledonary age. The data were taken along the pH curve starting at the pH 7.4 value of Fig. 9 b. • DPIP o FeCN () NADP(HzO) + NADP(ASC)
6.0 2 log(days after seeding)
Fig. 11: Logarithmic decline with age of the pH optimum starting around pH 7.5 for various partial reactions of isolated pumpkin chloroplasts.
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The appearance of the data when analyzed along the line of the pH optimum starting at a pH of 6.5 and 7.5 is depicted in Fig. 10. In a similar way to the pattern shown in Fig. 6, a progressive increase in activity to a maximal rate followed by a decline can be observed reflecting the respective growth phases of greening, expansion and aging in vivo. Other reactions The other partial reactions of photosynthesis show related but somewhat more complicated phenomena due to additional cyclic processes. Fig. 11 illustrates that the described pH shift might be common. In their uncoupled, strictly electrontransporting form all the major reactions show a similar decline of their pH optima, the optimum starting around 7.5 is depicted as an example.
Discussion The data obtained for changes in Hill activity with age give simple and meaningful results only if the concept of a migration of the pH-optima towards more acidic positions is introduced into the analysis of these reactions. All the observations reported above support such an interpretation but the cause of this shift remains unknown. Several possible explanations may be put forward. Firstly, it is well known that chloroplasts break up during the preparation procedure resulting in a mixture of whole and broken plastids even under the most careful conditions. Everyone of these fractions might possess its own pH-optimum for maximal activity. JACOBI and LEHMANN (1969) have shown that th~ pH of maximal activity becomes more acidic with decrease in size of the plastid fragments. Thus, one can envision that a chloroplast preparation from older cotyledons contains a higher amount of fragments than a suspension obtained from young cotyledons, a reasonable assumption born out by their structural degradation seen in the electron microscope (HARNISCHFEGER, 1973). Since structural decay occurs gradually, the fraction of small particles in the preparation should increase slowly, their pH optimum becoming more and more dominant. Functionally one would obtain the observed pH shift. In order to test this possibility experimentally, a separation of the various whole and broken plastid types by the counter-current-distribution method of KARLSTAM and ALBERTSSON (1972) was performed with chloroplast preparations from both young and old cotyledons. Superimposable distributions were obtained which is at variance with the above argument. Moreover, the more subjective method of microscopic inspection for intact and broken plastids leads to a similar negative conclusion. A second possible explanation for the observed shift can be derived in analogy to the results of KAHN (1971) with Euglena. He showed with extensive use of various inhibitors that this organism contains not one but two different compartments in its thylakoids with counteracting proton pumps. Their performance in relation to each Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.
Chloroplast Degradation in ruivo II
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other and to the surrounding fluid is dependent on the pH of the medium. It is conceivable that pumpkin thylakoids might also contain more than one compartment and that membrane rearrangement and decay upon aging leads to a slow but irreversible loss of one type of proton pump. The overall functional result would be, again, the observed pH shift. A third possibility to explain the pH shift can be derived from thermodynamic considerations. In order for a reaction to occur the emf is critical, and this holds true also for the redox steps of the electron transport chain. For the quinone-cytochrome step the Nernst equation reads as follows: E = Eo -
RT [Q] ~ In [QH 2 ]
-
RT [Fe++J - F - In [Fe+++J
+
RT 2.3F pH.
It follows that a change in the ratio of the redox components has to be counterbalanced by a shift in pH in order to achieve a stable emf value. Let e. g. [Qtotal] decline. Since [Qtotal] = [Q] + [QH2 ] and [QH2 ] can be considered constant during the reaction (non-equilibrium conditions because light forces the electrons toward cytochrome), this will result in a smaller Q/QH 2 ratio which has to be counteracted by a decrease in pH. A similar argument can be made for the cytochrome component. Observations supporting this notion have been published by LICHTENTHALER (1969). He showed that during plastid aging in vivo more and more plastoglobuli are formed from the decaying membranes. These lipid globules contained, besides fatty acids, carotenes and plastoquinone which is rendered inaccessible for electron transport in the thylakoid. Furthermore, the decline in cytochrome f content with age (HARNISCHFEGER, 1973) also points strongly in this direction and could well constitute a major component involved in the pH-shift. It may thus be concluded that the concept of a pH shift of the electron transport optima upon aging explains well the observed data and can be easily worked into the current knowledge of the photosynthetic apparatus. Its implications, however, for the evaluation of functional parameters in isolated plastids can only remain speculative. Acknowledgements I want to thank Ms. S. FORBACH for excellent technical assistance. These studies were made possible through a grant from the Deutsche Forschungsgemeinschaft.
Literature ARNON, D. 1.: Plant Phys. 24, 1 (1949). BARTELS, F.: Protoplasma 72,27 (1971). GOOD, N. E., and S. IZAwA: In: Methods in enzymology Vol. XXIVB, (A. San Pietro ed.) p. 53. Academic Press, New York (1972). HARNISCHFEGER, G.: ]. expo Bot" in press (1973).
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G. HARNISCHFEGER
JACOBI, G., and H. LEHMANN: In: Progress in Photosynthetic Research Vol. 1 (H. Metzner ed.) p. 159. Tubingen (1969). KAHN, J. S.: Biochim. Biophys. Acta 245,144 (1971). KARLsTAM, B., and P. A. ALBERTSSON: Biochim Biophys. Acta 255, 539 (1972). LICHTENTHALER, H. K.: Protoplasma 68, 315 (1969). POSTIUS, S., and G. JACOBI: Planta 99, 222 (1971). GOTZ HARNISCHFEGER, Lehrstuhl fur Biochemie der Pflanze, D-34 Gottingen, Untere Karspule 2.
Z. Pjlanzenphysiol. Bd. 71. S. 301-312. 1974.