tnHuence of Magnesium !ons on the Action of Photosystems ! and H at Different Wavetengths
Experiments w i t h Barley Chloroplasts o f Different C h l o r o p h y l l b Content Eckhard Loos and Ernst K e l l n e r
Institut fur Botanik, Universitat Regensburg, UniversitatsstraBe 31. D-8400 Regensburg Z. Naturforsch. 35c, 29S-302 (1980); received October 17, !979/January 23, 1980 Chlorophyll b, Barley, Magnesium Ions, Wavelength Action
Barley leaves grown under a natural light/dark regime have a chlorophyll content of 1300 p.g/g fresh weight and a chlorophyll a/b ratio of 2.5-3. When the plants are grown under cycles of 2 min light/! 18 min dark, the respective values are 50 and 5 - 9 . With chloroplasts with !ow chlo- rophyll b content variable fluorescence is depressed by about 30% by M g C L ; with those of high chlorophyll b content a threefold increase is seen instead. Action spectra tor variable fluorescence of chloroplasts of high chlorophylll b content show enhancement by M g ^ around 475 and 650 nm;
for the system 1-mediated methyl viologen reduction, a depression is seen at these wavelengths.
These effects are practically absent in chloroplasts with low chlorophyll b content. The data corro- borate the hypothesis that a chlorophyll b-containing pigment protein complex is required for re- gulation of energy transfer to system ! and 11 by magnesium ions.
Lntroduction
The photosynthetic apparatus o f higher plants is thought to have a tripartite organization: Besides photosystem I and H , there is a chlorophyll b-con- taining pigment protein complex transferring excita- tion energy to both photosystems [ 1 - 3 ] . Mg^+ ions are thought to promote the energy flow to system H [4, 5] by increasing the transfer o f light energy from the light harvesting complex to system II and by decreasing the transfer to system I. T h i s has been concluded from the M g ^ - m e d i a t e d increase in ac- tion around 480 n m for system II reactions with a corresponding decrease for system I reactions [3], 480 nm being an absorption m a x i m u m of chloro- phyll b /f? WW. If Mg^+ acts on the light-harvesting complex and thereby influences chlorophyll b action, these effects should be absent in chloroplasts lacking this complex and chlorophyll b. T h i s is tested here using plants grown i n intermittent light which are known to be d e v o i d o f the light harvesting pigment protein and to be l o w in chlorophyll b content [ 6 - 8 ] . Furthermore, the observations are extended into the red part o f the spectrum. T h i s should give better information on a specific involvement o f c h l o r o p h y l l b, since in this spectral region an eventual inter- ference from carotenoids is absent.
^&fv/%//<7M&- Tricine, tris-(hydroxymethyl)-methy!-g!ycine;
D C M U , 3-(3,4-dichloropheny!)-l, 1-dimethylurea; DCIP, 2,6-dichlorophenolindophenol.
Reprint requests to Dr. E. Loos.
0341-0382/80/0300-0298 $ 01.00/0
Materiats and Methods
Barley (//(vr/cMf?? i n v A ^ f , var. E i r l b c c k s U n i o n ) was grown in a greenhouse for 1 0 - 3 0 days and used for the experiments with chloroplasts o f high chlo- rophyll b content. In order to obtain c h l o r o p h y l l b- deflcicnt plants, the seedlings were kept in darkness for one week after sowing and then subjected to cycles of 2 m i n light/118 m i n dark for two days. In this case, light was provided by white fluorescent tubes (6000 lux), the temperature was maintained at 23 ° C .
Chloroplasts were isolated as described previously [3] except that for better yield the initial centrifuga- tion was 3 m i n at 4000 x ^ . Chloroplasts were finally washed and rcsuspended in a m e d i u m consisting o f
10 m M K C 1 and 20 m M T r i c i n c - K O H ( p H 8 . 0 ) . C h l o r o p h y l l concentrations were determined accord- ing to A r n o n [9].
Eor excitation spectra variable fluorescence was obtained with the following method: f i r s t , dark- adapted chloroplasts were exposed to a series o f flashes o f the different wavelengths and the fluores- cence o f each flash was registered. Reference flashes given at the beginning and the end o f such a series yielded the same fluorescence. T h i s showed that the intensity of the flashes was weak enough and the dark time between them long enough to avoid ac- cumulation o f reduced quencher (Q) w h i c h would give rise to an additional fluorescence, the variable fluorescence (Fv^ (/ [!0]); so, the first series o f flashes induced prompt fluorescence (/\,) only. T h e n
a continous background light was turned on, w h i c h served to b r i n g Q into a reduced state. W i t h this background light on, the same series o f Hashes was given w h i c h now excited a stronger fluorescence, com- posed o f Fo and Fy. V a r i a b l e fluorescence was ob- tained by subtracting the two sets o f measurements.
T h e flashes (20 ms duration) were obtained by means o f an electric shutter (compur M 3 ) i n con- j u n c t i o n w i t h a tungsten l a m p and a monochromator
(Bausch and L o m b ) . T h e slits were set for 5 n m half band w i d t h . T h e time between the flashes was about 45 s. T h e sample (3 m l ) was contained i n a cuvette o f 1 x 1 c m cross section. Chloroplasts were suspended to y i e l d a c h l o r o p h y l l concentration o f 5 u g / m l i n a m e d i u m containing 10 m M K C 1 , 20 m M T r i c i n e - K O H , ( p H 8) and 16 nM D C M U . Fluorescence was detected at right angles to the exciting beam by a photomultiplier ( H a m a m a t s u R 374) protected by an interference filter and a red glass (Schott D A L 680 n m , 6 m m R G 665). T h e signals were fed to a storage oscilloscope. Continuous background light was provided from a projector w i t h a heat-reflecting glass, a 5 c m water-filled cuvette, cut-off glasses (Schott G G 420, K I F 560) and a blue green glass ( C o m i n g 9782) i n the beam. M e t h y l viologen re- duction was monitored spectrophotometrically as absorption increase at 396 nm. T h e measuring beam fell through the sample cuvette o f 1 x 1 c m cross section o n a p h o t o m u l t i p l i e r ( H a m a m a t s u R 374) protected from stray light and fluorescence by glass filters ( C o m i n g 9782, Schott U G 2 ) . A c t i n i c light from two sides perpendicular to the measuring beam was provided by two slide projectors, each w i t h a heat-reflecting glass, a 5 c m layer o f water, an ap- propriate lens and an interference filter i n the beam.
Interference filters o f 435, 476, 653 and 698 n m were used w i t h h a l f band widths ranging from 11 to 19 n m . T h e reaction mixture contained 10 m M K C 1 , 20 m M T r i c i n e - K O H ( p H 8), 40 m M cysteine, 20 uM D C I P , 0.2 m M methyl viologen and 10 uM D C M U . T o remove oxygen the mixture was b u b b l e d for
1 m i n w i t h nitrogen, filled into the cuvette and plugged up.
Results
W h e n dark grown barley is exposed to cycles o f 2 m i n light/118 m i n darkness, c h l o r o p h y l l synthesis
T ! ' r
1 2 3 4 5
time [days]
Fig. 1. Kinetics of chlorophyll formation in intermittent light. At time zero, the plants were transferred from dark to cycles of 2 min light/118 min dark.
proceeds for about two days and then comes to a halt ( F i g . 1). T h e ratio o f c h l o r o p h y l l a / c h l o r o p h y l l b reached after two days ranged from 5 to 9 i n different experiments, the c h l o r o p h y l l content was about 50 ug/g fresh weight o f leaves. Chloroplasts isolated from such plants w i l l be called " c h l o r o - phyll b-deficient chloroplasts". Plants grown i n the greenhouse had a c h l o r o p h y l l a/b ratio o f 2.5 to 3, contained around 1 300 ug c h l o r o p h y l l / g fresh weight of leaves and were the source o f " n o r m a l c h l o r o - plasts".
Prow/?/ #M<V var/a^/i?y7tvorasc^^cc; ^//ff/ <?/Afg^+
In F i g . 2 is plotted the fluorescence excited by 20 ms test flashes superimposed on an increasingly intense background i l l u m i n a t i o n . T h e fluorescence obtained at zero intensity o f background light is considered to be prompt fluorescence (F^), to w h i c h adds at higher intensities fluorescence o f variable yield ( F J . A quite weak background light o f 0.1 to 0.2 W / m ^ is sufficient to obtain m a x i m a l fluores- cence yield. T h e light intensity employed i n action spectra measurements (r/^ methods) was 0.12 W / n P .
W i t h the normal chloroplasts Fy is increased by Mg^+ about threefold ( F i g . 2 A ) , whereas Fo is little affected. W i t h c h l o r o p h y l l b-deflcient chloroplasts, however, / \ is decreased i n the presence o f Mg^+ by about 30% ( F i g . 2 B ) . A n o t h e r difference is the rel- atively lower fraction c o m p r i z e d by Fy from the total fluorescence Fo +
0.2 0.3 tight i n t e n s i t y [W/m^]
Fig. 2. Fluorescence produced by a test flash (435 nm, 0.08 W/m^, 20 ms) v^ M j intensity of blue-green back- ground illumination in the presence (#) and absence (O) of 5 m M MgClg. A : Normal chloroplasts; B: Chlorophyll b- deficient chloroplasts.
V a r i a b l e fluorescence p r o b a b l y originates from system II [10, 11]; action spectra for Fy, therefore, should represent system II action. F i g . 3 A shows the result o f an experiment w i t h n o r m a l chloroplasts.
T h e actions have been n o r m a l i z e d at 435 n m . T h e most pronounced effect o f Mg^+ is to enhance the relative action o f wavelengths around 475 n m . T h i s
and the small increase i n action around 600 and 650 n m have been observed consistently w i t h two other chloroplast preparations. A c t i o n spectra w i t h chloroplasts from intermittent l i g h t - g r o w n plants ( F i g . 3 B ) are l a c k i n g the peak at 475 n m o b v i o u s l y because o f the deficiency i n c h l o r o p h y l l b. F u r t h e r is lost any significant influence o f Mg^+ on the shape o f the spectra.
Attempts to use m e t h y l viologen-mediated Og- uptake i n the presence o f D C M U w i t h an artificial electron donor system as a test reaction for system I were unsuccessful w i t h c h l o r o p h y l l b-deficient chlo- roplasts, because they exhibited a strong light-de- pendent oxygen uptake even without the d o n o r system. T h i s oxygen uptake m a y be system 1-me- diated, but m a y represent equally w e l l an unspecific photooxidation. System I activity c o u l d be mea- sured, however, spectroscopically as m e t h y l v i o l o g e n reduction under anaerobic conditions w i t h cysteine/
D C I P as electron donor. W i t h this reaction the rate o f reduction decreased d u r i n g exposure to l i g h t and u p o n shutting o f f the light a back reaction o f the reduced dye was seen. Instead o f measuring slopes it proved to be more practicable to take the absorption increase produced by a 10 s i l l u m i n a t i o n for system I activity.
Plots o f this response versus light intensity were slightly curved, i n d i c a t i n g the beginning o f light saturation. T h e action (= ratio response/incident light), therefore, v a r i e d w i t h the size o f the response.
T o make a v a l i d c o m p a r i s o n o f the actions o f dif- ferent wavelengths, the intensities o f the actinic beams were adjusted to produce equal amounts o f reduced dye. T h i s matching was done i n the blue
Table I. Effect of 5 m M MgClz on ratios of actions of system I - mediated methyl viologen reduction for the wavelength pairs 435/476 nm and 653/698 nm m chloroplasts from normal and intermittent light-grown barley.
Normal chloroplasts Chlorophyll b-deficient chloroplasts
- M g ' + + Mg'+ - M g ' + + Mg'+
Action 476 nm
Action 435 nm 0.69 0.54 0.58 0.50
Action 653 nm
Action 698 nm 1.75 1.22 1.47 1.51
B
w a v e l e n g t h [nm]
Fig. 3. Action spectra of variable fluorescence in the presence (+) and absence (O) of 5 m M M g C l , . A : chloroplasts; B: Chlorophyll b-deficient chloroplasts.
region for 435 and 476 n m and i n the red for 653 and 698 n m . T h e ratios o f actions obtained i n this way are listed i n T a b l e I. W i t h normal chloroplasts, Mg^+ lowers the ratio o f action at 476 n m versus that at 435 n m by about 20% and the ratio at 653 nm versus 698 n m by 30%. C h l o r o p h y l l b-deficient chlo- roplasts exhibit a somewhat smaller effect for the blue wavelengths; the ratio for the two red wave- lengths is hardly affected by Mg^+. T w o independent repetitions o f this experiment gave essentially the same results.
Discussion
Barley plants grown i n intermittent light show a relatively high chlorophyll a/b ratio between 5 and 9. T h i s agrees well with the figures given by Genge
^?%/. [12] for s i m i l a r l y grown barley seedlings. Even higher a/b ratio ( > 12) have been found upon cycles o f 1 ms light - 15 m i n dark [13] or w i t h bean and pea [6, 14].
V a r i a b l e fluorescence o f norma! chloroplasts is strongly increased by Mg^+, as has been reported by others [4, 5]. W i t h c h l o r o p h y l l b-deficient chloro- plasts, however, Mg^+ causes a small depression o f / \ . T h e reason for this is not known. W i t h chloro- phyll b-deficient chloroplasts from a barley mutant or intermittent light-grown pea, no or only little stimulation o f fluorescence has been observed upon addition o f Mg^+ [8, 15]. T h e smaller share o f from total fluorescence seen with chloroplasts with low chlorophyll b content ( F i g . 2) has also been found i n a pea system [14].
W i t h normal chloroplasts, the action spectrum for Fv ( F i g . 3 A) shows i n the presence o f Mg^+ higher action around 475 nm indicating involvement o f chlorophyll b. T h e smaller effects around 600 and 650 nm may be due to the smaller absorption coef- ficients o f chlorophyll b in this part o f the spectrum as compared to those o f chlorophyll a. F o r system 1 action, however, the wavelengths o f predominant chlorophyll b absorption are less effective i n the
presence o f Mg^+ (Table I). T h i s is especially c o n - spicous when c o m p a r i s o n is made for the wave- lengths 653 a n d 698 n m , probaly because the ratios o f c h l o r o p h y l l a / c h l o r o p h y l l b absorption are differ- i n g considerably at these wavelengths. T h i s influence o f Mg^+ o n the wavelength dependency o f system I and II reactions confirms previous results [3]. It is consistent w i t h the hypothesis that i n the presence o f Mg2+, transfer from the c h l o r o p h y l l b-containing light-harvesting c o m p l e x is increased to system II and decreased to system I. Since the occurence o f c h l o r o p h y l l b seems to be strictly coupled to that o f the light-harvesting pigment protein c o m p l e x [1, 8, 16], this complex seems plausible to undergo inter- action w i t h Mg2+. A n aggregation o f the p u r i f i e d pigment protein has recently been found to be i n - duced b y this i o n [17].
In chloroplasts w i t h l o w c h l o r o p h y l l b content, however, little or no effect o f Mg^+ is seen o n the
action o f wavelengths absorbed b y c h l o r o p h y l l b ( F i g . 3 B , T a b l e I). T h i s confirms the evidence that the influence o f Mg^+ is o n the action o f c h l o r p h y l l b.
F o r the smaller effect persisting o n the ratio o f System I actions 476/435 n m , the c h l o r o p h y l l b does not appear to be responsible, since there is no cor- responding effect o n the ratio o f actions 653/698 n m . T h e " c h l o r o p h y l l b-deficient" chloroplasts contain still some c h l o r o p h y l l b ; a n expected s m a l l effect could have been w i t h i n the limits o f experimental error and presumably d i d not occur due to a not yet fully assembled light harvesting c o m p l e x , unable to interact w i t h magnesium ions.
Thanks are due to D r . W . L o c k a u for s t i m u l a t i n g discussions.
[1] J. P. Thomber and H. R. Highkin, Eur. J. Biochem.
41, 109-116(1974).
[2] W. L. Butler and M . Kitajima, Biochim. Biophys.
Acta 396,72-85 (1975).
[3] E. Loos, Biochim. Biophys. Acta 440,314-321 (1976).
[4] P. Homann, Plant Physiol. 44,932-936 (1969).
[5] N. Murata, Biochim. Biophys. Acta 189, 171-181 (1969).
[6] J. H. Argyroudi-Akoyonoglou and G. Akoyonoglou, Plant Physiol. 46,247-249 (1970).
[7] R. G. Hiller, D. Pilger, and S. Genge, Plant Sci. Lett.
1,81-88 (1973).
[8] D. J. Davis, P. A. Armond, E. L. Gross, and C. J.
Amtzen, Arch. Biochem. Biophys. 175,64-70 (1976).
[9] D. J. Amon, Plant Physiol. 24,1 -15 (1949).
[10] L. N. M . Duysens and H. E. Sweers, Studies on Microalgae and Photosynthetic Bacteria (J. Ashida, edj, pp. 353-372, University of Tokyo press, Tokyo
[11] J. Lavorel, Biochim. Biophys. Acta 88,20-36 (1964).
[12] S. Genge, D. Pilger, and R. G. Hiller, Biochim. Bio- phys. Acta 347,22-30 (1973).
[13] G. Akoyonoglou, J. H. Argyroudi-Akoyonoglou, M.
R. Michel-Wolwertz, and C. Sironval, Physiol. Plant 19,1101-1104(1966).
[14] P. A. Armond, C. J. Amtzen, J.-M. Briantais, and C.
Vemotte, Arch. Biochem. Biophys. 175,54-63 (1976).
[15] J. R. Lieberman, S. Bose, and C. J. Amtzen, Biochim.
Biophys. Acta 502,417-429 (1978).
[16] O. Machold, H. Meister, H. Sagromsky, G . Hoeyer- Hansen, and D. V. Wettstein, Photosynthetica 11, 200-206 (1977).
[17] J. J. Burke, C. L. Ditto, and C. J. Amtzen, Arch.
Biochem. Biophys. 187,252-263 (1978).
