Photosynthesis Research 12:35-42 (1987)
© Martinus NijhoffPublishers, Dordrecht--Printed in the Netherlands
35
Regular paper
Photoinhibition of photosynthesis: effect of 02 and selective excitation of the photosystems in intact Lemna gibba plants REKHA CHATURVEDI & STEIN NILSEN The Phytotron, Department of Biology, University of Oslo, Post Box 1066, Blindern, 0316 Oslo 3, Norway Received 7 April 1986; accepted in revised form 30 September 1986
Keywords: monochromatic light, oxygen concentration, photoinhibition, photorespiration, photosynthesis, photosystems Abstract. Intact Lemna gibba plants were photoinhibited under anaerobic conditions on illumination with monochromatic light which selectively excited the photosystems. Photoinhibition was less when PS 1 was excited and greatest when mainly PS 2 was excited, which suggests that PS 2 was most damaged by photoinhibition induced in complete absence of 0 2 and CO:. The illumination of plants with monochromatic light exciting PS 1, at different 0 2 concentrations (in CO 2 deficient conditions), showed that PS 1 photoinhibition was increased at the low 0 2 concentrations. The damage to PS 1 was more evident at 2% 0 2 than at the higher 0 2 concentrations. CO 2 as well as 0 2 at atmospheric concentration, (air), was necessary for complete protection of the plant from photoinhibition when both photosystems were excited either separately or together. Abbreviations: I - irradiance, photon fluence rate, PCO - photosynthetic carbon oxidation cycle, PCR - photosynthetic carbon reduction cycle, PS 1 - photosystem l, PS 2 - photosystem 2
Introduction
Photoinhibition of photosynthesis is the consequence of exposure of plants to an irradiance (I) much higher than that used during growth, [3, 16, 28]. When the capacity of photosystems for dissipating the excitation energy is not large enough, the reaction centers are inactivated and photoinhibition occurs [27]. The illumination of plants in absence or low concentrations of CO 2 and 02 also results in photoinhibition of photosynthesis. In this situation photoinhibition is due to the damage caused by the unconsumed photochemical energy through the integrated operation of photosynthetic carbon reduction cycle (PCR) and photosynthetic carbon oxidation cycle (PCO) [5, 17, 26]. It has been shown that the PS 2 related reactions are mainly damaged in photoinhibition [2, 7, 28]. It is suggested that the primary site of damage is at the 32-kilo dalton herbicide binding protein, the QB apoprotein [1, 19]. PS 1 can also be damaged as a consequence of photoinhibition [27, 29, 31]. Photoinhibition ofPS 1 was equal to [31] or less than [26, 27, 34] that of PS 2. The proposed site of this inhibition is very close to the PS 1 reaction center [33]. Harvey and Bishop [10], Powles and Bj6rkman [29] and Satoh [32] found that
36 the photoinhibition of PS 1 is an oxygen requiring process. In their opinion, the generation of oxidised destructive entity may be involved in the damage of PS 1. Maintenance of some minimal level of photosynthetic carbon metabolism is necessary to prevent or minimize photoinhibition [25]. Pseudocyclic electron transport to some extent can also help in the dissipation of the excess of photochemical energy [27]. The main objective of this study was to establish the involvement of the photosystems in the photoinhibition of intact Lemna gibba plants and to study the effect of various 02 levels on the degree of damage.
Material and methods
Axenic cultures of Lemna gibba L. G3, obtained from Dr Elisabeth Tillberg, University of Stockholm, were precultured at I of 220 #tool m -2 s 1, in nutrient medium containing sucrose, as reported by Chaturvedi et al. [4]. These cultures were then transferred to nutrient medium without sucrose and grown at the same I for 8 days [22]. Photosynthetic measurements were made in a closed gas exchange system using an infrared gas analyser (Uras 2, Hartman and Braun, W. Germany) as described by Nilsen et al. [20]. The light sources consisted of a xenon lamp (Osram XBO 2500W housed in a Bauer B L l l + 3/1 chamber, W. Germany) and a slide projector (Prado universal, Leitz, W. Germany, with a 250 W, 24 V halogen lamp) with a built in calflex filter. In front of the xenon lamp was placed a 30cm water filter to absorb the infra red radiation. Light of different wavelengths, 477, 662 and 700 nm., were obtained with interference filters (Jena Glaswerk, Schott and Gen, W. Germany) with a half band width of 20 nm. The I was measured with a radiometer (Li-Cor Model 185, Lambda instruments Corp. USA) with a pyranometer sensor, corrected against an instrument which emits equal quanta at each wavelength [9]. The correction factor between pyranometer values (Wm 2) and #tool quanta m-2s 1 were 6.7 at 474nm, 4.2 at 662 nm and 4.0 at 700 nm. 21% 02 was obtained by removing CO2 from air in a column of soda asbestos, while the other oxygen concentrations (from 0.5 to 10%) were made by mixing 02 and N2 in two pumps (type SA 27 and G 27, W6sthoff, W. Germany). The experimental procedure was as follows: Lemna plants in the 4-frond stage were floated on 50 ml nutrient medium in an assimilation chamber. They were preilluminated in air for 30 minutes with the light to be used in the photoinhibitory treatment. The photosynthetic CO2 uptake rate was measured in white light at the I of 220 pmol m - ; s-1 (in all the experiments unless otherwise stated) after an adaptation of 5 minutes under these light and air conditions. The plants were then subjected to the 'photoinhibitory treatment' for 2 hours, after which the plants were once more placed under the measurement I in the presence of air for 5 minutes and the photosyn-
37 thetic rate was measured. The plant area was then determined with an optical area meter. Photoinhibitory treatment consisted of exposure of the plants to monochromatic light (477, 662 and 700 nm) and white light together with various gas mixtures: a) an atmosphere of N2 and I producing similar quanta, (160 #tool m 2s-l), or, b) an atmosphere of N2 or various concentrations of 02 (0.5% to 21% 02) at an I ensuring similar photosynthetic rates or, c) various gases (N> 2% 02, 21% 02 and air) at an I giving similar quanta (110 /~molm-2 s-1 ) or, d) N2 and darkness. Data were compared by analysis of variance (Anova 1 way), and the LSD (Least Significant Difference) was calculated to show significant difference between the means of various treatments based on raw data [35].
Results and discussion
Exposure of Lemna gibba plants to light in a N 2 atmosphere, resulted in photoinhibition (Tables la and lb). Monochromatic light at wavelength 477 nm was more effective in producing photoinhibition in absence of CO2 and 02 than either 662 nm or 700 nm. Since PS 2 is mainly excited by 477 nm [30], the data indicates that PS 2 is more sensitive to photoinhibitory damage than PS 1. The simultaneous excitation of both the photosystems (662 nm and white light) at the same quanta level, produced a more pronounced photoinhibition (Table 1a) than the excitation of PS 1 alone (700nm). When the irradiance of the monochromatic light was regulated to levels which in normal air gives similar photosynthetic rates (l.9/~mol COzm 2s-|), and was given during photoinhibitory treatment (Table lb), the PS 1 (700 nm) exciting light showed less pronounced photoinhibition than PS 2 exciting light (477 nm). This observation provides further support for the hypothesis that PS 2 is damaged more than PS 1 by photoinhibition [7, 14, 21, 28, 29]. Occurrence of photoinhibition on illumination can be attributed to the accumulation of excess photochemical energy in the absence of 02 and CO2 [5, 17, 26]. Observation of a higher percent photoinhibition when illuminating plant material with a higher I (Table la), in comparison to that observed when lower I (Table lb) were used, further confirms that N2 alone is not the cause of inhibtion of photosynthetic CO2 uptake, but light is also a contributing factor. The same conclusion was shown by Krause et al. [18]. The effect of exposure to light and darkness on the inhibition of photosynthesis under anaerobic conditions were different. In darkness the plants wilted and showed a very high inhibition of photosynthesis (data not shown), indicating that under anaerobic conditions, the damage caused to leaves in light and darkness are of different nature.
38 Table la. Effect of photoinhibitory treatment in monochromatic light at different wavelengths, at the same I. Photoinhibitory treatment consisted of exposure of plants for 120min to N2 and the monochromatic light. The photosynthetic measurements before and after the treatment were made in air and white light at an I of 220 #mol quanta m-2 s- 1 Mean of i S.D. of 3 parallel experiments. LSD for percent photoinhibition at 5% p-level = 2.5 based on raw data.
Photoinhibitory treatment
Net photosynthesis /~mol CO2 m 2s-l
Wavelength nm
I #mol m-2 s- ~
Before treatment
After treatment
White light 477 662 700
160 160 160 160
5.2 4.8 4.9 4.9
3.7 3.3 3.6 4.3
± 0.1 ± 0.2 __+0.2 ___0.4
% Photoinhibition
± _ ± ±
0.1 0.1 0.04 0.3
29.8 i31.6 ± 26.5 ± 12.9 +
1.2 1.6 2.3 0.4
Table lb. Effect of photoinhibitory treatment in monochromatic light at different wavelengths, at an I producing similar photosynthetic rates (1.9/~mol CO2m 2s-l). Photoinhibitory treatment consisted of 120rain. exposure to Nz and the monochromatic light. The photosynthetic measurements before and after the treatment were made in air and white light at an I of 220 #tool quanta m 2s-< Mean of ±S.D. of 3 parallel experiments. LSD for percent photoinhibition at 5% p-level = 1.5 based on raw data.
Photoinhibitory treatment
Net photosynthesis ,umol CO2 m 2s-I
Wavelength nm
1 #tool m 2s- l
Before treatment
After treatment
White light 477 662 700
50 52 42 160
4.6 4.9 4.9 4.9
4.0 3.8 4.3 4.3
± 0.I +_ 0.1 ___0.1 _+ 0.4
% Photoinhibition
_+ 0.1 ± 0.1 _+ 0.1 + 0.3
12.4 _+ 1.0 22.8 4- 1.I 13.5 ± 1.1 12.9 __+0.4
Different gas atmospheres in c o m b i n a t i o n with the selective excitation o f PS 1 a n d PS 2, were used d u r i n g the p h o t o i n h i b i t o r y treatment. Table 2 a n d Fig. 1 show that the degree of p h o t o i n h i b i t i o n was influenced by the photosystem excited a n d the type a n d c o n c e n t r a t i o n of gases used. M o n o c h r o m a t i c lights at 700, 662 a n d 477 n m produce different rates of photosynthetic CO2 u p t a k e at the same q u a n t a level, since they excite the photosystems to various extent. Table 2, therefore, includes the effect of the I level in a d d i t i o n to the effects o f the wavelengths a n d the gas c o m b i n a t i o n s . Figure 1 d e m o n s t r a t e s only the effect of 02 c o n c e n t r a t i o n d u r i n g the selective excitation of the photosystems, ~ince here the photosynthetic efficiency was kept at a similar level d u r i n g the treatment. P h o t o i n h i b i t i o n was gradually reduced in the presence of 02 when PS 2 was m a i n l y (477 nm) or partly (662 nm) operative (Table 2). This corresponds with the findings of Powles et al. [26], that O2 alleviates p h o t o i n h i b i t i o n . The involvem e n t of p h o t o r e s p i r a t i o n a n d s u b s e q u e n t refixation of the photorespired CO2 as suggested by O s m o n d [23], seem to be a reasonable e x p l a n a t i o n for this reduction in p h o t o i n h i b i t i o n in presence o f 02, especially since the a m o u n t of relief
39 Table 2. Effect of photoinhibitory treatment with monochromatic light at wavelengths 477,662 and 700 n m at the I of 110/~mol quanta m 2 s - ~in various gas atmospheres for 120 min. Photosynthetic measurements before and 5 minutes after treatment were made in white light at I of 450 #mol quanta m -2 s -1 in air. Mean + S.D. of 3 parallel experiments. (LSD between the rows at 5% p-level = 2.2). Atmosphere during photoinhibitory treatment
% Photoinhibition
N2 2% 02 in N 2 21% O 2 i n N 2 Air
Wavelength during treatment 477 n m 662 n m
700 n m
28 18 12 2
11 17 7 0
+ _+ _ ±
2 1 1 1
22 12 9 0
+ 2 _+ 2 _ 2 _ 0
_+ 44_
1 2 1 0
from photoinhibition corresponded with the increase in 02 concentration, hence with photorespiration. At 662 nm and white light, when both the photosystems were excited, pseudocyclic electron transport might be operative [11] in the presence of O2 and no CO2 [12], which have accounted for the consumption of photochemical energy in addition to that used by photorespiration. The largest reduction in photoinhibition occurred at oxygen concentrations above 2%, corresponding with the 28
O,o/
24
e--e O--O T--v u--o
2O
700nm,160 #mol 4 7 7 ,, 52 ,, 6 6 2 ,, 42 white light, 50
m-2s
-1
6
O
T~ 16 ,.c O
"5 12
o
,X
.ic Q_
°
X"
,I
0
I
2
I
4
I
6
I
8
i
10
i
12
i
14
;
1
18
~
0
212
0 2 concentration, %
Fig. 1. Effect of 120min. exposure of intact Lemna gibba plants, to various 02 concentrations and monochromatic light selectively exciting the photosystems (700, 477 and 662 nm) or white light, at an I producing similar photosynthetic CO 2 uptake rate. The photosynthetic CO~ uptake rate before and after photoinhibitory treatment was measured in white light at an I of 220 #mol m - 2 s ~. Mean of results form 3 parallel experiments is plotted in the figure. LSD (least significant difference) at 0.05 level, a m o n g the various oxygen concentrations and among different wavelengths is 0.8 and 1.3 respectively.
40 highest rate of pseudocyclic phosphorylation which begins to show saturation at 4% 02 and above [13], and the higher rate ofphotorespiration which increases with 02 concentration. When 02 and CO2 (air) are provided during the treatment, photoinhibition was negligible, which suggests that carbon metabolism via integrated PCR and PCO cycles provides suitable sinks for the photochemical energy generated by the photosystems [26]. The 02 levels above 2% significantly reduced photoinhibition when PS 1 was mainly excited (700nm), (Table 2 and Fig. 1). The low 02 levels were more inhibitory for PS 1 and photoinhibition was maximum at 2% 02 concentration (Fig. 1). Some inhibitory effect of low 02 concentration was also visible when the plant material was illuminated with the lights exciting some PS 1, that is, 662 nm and white light. These observations indicate an enhancing effect of low 02 on PS 1 inhibition. The presence of low 02 concentrations (up to 2%) were not at all helpful in reducing photoinhibition when PS 1 was excited, as the inhibition was even more than that in the complete absence o f O 2 and CO2 (Table 2 and Fig. 1). This supports the view that the photoinhibition of PS I is enhanced by oxygen [10, 29, 32]. The lower rates of photorespiration at low O2 levels [15, 36, 37], would cause some accumulation of excessive photochemical energy. This extra energy along with the energy rich radicals formed in the presence of oxygen [8, 32] might be responsible for~the higher percent photoinhibition observed when PS 1 is excited (700 nm) at low 02 levels. The 02 dependence of PS 1 photoinhibition has only been demonstrated in isolated chloroplasts [34] or in chloroplast fragments [29, 32]. The present study does not show oxygen dependence, but shows increased photoinhibitory effect on PS 1 excitation (700 nm) at low oxygen concentrations (Table 2 and Fig. 1). This sensitivity of PS 1 inhibition to low oxygen is also reflected whenever PS 1 is excited together with PS 2 (662 nm and white light). Such an effect was also noted by Cornic and Miginiac-Maslow [6] in whole chain electron transport measurements in broken chloroplasts. 0gren and 0quist [24] hinted that in intact Lemna gibba plants, PS 1 might be protected against photoinhibition. We suggest that absence of sensitivity of PS 1 photoinhibition to oxygen concentrations higher than 2% could be due to some kind of protective (reducing) mechanism which, in the intact plant systems, might become operative at the higher oxygen levels, and this mechanism could involve photorespiration. Satoh [32] pointed to the involvement of an oxidised entity (a peroxide, but not H202) which could be formed when oxygen combines with the photochemical product on the reducing side of PS 1. This peroxide is probably a strong inhibitor of the early steps in photosynthesis. Foyer and Hall [8] suggested that the generation of a highly destructive oxygen species, such as 102 and "OH would damage the reaction center of PS 1 and lead to photoinhibition. It can be inferred from the present study that PS 2 is mainly damaged on illuminating the Lemna gibba plants under anaerobic conditions. PS 1 photoinhibition is sensitive only to low 02 levels in the intact plant material. It is further suggested that pseudocyclic electron transport, in addition to photo-
41 respiration, could be considered as an additional mechanism which provide relief from photoinhibition when both the photosystems are excited.
Acknowledgements The authors wish to acknowledge Dr M.K. Haugstad and L.A. Oritsland for their valuable assistance in the preparation of this manuscript.
References 1. Arntzen CJ, Kyle D, Wettern M and Ohad I (1984) Photoinhibition: A Consequence of the accelerated breakdown of the apoprotein of the secondary electron acceptor of PS 2. In: Hallick R et al. (eds) Biosynthesis of the photosynthetic apparatus: Molecular biology, development and regulation, UCLA symposia on Molecular and Cellular Biology, New series 14:313-324 2. Bj6rkman O (1968) Further studies on differentiation of photosynthetic properties in sun and shade ecotypes of Solidago virgaurea. Physiol Plant 21:84-99 3. Bj6rkman O and Holmgren P (1963) Adaptability of the photosynthetic apparatus to light intensity in ecotypes from exposed and shaded habitats. Physiol Plant 16:889-914 4. Chaturvedi R, Haugstad M and Nilsen S (1982) The relationship between photosynthetic electron transport and photorespiratory 14CO2 release after DCMU treatment in the duckweed, Lemna gibba. Physiol Plant 56:23-27 5. Cornic G, Woo KC and Osmond CB (1982) Photoinhibition of CO2 dependent O 2 evolution by intact chloroplasts isolated from spinach leaves. Plant Physiol 70:1310-1315 6. Cornic G, Miginiac-Maslow M (1985) Photoinhibition of photosynsthesis in broken chloroplasts as a function of electron transfer rates during light treatment. Plant Physiol 78:724-729 7. Critchley C (1981) Studies on mechanism ofphotoinhibition in higher plants: 1. Effects of high light intensity on chloroplast activities in cucumber adapted to low light. Plant Physiol 67:1161-1165 8. Foyer CH and Hall DO (1980) Oxygen metabolism in the active chloroplasts. Trends Biochem Sci pp 88-191 9. Halldal P, Nordstr6m B and (}quist G (1975) Quanta-corrected spectrometer for recording different types of photobiological, photochemical and spectrophotometric measurements. Physiol Plant 35:5-10 I0. Harvey GW and Bishop NI (1978) Photolability of photosynthesis in two separate mutants of Scenedesmus obliquus. Plant Physiol 62:330 336 11. Heber U (1969) Conformational changes of chloroplast induced by illumination of leaves in vivo. Biochim Biophys Acta 180:302-319 12. Heber U (1973) Stoichiometry of reduction and phosphorylation during illumination of intact chloroplasts. Biochem Biophys Acta 305:140-152 13. Heber U and French CS (1968) Effects of oxygen on the electron transport chain of photosynthesis. Planta 79:99 112 14. Jones LW and Kok B (1966) Photoinhibition of chloroplasts reactions: 1. Kinetics and action spectra. Plant Physiol 41:103%1043 15. Kaminski Z, Gutowski R and Maleszewski S (1979) Photosynthesis in bean leaves treated with glycolic acid oxidase inhibitor. Z Pflanzen Physiol 91:17 24 16. Kok B, Gassner EB and Rurainski HJ (1965) Photoinhibition of chloroplasts reactions. Photochem Photobiol 4:215-227 17. Krause GH, Kirk M, Heber U and Osmond CB (1978) 02 dependent inhibition ofphotosyn-
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18. 19.
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thetic capacity in intact isolated chloroplasts and isolated cells from spinach leaves illuminated in absence of CO2. Planta 142:229-233 Krause GH, K6ster S and Wong SC (1985) Photoinhibition of photosynthesis under anaerobic conditions studied with leaves and chloroplasts of Spinaeia oleraeea. Planta 65:430--438 Kyle DJ, Ohad I and Arntzen CJ (1984) Membrane protein damage and repair: Selective loss of a quinone protein function in chloroplast membranes. Proc Natl Acad Sci USA 81:4070-4074 Nilsen S, Kristiansen E and Halldal P (1975) A system for continuous measurement of photosynthetic rate and dark respiration at a constant CO 2 level. Physiol Plant 35:59-6! Nilsen S and Danielsen S (1984) Photoinhibition of photosynthesis in Lemna gibba: The effect of 02, CO2 and selective inhibition of photosystem 2. Pflanzen Physiol 115:39-48 Nilsen S, Chaturvedi R and Dons C (1984) Photoinhibition of photosynthesis in Lemna gibba: The effect of different 02 and CO2 concentrations during the photoinhibitory treatment. Acta Horticulturae 129:129-135 Osmond CB (1981) Photorespiration and Photoinhibition. Some implications for the energetics of photosynthesis. Biochim Biophys Acta 639:77-98 Ogren E and Oquist G (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. II Photosynthetic electron transport. Physiol Plant 62:187-192 Powles SB (1984) Photoinhibition of photosynthesis induced by visible light. Ann Rev Plant Physiol 35:15-44 Powles SB, Osmond CB and Thorne SW (1979) Photoinhibition of intact attached leaves of C 3 plants illuminated in absence of both CO 2 and of photorespiration. Plant Physiol 64:982-988 Powles SB and Critchley C (1980) Effect of light intensity during growth on photoinhibition of intact attached bean leaflets. Plant Physiol 65:1181-1184 Powles SB and Thorne SW (1981) Effect of high light treatments in inducing photoinhibitlon of photosynthesis in intact leaves of low light grown Phaseolus vulgaris and Lastreopsei microsora. Planta 152:471-477 Powles SB and Bjorkman O (1982) Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander. Plapta 156:97-107 Ried A (1972) Improved action spectra of light reaction I and II. In: Forti G e t al. (eds) Proc 2rid Int Congr Photosyn Res, pp 763 772. The Hague: Dr W. Junk Satoh K (1970) Mechanism of photoinactivation in photosynthetic systems. I The dark reaction in inactivation. Plant and Cell Physiol 11:15-27 Satoh K (1970) Mechanism of photoinactivation in photosynthetic systems. II The occure~ce and properties of two different types of photoinactivation. Plant and Cell Physiol 11:29-38 Satoh K (1970) Mechanism ofphotoinactivation in photosynthetic systems. III Site and mode of inactivation in PS I. Plant and Cell Physiol 1I:18%197 Satoh K and Fork DC (1982) Photoinhibition of reaction centers of PS 1 and 2 in intact Bryopsis chloroplasts under anaerobic conditions. Plant Physiol 70:1004-1008 Sokal RR and Rohlf FJ (1981) Assumptions of analysis of variance. In: Biometry, pp 4 0 ~ 5 . San Francisco: WH Freeman & Company Tolbert NE (1973) Glycolate biosynthesis. In: Horecker BL and Stadtman ER (eds) Current topics in cellular regulation, pp 21-50. New York: Academic Press Inc. Tolbert NE (1977) Regulation of products of photosynthesis by photorespiration and red)Jction of carbon. In: Mitsui A (ed.) Biological Solar Energy Conversion, Vol. 12, 243-264. New York: Academic Press Inc.
© Martinus NijhoffPublishers, Dordrecht--Printed in the Netherlands
35
Regular paper
Photoinhibition of photosynthesis: effect of 02 and selective excitation of the photosystems in intact Lemna gibba plants REKHA CHATURVEDI & STEIN NILSEN The Phytotron, Department of Biology, University of Oslo, Post Box 1066, Blindern, 0316 Oslo 3, Norway Received 7 April 1986; accepted in revised form 30 September 1986
Keywords: monochromatic light, oxygen concentration, photoinhibition, photorespiration, photosynthesis, photosystems Abstract. Intact Lemna gibba plants were photoinhibited under anaerobic conditions on illumination with monochromatic light which selectively excited the photosystems. Photoinhibition was less when PS 1 was excited and greatest when mainly PS 2 was excited, which suggests that PS 2 was most damaged by photoinhibition induced in complete absence of 0 2 and CO:. The illumination of plants with monochromatic light exciting PS 1, at different 0 2 concentrations (in CO 2 deficient conditions), showed that PS 1 photoinhibition was increased at the low 0 2 concentrations. The damage to PS 1 was more evident at 2% 0 2 than at the higher 0 2 concentrations. CO 2 as well as 0 2 at atmospheric concentration, (air), was necessary for complete protection of the plant from photoinhibition when both photosystems were excited either separately or together. Abbreviations: I - irradiance, photon fluence rate, PCO - photosynthetic carbon oxidation cycle, PCR - photosynthetic carbon reduction cycle, PS 1 - photosystem l, PS 2 - photosystem 2
Introduction
Photoinhibition of photosynthesis is the consequence of exposure of plants to an irradiance (I) much higher than that used during growth, [3, 16, 28]. When the capacity of photosystems for dissipating the excitation energy is not large enough, the reaction centers are inactivated and photoinhibition occurs [27]. The illumination of plants in absence or low concentrations of CO 2 and 02 also results in photoinhibition of photosynthesis. In this situation photoinhibition is due to the damage caused by the unconsumed photochemical energy through the integrated operation of photosynthetic carbon reduction cycle (PCR) and photosynthetic carbon oxidation cycle (PCO) [5, 17, 26]. It has been shown that the PS 2 related reactions are mainly damaged in photoinhibition [2, 7, 28]. It is suggested that the primary site of damage is at the 32-kilo dalton herbicide binding protein, the QB apoprotein [1, 19]. PS 1 can also be damaged as a consequence of photoinhibition [27, 29, 31]. Photoinhibition ofPS 1 was equal to [31] or less than [26, 27, 34] that of PS 2. The proposed site of this inhibition is very close to the PS 1 reaction center [33]. Harvey and Bishop [10], Powles and Bj6rkman [29] and Satoh [32] found that
36 the photoinhibition of PS 1 is an oxygen requiring process. In their opinion, the generation of oxidised destructive entity may be involved in the damage of PS 1. Maintenance of some minimal level of photosynthetic carbon metabolism is necessary to prevent or minimize photoinhibition [25]. Pseudocyclic electron transport to some extent can also help in the dissipation of the excess of photochemical energy [27]. The main objective of this study was to establish the involvement of the photosystems in the photoinhibition of intact Lemna gibba plants and to study the effect of various 02 levels on the degree of damage.
Material and methods
Axenic cultures of Lemna gibba L. G3, obtained from Dr Elisabeth Tillberg, University of Stockholm, were precultured at I of 220 #tool m -2 s 1, in nutrient medium containing sucrose, as reported by Chaturvedi et al. [4]. These cultures were then transferred to nutrient medium without sucrose and grown at the same I for 8 days [22]. Photosynthetic measurements were made in a closed gas exchange system using an infrared gas analyser (Uras 2, Hartman and Braun, W. Germany) as described by Nilsen et al. [20]. The light sources consisted of a xenon lamp (Osram XBO 2500W housed in a Bauer B L l l + 3/1 chamber, W. Germany) and a slide projector (Prado universal, Leitz, W. Germany, with a 250 W, 24 V halogen lamp) with a built in calflex filter. In front of the xenon lamp was placed a 30cm water filter to absorb the infra red radiation. Light of different wavelengths, 477, 662 and 700 nm., were obtained with interference filters (Jena Glaswerk, Schott and Gen, W. Germany) with a half band width of 20 nm. The I was measured with a radiometer (Li-Cor Model 185, Lambda instruments Corp. USA) with a pyranometer sensor, corrected against an instrument which emits equal quanta at each wavelength [9]. The correction factor between pyranometer values (Wm 2) and #tool quanta m-2s 1 were 6.7 at 474nm, 4.2 at 662 nm and 4.0 at 700 nm. 21% 02 was obtained by removing CO2 from air in a column of soda asbestos, while the other oxygen concentrations (from 0.5 to 10%) were made by mixing 02 and N2 in two pumps (type SA 27 and G 27, W6sthoff, W. Germany). The experimental procedure was as follows: Lemna plants in the 4-frond stage were floated on 50 ml nutrient medium in an assimilation chamber. They were preilluminated in air for 30 minutes with the light to be used in the photoinhibitory treatment. The photosynthetic CO2 uptake rate was measured in white light at the I of 220 pmol m - ; s-1 (in all the experiments unless otherwise stated) after an adaptation of 5 minutes under these light and air conditions. The plants were then subjected to the 'photoinhibitory treatment' for 2 hours, after which the plants were once more placed under the measurement I in the presence of air for 5 minutes and the photosyn-
37 thetic rate was measured. The plant area was then determined with an optical area meter. Photoinhibitory treatment consisted of exposure of the plants to monochromatic light (477, 662 and 700 nm) and white light together with various gas mixtures: a) an atmosphere of N2 and I producing similar quanta, (160 #tool m 2s-l), or, b) an atmosphere of N2 or various concentrations of 02 (0.5% to 21% 02) at an I ensuring similar photosynthetic rates or, c) various gases (N> 2% 02, 21% 02 and air) at an I giving similar quanta (110 /~molm-2 s-1 ) or, d) N2 and darkness. Data were compared by analysis of variance (Anova 1 way), and the LSD (Least Significant Difference) was calculated to show significant difference between the means of various treatments based on raw data [35].
Results and discussion
Exposure of Lemna gibba plants to light in a N 2 atmosphere, resulted in photoinhibition (Tables la and lb). Monochromatic light at wavelength 477 nm was more effective in producing photoinhibition in absence of CO2 and 02 than either 662 nm or 700 nm. Since PS 2 is mainly excited by 477 nm [30], the data indicates that PS 2 is more sensitive to photoinhibitory damage than PS 1. The simultaneous excitation of both the photosystems (662 nm and white light) at the same quanta level, produced a more pronounced photoinhibition (Table 1a) than the excitation of PS 1 alone (700nm). When the irradiance of the monochromatic light was regulated to levels which in normal air gives similar photosynthetic rates (l.9/~mol COzm 2s-|), and was given during photoinhibitory treatment (Table lb), the PS 1 (700 nm) exciting light showed less pronounced photoinhibition than PS 2 exciting light (477 nm). This observation provides further support for the hypothesis that PS 2 is damaged more than PS 1 by photoinhibition [7, 14, 21, 28, 29]. Occurrence of photoinhibition on illumination can be attributed to the accumulation of excess photochemical energy in the absence of 02 and CO2 [5, 17, 26]. Observation of a higher percent photoinhibition when illuminating plant material with a higher I (Table la), in comparison to that observed when lower I (Table lb) were used, further confirms that N2 alone is not the cause of inhibtion of photosynthetic CO2 uptake, but light is also a contributing factor. The same conclusion was shown by Krause et al. [18]. The effect of exposure to light and darkness on the inhibition of photosynthesis under anaerobic conditions were different. In darkness the plants wilted and showed a very high inhibition of photosynthesis (data not shown), indicating that under anaerobic conditions, the damage caused to leaves in light and darkness are of different nature.
38 Table la. Effect of photoinhibitory treatment in monochromatic light at different wavelengths, at the same I. Photoinhibitory treatment consisted of exposure of plants for 120min to N2 and the monochromatic light. The photosynthetic measurements before and after the treatment were made in air and white light at an I of 220 #mol quanta m-2 s- 1 Mean of i S.D. of 3 parallel experiments. LSD for percent photoinhibition at 5% p-level = 2.5 based on raw data.
Photoinhibitory treatment
Net photosynthesis /~mol CO2 m 2s-l
Wavelength nm
I #mol m-2 s- ~
Before treatment
After treatment
White light 477 662 700
160 160 160 160
5.2 4.8 4.9 4.9
3.7 3.3 3.6 4.3
± 0.1 ± 0.2 __+0.2 ___0.4
% Photoinhibition
± _ ± ±
0.1 0.1 0.04 0.3
29.8 i31.6 ± 26.5 ± 12.9 +
1.2 1.6 2.3 0.4
Table lb. Effect of photoinhibitory treatment in monochromatic light at different wavelengths, at an I producing similar photosynthetic rates (1.9/~mol CO2m 2s-l). Photoinhibitory treatment consisted of 120rain. exposure to Nz and the monochromatic light. The photosynthetic measurements before and after the treatment were made in air and white light at an I of 220 #tool quanta m 2s-< Mean of ±S.D. of 3 parallel experiments. LSD for percent photoinhibition at 5% p-level = 1.5 based on raw data.
Photoinhibitory treatment
Net photosynthesis ,umol CO2 m 2s-I
Wavelength nm
1 #tool m 2s- l
Before treatment
After treatment
White light 477 662 700
50 52 42 160
4.6 4.9 4.9 4.9
4.0 3.8 4.3 4.3
± 0.I +_ 0.1 ___0.1 _+ 0.4
% Photoinhibition
_+ 0.1 ± 0.1 _+ 0.1 + 0.3
12.4 _+ 1.0 22.8 4- 1.I 13.5 ± 1.1 12.9 __+0.4
Different gas atmospheres in c o m b i n a t i o n with the selective excitation o f PS 1 a n d PS 2, were used d u r i n g the p h o t o i n h i b i t o r y treatment. Table 2 a n d Fig. 1 show that the degree of p h o t o i n h i b i t i o n was influenced by the photosystem excited a n d the type a n d c o n c e n t r a t i o n of gases used. M o n o c h r o m a t i c lights at 700, 662 a n d 477 n m produce different rates of photosynthetic CO2 u p t a k e at the same q u a n t a level, since they excite the photosystems to various extent. Table 2, therefore, includes the effect of the I level in a d d i t i o n to the effects o f the wavelengths a n d the gas c o m b i n a t i o n s . Figure 1 d e m o n s t r a t e s only the effect of 02 c o n c e n t r a t i o n d u r i n g the selective excitation of the photosystems, ~ince here the photosynthetic efficiency was kept at a similar level d u r i n g the treatment. P h o t o i n h i b i t i o n was gradually reduced in the presence of 02 when PS 2 was m a i n l y (477 nm) or partly (662 nm) operative (Table 2). This corresponds with the findings of Powles et al. [26], that O2 alleviates p h o t o i n h i b i t i o n . The involvem e n t of p h o t o r e s p i r a t i o n a n d s u b s e q u e n t refixation of the photorespired CO2 as suggested by O s m o n d [23], seem to be a reasonable e x p l a n a t i o n for this reduction in p h o t o i n h i b i t i o n in presence o f 02, especially since the a m o u n t of relief
39 Table 2. Effect of photoinhibitory treatment with monochromatic light at wavelengths 477,662 and 700 n m at the I of 110/~mol quanta m 2 s - ~in various gas atmospheres for 120 min. Photosynthetic measurements before and 5 minutes after treatment were made in white light at I of 450 #mol quanta m -2 s -1 in air. Mean + S.D. of 3 parallel experiments. (LSD between the rows at 5% p-level = 2.2). Atmosphere during photoinhibitory treatment
% Photoinhibition
N2 2% 02 in N 2 21% O 2 i n N 2 Air
Wavelength during treatment 477 n m 662 n m
700 n m
28 18 12 2
11 17 7 0
+ _+ _ ±
2 1 1 1
22 12 9 0
+ 2 _+ 2 _ 2 _ 0
_+ 44_
1 2 1 0
from photoinhibition corresponded with the increase in 02 concentration, hence with photorespiration. At 662 nm and white light, when both the photosystems were excited, pseudocyclic electron transport might be operative [11] in the presence of O2 and no CO2 [12], which have accounted for the consumption of photochemical energy in addition to that used by photorespiration. The largest reduction in photoinhibition occurred at oxygen concentrations above 2%, corresponding with the 28
O,o/
24
e--e O--O T--v u--o
2O
700nm,160 #mol 4 7 7 ,, 52 ,, 6 6 2 ,, 42 white light, 50
m-2s
-1
6
O
T~ 16 ,.c O
"5 12
o
,X
.ic Q_
°
X"
,I
0
I
2
I
4
I
6
I
8
i
10
i
12
i
14
;
1
18
~
0
212
0 2 concentration, %
Fig. 1. Effect of 120min. exposure of intact Lemna gibba plants, to various 02 concentrations and monochromatic light selectively exciting the photosystems (700, 477 and 662 nm) or white light, at an I producing similar photosynthetic CO 2 uptake rate. The photosynthetic CO~ uptake rate before and after photoinhibitory treatment was measured in white light at an I of 220 #mol m - 2 s ~. Mean of results form 3 parallel experiments is plotted in the figure. LSD (least significant difference) at 0.05 level, a m o n g the various oxygen concentrations and among different wavelengths is 0.8 and 1.3 respectively.
40 highest rate of pseudocyclic phosphorylation which begins to show saturation at 4% 02 and above [13], and the higher rate ofphotorespiration which increases with 02 concentration. When 02 and CO2 (air) are provided during the treatment, photoinhibition was negligible, which suggests that carbon metabolism via integrated PCR and PCO cycles provides suitable sinks for the photochemical energy generated by the photosystems [26]. The 02 levels above 2% significantly reduced photoinhibition when PS 1 was mainly excited (700nm), (Table 2 and Fig. 1). The low 02 levels were more inhibitory for PS 1 and photoinhibition was maximum at 2% 02 concentration (Fig. 1). Some inhibitory effect of low 02 concentration was also visible when the plant material was illuminated with the lights exciting some PS 1, that is, 662 nm and white light. These observations indicate an enhancing effect of low 02 on PS 1 inhibition. The presence of low 02 concentrations (up to 2%) were not at all helpful in reducing photoinhibition when PS 1 was excited, as the inhibition was even more than that in the complete absence o f O 2 and CO2 (Table 2 and Fig. 1). This supports the view that the photoinhibition of PS I is enhanced by oxygen [10, 29, 32]. The lower rates of photorespiration at low O2 levels [15, 36, 37], would cause some accumulation of excessive photochemical energy. This extra energy along with the energy rich radicals formed in the presence of oxygen [8, 32] might be responsible for~the higher percent photoinhibition observed when PS 1 is excited (700 nm) at low 02 levels. The 02 dependence of PS 1 photoinhibition has only been demonstrated in isolated chloroplasts [34] or in chloroplast fragments [29, 32]. The present study does not show oxygen dependence, but shows increased photoinhibitory effect on PS 1 excitation (700 nm) at low oxygen concentrations (Table 2 and Fig. 1). This sensitivity of PS 1 inhibition to low oxygen is also reflected whenever PS 1 is excited together with PS 2 (662 nm and white light). Such an effect was also noted by Cornic and Miginiac-Maslow [6] in whole chain electron transport measurements in broken chloroplasts. 0gren and 0quist [24] hinted that in intact Lemna gibba plants, PS 1 might be protected against photoinhibition. We suggest that absence of sensitivity of PS 1 photoinhibition to oxygen concentrations higher than 2% could be due to some kind of protective (reducing) mechanism which, in the intact plant systems, might become operative at the higher oxygen levels, and this mechanism could involve photorespiration. Satoh [32] pointed to the involvement of an oxidised entity (a peroxide, but not H202) which could be formed when oxygen combines with the photochemical product on the reducing side of PS 1. This peroxide is probably a strong inhibitor of the early steps in photosynthesis. Foyer and Hall [8] suggested that the generation of a highly destructive oxygen species, such as 102 and "OH would damage the reaction center of PS 1 and lead to photoinhibition. It can be inferred from the present study that PS 2 is mainly damaged on illuminating the Lemna gibba plants under anaerobic conditions. PS 1 photoinhibition is sensitive only to low 02 levels in the intact plant material. It is further suggested that pseudocyclic electron transport, in addition to photo-
41 respiration, could be considered as an additional mechanism which provide relief from photoinhibition when both the photosystems are excited.
Acknowledgements The authors wish to acknowledge Dr M.K. Haugstad and L.A. Oritsland for their valuable assistance in the preparation of this manuscript.
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