Aloysius Wild

Studies on the mechanism of photosystem II photoinhibition II. The involvement of toxic oxygen species

In a previous paper it was shown that photoinhibition of reaction centre II of spinach thylakoids was predominantly caused by the degradation of D1-protein. An initial inactivation step at the QB-site was distinguished from its breakdown. The present paper deals with the question as to whether this loss of QB-function is caused by oxygen radical attack. For this purpose the photoinhibition of thylakoids was induced at 20°C in the presence of either superoxide dismutase and catalase or the antioxidants glutathione and ascorbic acid. This resulted in comparable though not total protection of D1-protein, photochemistry and fluorescence from photoinhibition. The combined action of both the enzymatic and the non-enzymatic radical scavenging systems brought about an even more pronounced protective effect against photoinhibition than did either of the two systems singularly at saturating concentrations. The results signify a major contribution of activated oxygen species to the degradation process of D1-protein and the related phenomena of photoinhibition. Thylakoids treated with hydroxyl radicals generated through a Fenton reaction showed a loss of atrazine binding sites, electron transport capacity and variable fluorescence in a similar manner, though not to the same extent, as usually observed following photoinhibitory treatment.

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C3 (Sinapis alba) and C4 (Zea mays) plants under atmospheric conditions (400 ppm CO2, 21% O2). This photosynthesis inhibition is proceeding slower in C4 leaves. Under non-photorespiratory conditions (1000 ppm CO2, 2% O2) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH4+. The NH4+-accumulation is lower in C4 plants than in C3 plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH4+-accumulation is greatly increased. This indicates that NH4+-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO2-fixation.

Studies on the mechanism of inhibition by phosphinothricin of glutamine synthetase isolated from Triticum aestivum L.

Summary The activity of glutamine synthetase (EC 6.3.1.2) isolated from roots and leaves of wheat is strongly inhibited by phosphinothricin. As revealed by gel filtration and kinetic studies the mechanism of inhibition appears to be irreversible. The results indicate that the rate limiting step of the reaction of the inhibitor with the chloroplast glutamine synthetase is not the initial, but the following reaction. However, of root glutamine synthetase the irreversible inhibition follows a second order reaction depending on the concentration of the enzyme and of phosphinothricin and the rate limiting step is the formation of the initial enzyme-inhibitor complex. According to the kinetic data, the glutamine synthetase of the root appears to be more strongly inhibited by phosphinothricin than the chloroplast glutamine synthetase.

The influence of brassinosteroid on growth and parameters of photosynthesis of wheat and mustard plants.

The growth response of wheat (Triticum aest. L.) and mustard seedlings (Sinapis alba L.) treated with 10(-6) mol · l(-1) brassinosteroid (BR) foliar spray was measured. BR-treatment resulted in a general promotion of plant growth. We found the accumulation of photosynthates to be stimulated in the treated plants, as indicated by enhanced fresh and dry weights of leaves and shoots. BR also promoted the synthesis of soluble proteins and soluble reducing sugars, whereas the chlorophyll content was hardly affected. CO(2)-fixation in vivo as well as the (in vitro) RubPC-ase activity of BR-treated leaves were enhanced. In the developing wheat leaves we detected no difference in the ratio fraction-I-protein (F-I-P)/total soluble protein between BR-treated and control plants whereas in the expanded leaves this ratio was lowered under BR-treatment.

The Effect of Phosphinothricin (Glufosinate) on Photosynthesis I. Inhibition of Photosynthesis and Accumulation of Ammonia

Phosphinothricin (glufosinate) is an irreversible inhibitor of glutamine synthetase in plants. Enzyme inhibition results in substantial light-dependent accumulation of ammonia, inhibition of photosynthesis and ultimately the death of the plants. First of all the metabolic processes in which NH3 is liberated were established. Comparative investigations under photorespiratory and non- photorespiratory conditions with Sinapis alba plants revealed that about 60% of the accumulated NH3 derives from photorespiration. Trials with PPT and KNO3 showed that nitrate assimilation is insignificant as a source of NH3, and so the remaining 40% is accumulated in catabolic or anabolic processes. Under photorespiratory conditions CO2 fixation after treatment with 1 mᴍ PPT finally fell below the compensation point, and this was independent of the addition of nitrate. In non- photorespiratory conditions, by contrast, photosynthesis continued at a level of over 80% for several hours in entire plants and in severed leaves, although at the same time substantial amounts of ammonia had accumulated.

The Effect of Phosphinothricin on the Assimilation of Ammonia in Plants

The effects of ᴅʟ-phosphinothricin and L-methionine sulfoximine on the enzymes of nitrogen assimilation were studied. Furthermore we investigated the accumulation of ammonia and the photosynthetic activity after the treatment of mustard plants with phosphinothricin. Phosphino-thricin was a specific and very strong inhibitor of glutamine synthetase. Major differences, however, were found between the phosphinothricin affinity of the leaf enzyme and that of the root of mustard plants. The leaf enzyme was 50% inhibited at a concentration of 10-4 m phosphinothricin (pI50 = 4), whereas the root enzyme already showed the same effect at a concentration of 2 × 10-5m (pI50 = 4.7). In addition Ki values of about 0.03 mм for the leaf enzyme and 0.002 mм for the root enzyme respectively were determined. Phosphinothricin treatment of plants caused an ammonia accumulation in tissues. The accumulation was light dependent. At the beginning of the light period the major sources of ammonia accumulation could be the nitrogen assimilation as well as catabolic processes of nitrogen compounds. A clear contribution of photorespiration was only found when higher concentrations of ammonia were reached. The application of phosphinothricin induced a strong reduction of CO2 assimilation.

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, QB-independent electron flow and QB-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and QB-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The QB-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the QB-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of QA--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the QB-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.

The effect of phosphinothricin (glufosinate) on photosynthesis. II: The causes of inhibition of photosynthesis

It was shown in the previous study that phosphinothricin (glufosinate) causes an accumulation of ammonia and inhibition of photosynthesis. The extent to which there is a connection between these two processes is now investigated in the present study. First of all. the role of NH3 per se in the impairment of photosynthesis was to be clarified. For this purpose, the inhibition of photosynthesis was investigated in relation to exogenously applied ammonia in chloroplasts, protoplasts and entire leaves. The comparison with the experimental results in leaves in which the ammonia was formed endogenously (by action of phosphinothricin) shows that the ammonia toxicity at least cannot be solely responsible for the inhibition of photosynthesis. This is confirmed by the finding that photosynthesis is maintained under non-photorespiratory conditions, although considerable amounts of NH3 are concentrated at the same time in the plants. A process connected with photorespiration appears to play the essential role. Simultaneous administration of phosphinothricin and glutamine largely prevented the impairment of photosynthesis. This indicates that the main cause for the inhibition of photosynthesis by phosphinothricin is based on a depletion of glutamine. Three subsequent reactions which may cause the inhibition of photosynthesis are discussed: 1) an inhibition of protein biosynthesis, 2) a toxic accumulation of glyoxylate in the photorespiratory cycle and 3) a deficiency of intermediates of the Calvin cycle in consequence of the interrupted photorespiration.

Changes in the stoichiometry of photosystem II components as an adaptive response to high-light and low-light conditions during growth

The effect of different growth light intensities (60 W·m-2, 6 W·m-2) on the performance of the photosynthetic apparatus of mustard plants (Sinapis alba L.) was studied. A distinct decrease in photosystem II content per chlorophyll under low-light conditions compared to high-light conditions was found. For P-680 as well as for Oᴀ and Oв protein the molar ratio between high-light and low-light plants was 1.4 whereas the respective concentrations per chlorophyll showed some variations for P-680 and Oᴀ on the one and Oв protein on the other hand. In addition to the study of photosystem II components, the concentrations of PQ, Cyt f, and P-700 were measured. The light regime during growth had no effect on the amount of P-700 per chlorophyll but there were large differences with respect to PQ and Cyt f. The molar ratio for Cyt f and PQ between high- and low-light leaves was 2.2 and 1.9, respectively. Two models are proposed, showing the functional organization of the pigment system and the electron transport chain in thylakoids of high-light and low-light leaves of mustard plants.

The Variability of the Photosynthetic Unit in Chlorella II. The Effect of Light Intensity and Cell Development on Photosynthesis, P-700 and Cytochrome f in Homocontinuous and Synchronous Cultures of Chlorella.

The effect of light intensity of steady state photosynthesis, the contents of P-700 and cytochrome f was studied in homocontinuously grown Chlorella cells. The adaptation to higher light intensities drastically provokes the decrease of chlorophylls per dry weight, per packed cell volume and per cell. The light saturating curves show that high light grown cells produce threefold more oxygen per chlorophyll, whereas the oxygen evolution per cell is reduced by half. The rates of saturated photosynthesis per chlorophyll are directly correlated with increasing light intensities. The content of P-700 is slightly altered by light intensity. On the other hand, cytochrome f is changed in very strict accordance with the alterations of biomass productivity and photosynthetic capacity. The mathematical equation for the relationship between maximal photosynthesis and cytochrome f concentration is found to be linear with a correlation coefficient of 0.96. In addition to the results of homocontinuously grown Chlorella cells, the relationship between photosynthesis and the content of cytochrome f was analyzed in synchronous cultures. After the onset of light the photosynthetic activity rises as well as the content of cytochrome f. During the dark period, however, when the photosynthetic capacity is low, there still remains a high amount of cytochrome f. The results are discussed on the basis of the variability of the photosynthetic unit and the in-tactness of the thylakoid membrane. It is proposed to introduce the term of the «physiological photosynthetic unit» defined by the ratio of chlorophyll to cytochrome f. Its size is proved to be used as an indicator of the rate limiting step in the photosynthetic electron transport.