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  • Author: Georg H. Schmid x
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Abstract

Departement de Botanique, Service de Photobiologie, Universite de Liege, Sart-Tilman, Belgium and Georg H. Schmid

Abstract

The mykotrophic orchid Neottia nidus-avis does not evolve oxygen in the light but is able to perform photophosphorylation. The low temperature fluorescence emission spectrum lacks the 680 and 690 nm bands. Hence, the spectroscopic chlorophyll a forms which are attributed to photosystem II do not occur in plastids of this orchid. The low temperature excitation spectrum of photosystem I fluorescence exhibits a maximum at 666 nm. The position of this maximum appears not to be influenced by energy transfer and corresponds to the absorption maximum of the chlorophyll form which emits the photosystem I fluorescence. Energy migration, however, occurs from carotenoids whose absorption spectrum is shifted to longer wavelengths and which cause the yellow-brown color of the Neottia plastids. Room temperature fluorescence emission shows after the onset of light no variable part.

Despite the fact that plastids of the tobacco mutant NC 95 at most evolve only traces of oxygen the low temperature emission spectrum shows the three bands which are usually observed with fully functioning chloroplasts. However, the two bands at 680 and 690 nm are distinctly lower than with the wild type. The variable portion of room temperature fluorescence is barely detectable. In line with the very low capacity for oxygen evolution, rates of electron transport partial reactions in the region of photosystem II are extremely low. In agreement with this observation no 690 nm absorption change signal is detected. However, a normal P+700 signal is seen. In the presence of electron donors like reduced phenazine methosulfate the decay time of the P+700 signal is faster than with the wild type.

The yellow tobacco mutant Su/su var. aurea which exhibits at high light intensities higher rates of photosynthesis than the wild type shows at low temperature an emission spectrum with stronger photosystem II bands than the wild type.

Abstract

The Kok effect of photosynthesis was investigated in different tobacco mutants. It was found that the breaks in the light intensity curve were always at or around 1000 lux in all plants tested regardless of the unit sizes which differed by a factor of 10. It was concluded that the photo­ receptor responsible for the effect must be present in the wild type and the chlorophyll deficient mutants in the same amount and is probably not chlorophyll. Due to the fact that the light dependency of the Hill reaction in isolated tobacco chloroplasts also shows a break at or around the “Kok intensity” it was concluded that probably a structural change of the photochemical apparatus around 1000 lux contributes to the effect. Measurement of 180 2-uptake by mass spectrometry at low light intensity shows at low CO2-concentration an enhancement of 180 2-uptake again at/around 1000 lux indicating that photorespiration starts to function at the “Kok intensity”. Due to the fact that 180 2-uptake remains constant at high CO2-concentrations the break in the photosynthetic light intensity curve cannot be due to an inhibition of “dark respiration” at low light intensities.

Photorespiratory activity was measured in entire plants of five tobacco variants. These tobacco variants are: the green type N. tabacum var. John William’s Broadleaf (su/su Aur/aur or su/su Aur/Aur) the chlorophyll-deficient tobacco mutant Su/su (Su/su Aur/Aur) and the chlorophyll- deficient mutant Su/su var. Aurea (Su/su Aur/aur). Furthermore, two recently characterized phenotypes originating from N. tabacum var. Consolation namely “consolation green” (Aa Bb) and “consolation yellow-green” (aa bb). In entire plants of these phenotypes photorespiration was measured as 18O2-uptake in the light. This uptake was compared with the enhancement of CO2- fixation in the Warburg effect i. e. when the oxygen partial pressure is lowered from 21% Oz to 3% O2. The principal conclusion from these measurements is firstly that under the assay conditions which are identical for all 5 phenotypes (330 ppm CO2, 14000 lux white light and 25 °C) all five phenotypes yield considerable differences in photorespiratory activity. Furthermore, we were able to show that in the different phenotypes the global O2-uptake in the light is repartitioned to different degrees among different metabolic pathways. Thus, in JWB which is under the assay conditions the only fast growing species, only half of the measured 18O2-uptake belongs to glycolate metabolism or photorespiration proper, the other half belongs to a Mehler type reaction in which excess reducing power is eliminated apparently already at the level of photosynthetic electron transport. In the chlorophyll-deficient mutant Su/su, however, the observed 18O2-uptake in the light belongs under the assay conditions exclusively to glycolate metabolism (no Mehler type reaction). The chlorophyll-deficient mutant Su/su var. Aurea behaves more like JWB, that is, part of its 18O2-uptake is due to a Mehler type reaction and only the remainder is involved in CO2- metabolism, which has been already found out previously by genetic analysis. In addition photorespiration depends in Su/su more on the temperature than in the other phenotypes tested. One of the implications of our results could be that it makes a difference to the plant whether excess reducing power is disposed of at the level of the photosynthetic electron transport chain (via a Mehler type reaction) or at the level of CO2-metabolism.

When Euglena gracilis is dark adapted for 10 min or more, oxygen evolution as the consequence of short (5 μsec) saturating light flashes does not show the picture of a damped oscillation with a periodicity of 4, as known from the literature. The overall picture of this flash pattern is given by the fact that O2-evolution in the first two flashes is practically zero and rises from there onward in a continuous manner to the steady state with barely any visible oscillation at all. However, a second flash sequence fired one to two minutes after this first sequence induces an oxygen evolution pattern which is barely distinguishable from the well known usual Chlorella vulgaris pattern. The phenomenon is not influenced by changes in the oxygen tension nor do additions of chemicals like CCCP, sodium azide, or reducing agents like hydroxylamine or hydrogen peroxide substantially alter the described behavior. Deactivation experiments give the overall impression that the deactivation of the S-states is slower than with Chlorella. Hydroxylamine strongly accelerates the deactivation. The analysis of the S-state distribution in a four and five state Kok-model suggests that dark adapted Euglena is in a more reduced condition than dark adapted Chlorella. It looks as if dark adapted Euglena were in a condition which would correspond to 60 percent S-1, 30 percent S0 and 10 percent S1. The experimental flash sequence of such dark adapted cells fits best a synthetic sequence when the misses are in the region of 20-25 percent, with double hitting playing practically no role at all (the first two flashes are zero!). The impression that dark adapted Euglena starts its oxygen evolution from a more reduced state is strengthened by the analysis of room temperature fluorescence induction (Kautsky effect). It can be shown that the fluorescence induction curve of Euglena corresponds to that of Chlorella cells provided the latter have been briefly treated with a strong reductant such as sodium dithionite.

Photosynthetic O2-evolution patterns were determined in cells of Chlorella vulgaris 211 - 11 h, grown under air enriched with 2% CO2 (High CO2- cells) and under ordinary air (Low CO2-cells). Oxygen evolution in these algae was measured as consequence of short saturating light flashes with the three electrode system according to Schmid and Thibault, Z. Naturforsch. 34c, 414 (1979). It was shown that Low CO2-cells had the usual Joliot-Kok pattern of O2 evolution whereas High CO2-cells exhibited a more reduced pattern characterized by the fact that maximal flash yield was observed under the 4th flash and that damping due to misses was more important than in Low CO2-cells. Moreover, the amplitudes of the amperometric signals in High CO2-cells were consistently lower. The observations clearly speak in favor of a more reduced condition of the S-state system, when the cells are grown under high CO2. This was confirmed by the fact that higher concentrations of hydroxylamine had to be added to bring Low CO2-cells into the maximally reduced condition namely S-2, than was the case with High CO2-cells. Our observations suggest that the redox condition of the S-state system of photosystem II in Chlorella vulgaris is affected either by changes of CO2 concentrations during algal growth or that the S-state system is only maintained in the 4 state Kok condition when the enzyme carbonic anhydrase is present.

Ammonia was excreted at high rates in the presence of L-methionine sulfoximine (L-MSO) from Chlorella cells which have been grown and analyzed at normal CO2 partial pressure (330 ppm ). If these cells are analyzed at high CO2-concentration (3% CO2 in air) only little ammonia is excreted in the presence of L-MSO. In the absence of L-MSO no ammonia is excreted under either condition. In agreem ent with this observation Chlorella cells grown under high CO2 partial pressure (3% CO2 in air) but tested under normal CO2 partial pressure excreted only very little ammonia. Under these conditions neither “High CO2-cells” nor “Low CO2-cells” exhibited any glycolate excretion. However, glycolate excretion was observed in the presence of a-HPMS (a-hydroxy-2-pyridyl methanesulfonate) an inhibitor of glycolate dehydrogenase or INH (isonicotinyl hydrazide) an inhibitor of the glycine-serine am inotransferase, irrespective of the presence or absence of L-M SO. INH inhibited ammonia excretion. The above described high ammonia excretion in “Low CO2-cells” in the presence of L-MSO was suppressed or substantially reduced by 0.1 mм ethoxyzolamide an inhibitor of carbonic anhydrase which, however, at the same time caused a substantial excretion of glycolate into the medium. The same qualitative effect of ethoxyzolam ide was observed in “High CO2-cells” (tested under normal CO2 partial pressure) although the amount of glycolate excreted in this type of culture was very small. It was generally noted that glycolate excretion caused by ethoxyzolamide was stoichiometrically always more important than the rate of ammonia excretion which was inhibited. This shows that excretion and therefore most probably also the formation of glycolate are enhanced by ethoxyzolamide. The experiments seem to show that due to the inhibition of carbonic anhydrase the affinity of the ribulose-1,5-bisphosphate carboxylase/oxygenase system is increased towards oxygen, which leads to a stimulation of the photorespiratory carbon cycle.