Photoinhibition and zeaxanthin formation in intact leaves : a possible role of the xanthophyll cycle in the dissipation of excess light energy

Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O₂, 0% CO₂) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F_v, or maximum fluorescence, F_m, and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of β-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O₂ and 0% CO₂ resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F_v/F_m, determined with the modulated fluorescence technique at room temperature, and the photon yield of O₂ evolution, similar to previous findings (O Björkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

Impact of iron supply on the kinetics of recovery of photosynthesis in Cd-stressed poplar (Populus glauca)

Background and Aims

Cadmium (Cd) causes Fe-deficiency-like symptoms in plants, and strongly inhibits photosynthesis. To clarify the importance of Cd-induced Fe deficiency in Cd effects on photosynthesis, the recovery processes were studied by supplying excess Fe after the Cd symptoms had developed.

Methods

Fe-citrate at 10 µm or 50 µm was given with or without 10 µm Cd(NO₃)₂ to hydroponically cultured poplars (Populus glauca ‘Kopeczkii’) with characteristic Cd symptoms. Ion, chlorophyll and pigment contents, amount of photosynthetic pigment–protein complexes, chlorophyll fluorescence and carbon assimilation were measured together with the mapping of healing processes by fluorescence imaging.

Key Results

In regenerated leaves, the iron content increased significantly, while the Cd content did not decrease. As a result, the structural (increase in the amount of photosynthetic pigments and pigment–protein complexes, decrease in the F₆₉₀/F₇₄₀ ratio) and functional (elevation of CO₂ fixation activity and ΔF/F_m′) recovery of the photosynthetic machinery was detected. Cd-induced, light-stress-related changes in non-photochemical quenching, activity of the xanthophyll cycle, and the F₄₄₀?/F₅₂₀ ratio were also normalized. Imaging the changes in chlorophyll fluorescence, the recovery started from the parts adjacent to the veins and gradually extended to the interveinal parts. Kinetically, the rate of recovery depended greatly on the extent of the Fe supply, and chlorophyll a/b ratio and ΔF/F_m′ proved to be the most-rapidly reacting parameters.

Conclusions

Iron deficiency is a key factor in Cd-induced inhibition of photosynthesis.Key words: Cadmium, chlorophyll–protein, iron deficiency, poplar, Populus glauca Haines 1906 var. Kopeczkii, fluorescence imaging, chlorophyll fluorescence induction     

Promotion of crown-gall tumor growth by lysopine, octopine, nopaline, and carnosine

The growth of crown-gall tumors on primary bean leaves (Phaseolus vulgaris L. cv. “Pinto”) was promoted by the addition of d-lysopine, d-octopine, l-carnosine, or nopaline. Assayed on tumors induced by Agrobacterium tumefaciens strain B6, the relative activity was octopine = carnosine > lysopine nopaline; assayed on tumors induced by A. tumefaciens strain T-37, which induces tumors which form nopaline, the relative activity was nopaline = octopine = carnosine > lysopine. From one to three applications of carnosine or octopine gave equal additive increments in tumor growth, showing that a continual supply of these substances is required to maintain an increased rate of growth. At concentrations above 0.1 mm, pairs of these growth-promoting substances were less active than when applied singly. Inhibition of octopine-induced growth was obtained by applying 0.01 mm carnosine with 1 mm octopine and partial inhibition was obtained when carnosine was added 10 hr after octopine. Equimolar mixtures of lysopine, octopine, and carnosine, however, were at least as active in promoting tumor growth as any of the compounds added singly at equivalent concentrations. The activity of 0.1 to 0.5 mm lysopine, octopine, and carnosine was inhibited, respectively, by 1 mml-lysine, l-arginine, and l-histidine and this inhibition was limited in each case to the basic amino acid corresponding to that of the growth factor. Arginine fully inhibited octopine-induced tumor growth when applied as much as 6 hr after octopine, indicating that this inhibition was not due to prevention of octopine uptake. Although four separate substances were found which promoted tumor growth, the molecular specificity required for activity of each compound was high. Evidence is presented which suggests that a tumor growth-promoting substance extracted from tumorous leaves is a carnosine-like derivative of l-histidine.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydrolyase (deaminating) in a species of Corynebacterium

1. Three bacterial isolates capable of growth on l-threonine medium only when supplemented with branched-chain amino acids, and possessing high l-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that l-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of l-threonine 3-dehydrogenase or l-threonine aldolase in threonine catabolism. 3. l-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [S_0.5], was 40mm and the Hill coefficient (h) was 2.0. l-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60μm, but did not affect the Hill constant. At the fixed l-threonine concentration of 10mm, the effect of l-valine was biphasic, progressive activation occurring at concentrations up to 2mm-l-valine, but was abolished by higher concentrations. Substrate-saturation plots for the l-valine-activated enzyme exhibited normal Michaelis–Menten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the `biosynthetic' isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of l-threonine dehydratase was constitutive and was not subject to multivalent repression by l-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of l-threonine, apparently initiated by a `biosynthetic' l-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Light and calcium interactions in chlorella inhibited by sodium chloride

Analysis of NaCl toxicity in Chlorella sorokiniana showed decreased growth rates, increased dry weight per cell, increased intracellular Na⁺ and Cl⁻, more total chlorophyll per cell, a decreased chlorophyll a to chlorophyll b ratio, increased rates of O₂ evolution, and decreased rates of CO₂ fixation when the extracellular concentration of NaCl was increased from zero to 0.3 m. Cultures did not grow at concentrations greater than 0.3 m NaCl unless 10 mm calcium salts were present. Inclusion of that concentration of Ca²⁺ extended the tolerance to 0.5 m NaCl before growth stopped. Increasing the light intensity from 1.2 to 9.4 mw/cm² increased growth rates for cultures in 0.10 to 0.45 m NaCl. At 14 mw/cm² added Ca²⁺ reduced growth rates of cultures in 0.3 m NaCl compared to controls without added Ca²⁺. Maximal growth rates for cultures in NaCl media were achieved by addition of 10 mm CaSO₄ and maintenance of the light intensity at 9.4 mw/cm². The maximal growth rate of the organism was 9.6 doublings/day achieved at 2.7 mw/cm² for control cultures. In 0.3 m NaCl the growth rate was 4.3 doublings/day at 2.7 mw/cm² and 8.2 doublings/day at 9.4 mw/cm² with 10 mm CaSO₄ added.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

Evidence for active Phloem loading in the minor veins of sugar beet

Phloem loading in source leaves of sugar beet (Beta vulgaris, L.) was studied to determine the extent of dependence on energy metabolism and the involvement of a carrier system. Dinitrophenol at a concentration of 4 mm uncoupled respiration, lowered source leaf ATP to approximately 40% of the level in the control leaf and inhibited translocation of exogenously supplied ¹⁴C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mm inhibited rather than promoted CO₂ production, indicating a mechanism of inhibition other than uncoupling of respiration. The 8 mm dinitrophenol also reduced ATP to approximately 40% of the level in the control source leaf and reduced translocation of exogenous sucrose to approximately 10% of the control. Application of 4 mm ATP to an untreated source leaf promoted the translocation rate by approximately 80% over the control, while in leaves treated with 4 mm dinitrophenol, 4 mm ATP restored translocation to the control level. No recovery of translocation was observed when ATP was applied to leaves treated with 8 mm dinitrophenol. The results indicate an energy-requiring process for both phloem loading and translocation in the source leaf.     

Isolation of active mitochondria from tomato fruit

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl₂, 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 × g for 15 minutes. The supernatant is then centrifuged at 11,000 × g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl₂, 10 mm tris-HCl, 10 mm KH₂PO₄ and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and `Honey Dew' melon.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Changes in susceptibility to photoinhibition and recovery during the growth season

Kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) plants grown in an outdoor enclosure were exposed to the natural conditions of temperature and photon flux density (PFD) over the growing season (October to May). Temperatures ranged from 14 to 21° C while the mean monthly maximum PFD varied from 1000 to 1700 mol · m^–2 · s^–1, although the peak PFDs exceeded 2100 mol · m^–2 · s^–1. At intervals, the daily variation in chlorophyll fluorescence at 692 nm and 77K and the photon yield of O₂ evolution in attached leaves was monitored. Similarly, the susceptibility of intact leaves to a standard photoinhibitory treatment of 20° C and a PFD of 2000 mol · m^–2 · s^–1 and the ability to recover at 25° C and 20 mol · m^–2 · s^–2 was followed through the season. On a few occasions, plants were transferred either to or from a shade enclosure to assess the suceptibility to natural photoinhibition and the capacity for recovery. There were minor though significant changes in early-morning fluorescence emission and photon yield throughout the growing season. The initial fluorescence, F_o, and the maximum fluorescence, F_m, were, however, significantly and persistently different from that in shade-grown kiwifruit leaves, indicative of chronic photoinhibition occurring in the sun leaves. In spring and autumn, kiwifruit leaves were photoinhibited through the day whereas in summer, when the PFDs were highest, no photoinhibition occurred. However, there was apparently no non-radiative energy dissipation occurring then also, indicating that the kiwifruit leaves appeared to fully utilize the available excitation energy. Nevertheless, the propensity for kiwifruit leaves to be susceptible to photoinhibition remained high throughout the season. The cause of a discrepancy between the severe photoinhibition under controlled conditions and the lack of photoinhibition under comparable, natural conditions remains uncertain. Recovery from photoinhibition, by contrast, varied over the season and was maximal in summer and declined markedly in autumn. Transfer of shade-grown plants to full sun had a catastrophic effect on the fluorescence characteristics of the leaf and photon yield. Within 3 d the variable fluorescence, F_v, and the photon yield were reduced by 80 and 40%, respectively, and this effect persisted for at least 20 d. The restoration of fluorescence characteristics on transfer of sun leaves to shade, however, was very slow and not complete within 15 d.Abbreviations and Symbols F_o, F_m, F_v initial, maximum, variable fluorescence - F_i F_v at t = 0 - F F_v at t = - PFD photon flux density - PSII photosystem II - leaf absorptance ratio - (_a photon yield of O₂ evolution (absorbed basis) - _{i a} at t = 0 - _a at t = We thank Miss Linda Muir and Amanda Yeates for their technical assistance in this study.     

Photosynthetic capacity of the capsule wall and its contribution to carbon fixation and seed yield in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

Defoliation occurs in castor due to several reasons, but the crop has propensity to compensate for the seed yield. Photosynthetic efficiency in terms of functional (gas exchange and chlorophyll fluorescence) and structural characteristics (photosynthetic pigment profiles and anatomical properties) of castor capsule walls under light- and dark-adapted conditions was compared with that of leaves. Capsule wall showed high intrinsic efficiency of photosystem II (F _v/F _m, 0.82) which was comparable to leaves (F _v/F _m, 0.80). With increasing photon flux densities (PFD), actual quantum yields and photochemical quenching coefficients of the capsule walls were similar to that in leaves, while electron transport rates reached a maximum corresponding to about 118 % of the leaves. However, maximum net photosynthetic rate of the capsule walls (2.60 µmol CO₂ m^?2 s^?1) was less than one-fourth of the leaves (15.67 µmol CO₂ m^?2 s^?1) at the CO₂ concentration of 400 µmol mol^?1, and the difference was attributed to about 80 % lower stomatal density and the 75 % lower total chlorophyll content of capsule walls than the leaves. Furthermore, seed weight in dark-adapted capsules was 2.70–12.42 % less as compared to the capsules developed under light. The results indicate that castor capsule walls are photosynthetically active (about 15–30 % of the leaves) and contribute significantly to carbon fixation and seed yield accounting for 10 % photoassimilates towards seed weight.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of temperature

Photoinhibition of photosynthesis was induced in attached leaves of kiwifruit grown in natural light not exceeding a photon flux density (PFD) of 300 mol·m^-2·s^-1, by exposing them to a PFD of 1500 mol·m^-2·s^-1. The temperature was held constant, between 5 and 35° C, during the exposure to high light. The kinetics of photoinhibition were measured by chlorophyll fluorescence at 77K and the photon yield of photosynthetic O₂ evolution. Photoinhibition occurred at all temperatures but was greatest at low temperatures. Photoinhibition followed pseudo first-order kinetics, as determined by the variable fluorescence (F _v) and photon yield, with the long-term steady-state of photoinhibition strongly dependent on temperature wheareas the observed rate constant was only weakly temperature-dependent. Temperature had little effect on the decrease in the maximum fluorescence (F _m) but the increase in the instantaneous fluorescence (F _o) was significantly affected by low temperatures in particular. These changes in fluorescence indicate that kiwifruit leaves have some capacity to dissipate excessive excitation energy by increasing the rate constant for non-radiative (thermal) energy dissipation although temperature apparently had little effect on this. Direct photoinhibitory damage to the photosystem II reaction centres was evident by the increases in F _o and extreme, irreversible damage occurred at the lower temperatures. This indicates that kiwifruit leaves were most susceptible to photoinhibition at low temperatures because direct damage to the reaction centres was greatest at these temperatures. The results also imply that mechanisms to dissipate excess energy were inadequate to afford any protection from photoinhibition over a wide temperature range in these shade-grown leaves.Abbreviations and symbols fluorescence yield correction coefficient - F _o, F _m, F _v instantaneous, maximum, variable fluorescence - K _D, K _F, K _P, K _T rate constants for non-radiative energy dissipation, fluorescence, photochemistry, energy transfer to photosystem I - PFD photon flux density - PSI, II photosystem I, II - _i photon yield of photosynthesis (incident light)     

Effect of daily photon receipt on the susceptibility of dwarf bean (Phaseolus vulgaris L.) leaves to photoinhibition of photosynthesis

Bean (Phaseolus vulgaris L.) plants were grown at two light periods of 8 and 13 h with a similar photon flux density (PFD) giving a daily photon receipt (DPR) of 17.9 and 38.2 mol · m–2, respectively. Shoot growth and leaf area development were followed at regular intervals and diurnal whole-plant photosynthesis measured. Single mature trifoliate leaves were exposed to photoinhibitory treatments at PFDs of 800 and 1400 mol · m–2 · s–1 and at temperatures of 12 and 20°C. Chlorophyll fluorescence and photon yields were measured at regular intervals throughout each treatment. Plants grown in 13 h had significantly greater leaf areas than those grown in 8 h. There were no differences in maximum rates of photosynthesis, photon yields and only minor but significant differences in F_v/F_m for plants in the two treatments, showing photosynthetic characteristics were dependent on PFD but not DPR. A significant decline in photosynthesis and F_v/F_m occurred over the 13-h but little change in photosynthesis for plants in the 8 h, indicating some feedback inhibition of photosynthesis was occurring. Plants grown in 8 h were consistently more susceptible to photoinhibition of photosynthesis at all treatments than 13-h plants. Nevertheless, photoinhibition was exacerbated by increases in PFD, and by decreases in temperature for leaves from both treatments. However, for plants from the 8-h day, exposing leaves to 12°C and 1400 mol · m–2 · s–1 caused photo-oxidation and severe bleaching but no visible damage on leaves from 13-h-grown plants. Closure of the photosystem II reaction-centre pool was partially correlated with increasing extents of photoinhibition but the relationship was similar for plants from both treatments. There remains no clear explanation for their wide differences in susceptibility to photoinhibition.Abbreviations and Symbols DPR daily photon receipt - F₀ and F_m initial and maximal fluorescence - F_v/F_m fluorescence ratio in dark-treated leaves - F/F_m intrinsic efficiency of PSII during illumination - PFD photon flux density - _i photon yield (incident basis) - _psi quantum yield of PSII electron transport - P_max maximum rate of photosynthesis - q_N non-photochemical quenching coefficient - q_P photochemical quenching coefficient Many thanks to my colleague William Laing who spent a considerable effort in developing the programme to run the photosynthesis apparatus. I am also indebted to one reviewer with whom I corresponded to resolve some issues in the paper. This project was funded by the New Zealand Foundation for Research, Science and Technology.     

Light and the maintenance of photosynthetic competence in leaves of Populus balsamifera L. during short-term exposures to high concentrations of sulfur dioxide

Leaves of Populus balsamifera grown under full natural sunlight were treated with 0, 1, or 2 l SO₂·1^-1 air under one of four different photon flux densities (PFD). When the SO₂ exposures took place in darkness or at 300 mol photons·m^-2·s^-1, sulfate accumulated to the levels predicted by measurements of stomatal conductance during SO₂ exposure. Under conditions of higher PFD (750 and 1550 mol·m^-2·s^-1), however, the predicted levels of accumulated sulfate were substantially higher than those obtained from anion chromatography of the leaf extracts. Light-and CO₂-saturated capacity as well as the photon yield of photosynthetic O₂ evolution were reduced with increasing concentration of SO₂. At 2 l SO₂·1^-1 air, the greatest reductions in both photosynthetic, capacity and photon yield occurred when the leaves were exposed to SO₂ in the dark, and increasingly smaller reductions in each occurred with increasing PFD during SO₂ exposure. This indicates that the inhibition of photosynthesis resulting from SO₂ exposure was reduced when the exposure occurred under conditions of higher light. The ratio F _v/F _M (variable/maximum fluorescence emission) for photosyntem II (PSII), a measure of the photochemical efficiency of PSII, remained unaffected by exposure of leaves to SO₂ in the dark and exhibited only moderate reductions with increasing PFD during the exposure, indicating that PSII was not a primary site of damage by SO₂. Pretreatment of leaves with SO₂ in the dark, however, increased the susceptibility of PSII to photoinhibition, as such pretreated leaves exhibited much greater reductions inF _V/F _M when transferred to moderate or high light in air than comparable control leaves.Abbreviations and symbols A₁₂₀₀ photosynthetic capacity (CO₂-saturated rate of O₂ evolution at 1200 mol photons·m^-2·s^-1) - F_o instantaneous fluorescence emission - F_M maximum fluorescence emission - F_V variable fluorescence emission - PFD photon flux density (400–700 nm) - PSII photosystem II     

Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The K_m for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the K_m for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the K_i being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, K_m values for the amino alcohol and NAD⁺ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0·1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed.     

Immobilized thylakoids in a cross-linked albumin matrix: effects of cations studied by electron microscopy, fluorescence emission, photoacoustic spectroscopy, and kinetic measurements

Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O₂ evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O₂ production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na⁺ (or K⁺), 1 mm Mg²⁺, and 0.1 mm Mn²⁺ are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F₇₃₅ to F₆₉₅ ratio increases and the PA₆₇₆ to PA₄₄₀ ratio decreases.     

Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes,fluorescence and photosynthesis in leaves of Hedera canariensis

The role of the xanthophyll cycle in regulating the energy flow to the PS II reaction centers and therefore in photoprotection was studied by measurements of light-induced absorbance changes, Chl fluorescence, and photosynthetic O₂ evolution in sun and shade leaves of Hedera canariensis. The light-induced absorbance change at 510 nm (A₅₁₀) was used for continuous monitoring of zeaxanthin formation by de-epoxidation of violaxanthin. Non-radiative energy dissipation (NRD) was estimated from non-photochemical fluorescence quenching (NPQ).High capacity for zeaxanthin formation in sun leaves was accompanied by large NRD in the pigment bed at high PFDs as indicated by a very strong NPQ both when all PS II centers are closed (F'_m) and when all centers are open (F'_o). Such F_o quenching, although present, was less pronounced in shade leaves which have a much smaller xanthophyll cycle pool.Dithiothreitol (DTT) provided through the cut petiole completely blocked zeaxanthin formation. DTT had no detectable effect on photosynthetic O₂ evolution or the photochemical yield of PS II in the short term but fully inhibited the quenching of F_o and 75% of the quenching of F_m, indicating that NRD in the antenna was largely blocked. This inhibition of quenching was accompanied by an increased closure of the PS II reaction centers.In the presence of DTT a photoinhibitory treatment at a PFD of 200 mol m^-2 s^-1, followed by a 45 min recovery period at a low PFD, caused a 35% decrease in the photon yield of O₂ evolution, compared to a decrease of less than 5% in the absence of DTT. The F_v/F_m ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT F_o rose by 15–20% whereas no change was detected in control leaves.The results support the conclusion that the xanthophyll cycle has a central role in regulating the energy flow to the PS II reaction centers and also provide direct evidence that zeaxanthin protects against photoinhibitory injury to the photosynthetic system.Abbreviations F, F_m, F_o, F_v Fluorescence yield at actual degree of PS II center closure, when all centers are closed, when all centers are open, variable fluorescence - NPQ non-photochemical fluorescence quenching - NRD non-radiative energy dissipation - PFD photon flux density - Q_A primary acceptor PS II     

Underestimated chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence measurements on <Emphasis Type="Italic">Buxus microphylla</Emphasis> red winter leaves

Leaves under stressful conditions usually show downregulated maximum quantum efficiency of photosystem II [inferred from variable to maximum chlorophyll (Chl) a fluorescence (F_v/F_m), usually lower than 0.8], indicating photoinhibition. The usual method to evaluate the degree of photoinhibition in winter red leaves is generally by measuring the F_v/F_m on the red adaxial surface. Two phenotypes of overwintering Buxus microphylla ‘Wintergreen’ red leaves, with different measuring site and leaf thickness, were investigated in order to elucidate how red pigments in the outer leaf layer affected the Chl a fluorescence (F_v/F_m) and photochemical reflectance index. Our results showed that the F_v/F_m measured on leaves with the same red surface, but different leaf thickness, exhibited a slightly lower value in half leaf (separated upper and lower layers of leaves by removing the leaf edge similarly as affected by winter freezing and thawing) than that in the intact leaf (without removing the leaf edge), and the F_v/F_m measured on the red surface was significantly lower than that on the inner or backlighted green surface of the same thickness. Our results suggest that the usual measurement of F_v/F_m on red adaxial surface overestimates the actual degree of photoinhibition compared with that of the whole leaf in the winter.