The high light-inducible polypeptides in Synechocystis PCC6803. Expression and function in high light.

There are five Synechocystis PCC6803 genes encoding polypeptides with similarity to the Lhc polypeptides of plants. Four of the polypeptides, designated HliA-D (Dolganov, N. A. M., Bhaya, D., and Grossman, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 636-640) (corresponding to ScpC, ScpD, ScpB, and ScpE in Funk, C., and Vermaas, W. (1999) Biochemistry 38, 9397-9404) contain a single transmembrane domain. The fifth polypeptide (HemH) represents a fusion between a ferrochelatase and an Hli-like polypeptide. By using an epitope tag to identify specifically the different Hli polypeptides, the accumulation of each (excluding HemH) was examined under various environmental conditions. The levels of all of the Hli polypeptides were elevated in high light and during nitrogen limitation, whereas HliA, HliB, and HliC also accumulated to high levels following exposure to sulfur deprivation and low temperature. The temporal pattern of accumulation was significantly different among the different Hli polypeptides. HliC rapidly accumulated in high light, and its level remained high for at least 24 h. HliA and HliB also accumulated rapidly, but their levels began to decline 9-12 h following the imposition of high light. HliD was transiently expressed in high light and was not detected 24 h after the initiation of high light exposure. These results demonstrate that there is specificity to the accumulation of the Hli polypeptides under a diverse range of environmental conditions. Furthermore, mutants for the individual and combinations of the hli genes were evaluated for their fitness to grow in high light. Although all of the mutants grew as fast as wild-type cells in low light, strains inactivated for hliA or hliC/hliD were unable to compete with wild-type cells during co-cultivation in high light. A mutant lacking all four hli genes gradually lost its photosynthesis capacity and died in high light. Hence, the Hli polypeptides are critical for survival when Synechocystis PCC6803 is absorbing excess excitation energy and may allow the cells to cope more effectively with the production of reactive oxygen species.

Photoprotection in a zeaxanthin- and lutein-deficient double mutant of Arabidopsis.

When light absorption by a plant exceeds its capacity for light utilization, photosynthetic light harvesting is rapidly downregulated by photoprotective thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). To address the involvement of specific xanthophyll pigments in NPQ, we have analyzed mutants affecting xanthophyll metabolism in Arabidopsis thaliana. An npq1 lut2 double mutant was constructed, which lacks both zeaxanthin and lutein due to defects in the violaxanthin de-epoxidase and lycopene in-cyclase genes. The npq1 lut2 strain had normal Photosystem II efficiency and nearly wild-type concentrations of functional Photosystem II reaction centers, but the rapidly reversible component of NPQ was completely inhibited. Despite the defects in xanthophyll composition and NPQ, the npq1 lut2 mutant exhibited a remarkable ability to tolerate high light.

A pigment-binding protein essential for regulation of photosynthetic light harvesting.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants.

Collectively, the xanthophyll class of carotenoids perform a variety of critical roles in light harvesting antenna assembly and function. The xanthophyll composition of higher plant photosystems (lutein, violaxanthin, and neoxanthin) is remarkably conserved, suggesting important functional roles for each. We have taken a molecular genetic approach in Arabidopsis toward defining the respective roles of individual xanthophylls in vivo by using a series of mutant lines that selectively eliminate and substitute a range of xanthophylls. The mutations, lut1 and lut2 (lut = lutein deficient), disrupt lutein biosynthesis. In lut2, lutein is replaced mainly by a stoichiometric increase in violaxanthin and antheraxanthin. A third mutant, aba1, accumulates normal levels of lutein and substitutes zeaxanthin for violaxanthin and neoxanthin. The lut2aba1 double mutant completely lacks lutein, violaxanthin, and neoxanthin and instead accumulates zeaxanthin. All mutants were viable in soil and had chlorophyll a/b ratios ranging from 2.9 to 3.5 and near wild-type rates of photosynthesis. However, mutants accumulating zeaxanthin exhibited a delayed greening virescent phenotype, which was most severe and often lethal when zeaxanthin was the only xanthophyll present. Chlorophyll fluorescence quenching kinetics indicated that both zeaxanthin and lutein contribute to nonphotochemical quenching; specifically, lutein contributes, directly or indirectly, to the rapid rise of nonphotochemical quenching. The results suggest that the normal complement of xanthophylls, while not essential, is required for optimal assembly and function of the light harvesting antenna in higher plants.

Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion.

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.

The roles of specific xanthophylls in photoprotection.

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the alpha- or beta-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (alpha-carotene branch), zeaxanthin, and antheraxanthin (beta-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), alpha-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching.

The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light.

Nigericin insensitive post-illumination reduction in fluorescence yield in Dunaliella tertiolecta (chlorophyte).

Cells of the green alga Dunaliella tertiolecta grown in a light/dark cycle were exposed to high light for about 15 min. In light, energy-dependent quenching reduced fluorescence emission and decreased PS II efficiency. Within 3 minutes after darkening fluorescence quenching largely relaxed. However, PS II fluorescence emission decreased again after further darkening. Fo and Fm decreased to the same relative extent and the PS II efficiency was not reduced. This Reduction in Fluorescence yield in Darkness, termed RFD for the purpose of this paper, lasted about 20 min. The deepoxidation state of xanthophylls remained unchanged during and after the 15-min exposure to high light. We show that RFD is insensitive to the uncoupler nigericin and thus unrelated to energy-dependent quenching. RFD correlated with a reduction of the PQ pool after darkening and low levels of far red or blue light (430 nm more than 460 nm) prevented RFD. This is in contrast to observations in higher plants, where a post-illumination reduction of the PQ pool causes and increase in Fo (Groom et al. (1993) Photosynth Res 36: 205-215). Changes in the adenylate energy charge were not correlated with RFD. Antimycin A and cyanide, both inhibitors of the PQ-oxidase, caused an increase in RFD whereas SHAM, an inhibitor of the chloroplastic glycolate-quinone oxidoreductase, caused a decrease. Low CO2 concentrations, known to increase the oxygenase activity of Rubisco and to generate glycolate and P-glycolate in light, caused an increase in RFD. We propose that accumulated glycolate and P-glycolate reduce the PQ pool in darkness, leading to the formation of RFD. During RFD, 77 K fluorescence emission from PS II was more reduced than that from PS I, thus resembling a state I, state II transition. However, the reduction in fluorescence yield during RFD is much larger than the reduction previously attributed to state transitions and it is unclear whether RFD and state transitions are identical. The formation and relaxation of RFD increased with higher temperatures and the extent of RFD was largest at the growth temperature (25°C). RFD has to be taken into account when fluorescence is measured after darkening as it may be mistaken for energy-dependent quenching.

Influence of insulin on peripheral uptake of branched chain amino acids in the 60-hour fasted state.

The influence of insulin on branched chain amino acid (BCAA) metabolism was investigated in healthy subjects faster for 60-64 h, using the euglycemic insulin clamp technique and hepatic venous catheterization. As compared to the postabsorptive state, fasting resulted in a 50-80% decrease in glucose disposal during the clamps, indicating insulin resistance. However, the arterial concentrations of BCAA, which were increased by 200-220% after the fast, decreased to a similar extent during hyperinsulinemia, regardless of the fasting situation. The splanchnic exchange of BCAA was unaltered both in response to fasting itself and to fasting and hyperinsulinemia. The results suggest that insulin resistance during fasting does not influence BCAA metabolism. Furthermore, the changes in BCAA concentrations after a prolonged fast are due to altered peripheral metabolism of BCAA.

Brain uptake and release of amino acids in nondiabetic and insulin-dependent diabetic subjects: important role of glutamine release for nitrogen balance.

We measured net uptake and release of amino acids in the brain of 7 nondiabetic and six diabetic subjects. Duration of insulin-dependent diabetes (IDDM) was 19.4 +/- 2.1 years. Arteriojugular vein measurements were performed before and after 120 minutes of insulin infusion and ensuing Biostator-regulated normoglycemia. Cerebral blood flow was measured during normoglycemia by 11-CH3-F and positron emission tomography. During hyperglycemia in the IDDM subjects, arterial concentrations of valine and leucine were higher, and those of glutamic acid and arginine lower, than in nondiabetic subjects. Insulin infusion lowered levels of most amino acids in both groups. Insulin treatment did not significantly affect the uptake or release of amino acids. Significant net uptake of branched-chain amino acids was noted in both groups, as well as uptake of lysine and phenylalanine in the IDDM subjects. The sum of measured differences was not different from zero in either group. Nitrogen balance depended on impressive release of glutamine from the brain (-963 +/- 147 and -960 +/- 303 nmol/100 g/min), which amounted to 73% and 69% of net release in nondiabetic and IDDM subjects, respectively. We conclude that balance between uptake and release of amino acids is similar in nondiabetic and in long-term IDDM subjects.

Growth of cotton under continuous salinity stress: influence on allocation pattern, stomatal and non-stomatal components of photosynthesis and dissipation of excess light energy.

Cotton (Gossypium hirsutum L.) plants were grown in flowing-culture solutions containing 0%, 26% and 55% natural seawater under controlled and otherwise identical conditions. Leaf Na(+) content rose to 360 mM in 55% seawater, yet the K(+) content was maintained above 100 mM. The K(+)/Na(+) selectivity ratio was much greater in the saline plants than in the control plants. All plants were healthy and able to complete the life cycle but relative growth rate fell by 46% in 26% seawater and by 83% in 55% seawater. Much of this reduction in growth was caused by a decreased allocation of carbon to leaf growth versus root growth. The ratio of leaf area/plant dry weight fell by 32% in 26% seawater and by 50% in 55 % seawater while the rate of carbon gain per unit leaf area fell by only 20% in 26% seawater and by as much as 66% in 55% seawater. Partial stomatal closure accounted for nearly all of the fall in the photosynthesis rate in 26% seawater but in 55% seawater much of the fall also can be attributed to non-stomatal factors. As a result of the greater effect of salinity on stomatal conductance than on CO2-uptake rate, photosynthetic water-use efficiency was markedly improved by salinity. This was also confirmed by stablecarbon-isotope analyses of leaf sugar and of leaf cellulose and starch. - Although non-stomatal photosynthetic capacity at the growth light was reduced by as much as 42% in 55% seawater, no effects were detected on the intrinsic photon yield of photosynthesis nor on the efficiency of photosystem II photochemistry, chlorophyll a/b ratio, carotenoid composition or the operation of the xanthophyll cycle. Whereas salinity caused in increase in mesophyll thickness and content of chloroplast pigments it caused a decrease in total leaf nitrogen content. The results indicate that the salinity-induced reduction in non-stomatal photosynthetic capacity was not caused by any detrimental effect on the photosynthetic apparatus but reflects a decreased allocation to enzymes of carbon fixation. - Rates of energy dissipation via CO2 fixation and photorespiration, calculated from gas-exchange measurements, were insufficient to balance the rate of light-energy absorption at the growth light. Salinity therefore would have been expected to cause the excess excitation energy to rise, leading to an increased nonradiative dissipation in the pigment bed and resulting increases in non-photochemical fluorescence quenching and zeaxanthin formation. However, no such changes could be detected, implying that salinity may have increased energy dissipation via a yet unidentified energy-consuming process. This lack of a response to salinity stress is in contrast to the responses elicited by short-term water stress which caused strong non-photochemical quenching and massive zeaxanthin formation.

Chloroplast movements in leaves: Influence on chlorophyll fluorescence and measurements of light-induced absorbance changes related to ΔpH and zeaxanthin formation.

Light-induced chloroplast movements were found to cause changes in chlorophyll fluorescence emission, closely matching those in leaf absorptance, both in terms of the kinetics and the maximum extent of the changes observed in different species. The results demonstrate that chloroplast movements can have a significant effect on the efficiency of light utilization in photosynthesis. They further show that chloroplast movements need to be taken into account in measurements of fluorescence quenching and especially in measurements of light-induced optical changes used to monitor zeaxanthin formation and ΔpH associated light scattering in leaves. Means of minimizing and of adjusting for the influences of chloroplast movements in such measurements are discussed.

Carotenoid distribution and deepoxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize.

In response to excess light, the xanthophyll violaxanthin (V) is deepoxidized to zeaxanthin (Z) via antheraxanthin (A) and the degree of this deepoxidation is strongly correlated with dissipation of excess energy and photoprotection in PS II. However, little is known about the site of V deepoxidation and the localization of Z within the thylakoid membranes. To gain insight into this problem, thylakoids were isolated from cotton leaves and bundle-sheath strands of maize, the pigment protein-complexes separated on Deriphat gels, electroeluted, and the pigments analyzed by HPLC. In cotton thylakoids, 30% of the xanthophyll cycle pigments were associated with the PS I holocomplex, including the PS I light-harvesting complexes and PS I core complex proteins (CC I), and about 50% with the PS II light-harvesting complexes (LHC II). The Chl was evenly distributed between PS I and PS II. Less than 2% of the neoxanthin, about 18% of the lutein, and as much as 76% of the ß-carotene of the thylakoids were associated with PS I. Exposure of pre-darkened cotton leaves to a high photon flux density for 20 min prior to thylakoid isolation caused about one-half of the V to be converted to Z. The distribution of Z among the pigment-protein complexes was found to be similar to that of V. The distribution of the other carotenoids was unaffected by the light treatment. Similarly, in field-grown maize leaves and in the bundle-sheath strands isolated from them, about 40% of the V present at dawn had been converted to Z at solar noon. Light treatment of isolated bundle-sheath strands which initially contained little Z caused a similar degree of conversion of V to Z. As in cotton thylakoids, about 30% the V+A+Z pool in bundle-sheath thylakoids from maize was associated with the PS I holocomplex and the CC I bands and 46% with the LHC II bands, regardless of the extent of deepoxidation. These results demonstrate that Z is present in PS I as well as in PS II and that deepoxidation evidently takes place within the pigment-protein complexes of both photosystems.

Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L.

The temperature dependence of the rate of de-epoxidation of violaxanthin to zeaxanthin was determined in leaves of chilling-sensitive Gossypium hirsutum L. (cotton) and chilling-resistant Malva parviflora L. by measurements of the increase in absorbance at 505 nm (ΔA 505) and in the contents of antheraxanthin and zeaxanthin that occur upon exposure of predarkened leaves to excessive light. A linear relationship between ΔA 505 and the decrease in the epoxidation state of the xanthophyll-cycle pigment pool was obtained over the range 10-40° C. The maximal rate of de-epoxidation was strongly temperature dependent; Q10 measured around the temperature at which the leaf had developed was 2.1-2.3 in both species. In field-grown Malva the rate of de-epoxidation at any given measurement temperature was two to three times higher in leaves developed at a relatively low temperature in the early spring than in those developed in summer. Q10 measured around 15° C was in the range 2.2-2.6 in both kinds of Malva leaves, whereas it was as high as 4.6 in cotton leaves developed at a daytime temperature of 30° C. Whereas the maximum (initial) rate of de-epoxidation showed a strong decrease with decreased temperature the degree of de-epoxidation reached in cotton leaves after a 1-2 · h exposure to a constant photon flux density increased with decreased temperature as the rate of photosynthesis decrease. The zeaxanthin content rose from 2 mmol · (mol chlorophyll)(-1) at 30° C to 61 mmol · (mol Chl)(-1) at 10° C, corresponding to a de-epoxidation of 70% of the violaxanthin pool at 10° C. The degree of de-epoxidation at each temperature was clearly related to the amount of excessive light present at that temperature. The relationship between non-photochemical quenching of chlorophyll fluorescence and zeaxanthin formation at different temperatures was determined for both untreated control leaves and for leaves in which zeaxanthin formation was prevented by dithiothreitol treatment. The rate of development of that portion of non-photochemical quenching which was inhibited by dithiothreitol decreased with decreasing temperature and was linearly related to the rate of zeaxanthin formation over a wide temperature range. In contrast, the rate of development of the dithiothreitol-resistant portion of non-photochemical quenching was remarkably little affected by temperature. Evidently, the kinetics of the development of non-photochemical quenching upon exposure of leaves to excessive light is therefore in large part determined by the rate of zeaxanthin formation. For reasons that remain to be determined the relaxation of dithiothreitolsensitive quenching that is normally observed upon darkening of illuminated leaves was strongly inhibited at low temperatures.

Splanchnic and muscle fructose metabolism during and after exercise.

Regional substrate exchange was studied in 12 healthy males during 90 min of bicycle exercise at 30% of maximal O2 consumption with a 20-min recovery. Six subjects received an intravenous fructose infusion (8.5 mmol/min) from 40 min of exercise to the end of recovery. Splanchnic glucose output, muscle glucose uptake, arterial glucose, and insulin were uninfluenced by the infusion. The respiratory exchange ratio rose to 0.93 +/- 0.04, and arterial free fatty acids fell by 50% (P less than 0.05). Fructose was taken up by splanchnic tissues (45% of administered load), leg muscle (28%), and resting muscle (28%). During infusion, arterial lactate and pyruvate rose two- to threefold, and these substrates were released from splanchnic tissues and taken up by exercising and resting muscle. Splanchnic release of lactate, pyruvate, and glucose accounted for 78% of fructose uptake at 90 min of exercise. Uptake of fructose, lactate, and pyruvate accounted for 55% and together with glucose for 103% of the total oxidative metabolism by exercising muscle. The regional fructose uptakes and lactate exchanges persisted throughout recovery. The present results indicate that fructose infusion during leg exercise 1) results in increased carbohydrate oxidation from fructose, lactate, and pyruvate in exercising muscle, 2) exerts a glycogenic effect in resting muscle and liver during exercise and in liver and muscle recovering from exercise, and 3) does not interfere with glucose metabolism, and that fructose transport into muscle differs from that of glucose.

Gut exchange of glucose and lactate in basal state and after oral glucose ingestion in postoperative patients.

Glucose uptake by the intestine and its conversion into 3-carbon compounds in the human intestine in the basal state and after an oral glucose load are not understood. Consequently, we studied the arterial and portal venous concentration differences (A-PV) for glucose and glucogenic substrates in the basal state and 3 h after the ingestion of a 100-g glucose load with the catheter technique. Five patients were studied 3-11 days after surgery for gallbladder disease or cancer of the colon or liver. A-PV for glucose in the basal state was 0.12 +/- 0.02 mM (P less than 0.01), indicating net glucose uptake by extrahepatic splanchnic tissues. No net exchange of lactate or pyruvate was detected, but there was release of alanine and uptake of glutamine. After glucose ingestion, glucose was released by the gut, reflecting absorption of the load (mean A-PV for glucose -2.10 +/- 0.04 mM, P less than 0.01). The arterial glucose concentration rose gradually from 4.6 +/- 0.1 mM before glucose ingestion to a plateau at 9.5 +/- 0.7 mM from 90 to 180 min. Glucose ingestion was accompanied by net lactate and alanine release (A-PV -0.16 +/- 0.06 mM and -48 +/- 7 microM, respectively), whereas A-PV for pyruvate did not change. We conclude that, in postoperative patients, there is a significant net glucose uptake by the gastrointestinal tract in the basal state. Glucose ingestion is accompanied by a small release of lactate and alanine from the intestine. However, the estimated net gut formation of lactate and alanine can play only a minor role in the disposal of an oral glucose load.

Cerebral blood flow and substrate utilization in insulin-treated diabetic subjects.

We compared regional cerebral blood flow (rCBF) and arteriojugular vein differences of glucose, ketones, glycerol, lactate, pyruvate, and O2 in eight subjects with well-controlled insulin-dependent diabetes mellitus (IDDM) and in six healthy volunteers. Duration of diabetes was 19.4 +/- 2.1 yr. Measurements were performed before and after 120 min of insulin infusion and concomitant Biostator-controlled normoglycemia. Net uptake of ketones was seen in IDDM subjects before but not after insulin. Net uptake of glucose did not differ significantly between groups. During normoglycemia the molar ratio of O2 to glucose uptake was lower in IDDM than in nondiabetic subjects (4.68 vs. 5.50; P less than 0.05; Wilcoxon test). Small but significant release of lactate and pyruvate was seen in IDDM but not in nondiabetic subjects. The rCBF was measured by 11CH3F and position emission tomography. Global mean CBF was higher in IDDM subjects (64.9 +/- 5.9 vs. 49.3 +/- 2.7 ml.100 g-1.min-1, means +/- SE in nondiabetic subjects, P less than 0.05). rCBF was enhanced in many cortical and subcortical areas, whereas it was decreased in the head of the caudate nucleus. Neuropsychological testing did not reveal obvious cognitive dysfunction. The results imply that a larger fraction of glucose is nonoxidatively metabolized in the IDDM subjects and furthermore indicate an abnormal rCBF pattern in these subjects.

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts.

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F(o)) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the ;high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F(o) quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.

Leaf Xanthophyll content and composition in sun and shade determined by HPLC.

As a part of our investigations to test the hypothesis that zeaxanthin formed by reversible de-epoxidation of violaxanthin serves to dissipate any excessive and potentially harmful excitation energy we determined the influence of light climate on the size of the xanthophyll cycle pool (violaxanthin + antheraxanthin + zeaxanthin) in leaves of a number of species of higher plants. The maximum amount of zeaxanthin that can be formed by de-epoxidation of violaxanthin and antheraxanthin is determined by the pool size of the xanthophyll cycle. To quantitate the individual leaf carotenoids a rapid, sensitive and accurate HPLC method was developed using a non-endcapped Zorbax ODS column, giving baseline separation of lutein and zeaxanthin as well as of other carotenoids and Chl a and b.The size of the xanthophyll cycle pool, both on a basis of light-intercepting leaf area and of light-harvesting chlorophyll, was ca. four times greater in sun-grown leaves of a group of ten sun tolerant species than in shade-grown leaves in a group of nine shade tolerant species. In contrast there were no marked or consistent differences between the two groups in the content of the other major leaf xanthophylls, lutein and neoxanthin. Also, in each of four species examined the xanthophyll pool size increased with an increase in the amount of light available during leaf development whereas there was little change in the content of the other xanthophylls. However, the α-carotene/ß-carotene ratio decreased and little or no α-carotene was detected in sun-grown leaves. Among shade-grown leaves the α-carotene/ß-carotene ratio was considerably higher in species deemed to be umbrophilic than in species deemed to be heliophilic.The percentage of the xanthophyll cycle pool present as violaxanthin (di-epoxy-zeaxanthin) at solar noon was 96-100% for shade-grown plants and 4-53% for sun-grown plants with zeaxanthin accounting for most of the balance. The percentage of zeaxanthin in leaves exposed to midday solar radiation was higher in those with low than in those with high photosynthetic capacity.The results are consistent with the hypothesis that the xanthophyll cycle is involved in the regulation of energy dissipation in the pigment bed, thereby preventing a buildup of excessive excitation energy at the reaction centers.

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 (ΔA510) 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 Fo 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 Fo and 75% of the quenching of Fm, 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 µmol 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 Fv/Fm ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT Fo 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.