Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).     

From flour to flower: how Polycomb group proteins influence multiple aspects of plant development

Cell identity and differentiation are determined by patterns of regulatory gene expression. Spatially and temporally regulated homeotic gene expression defines segment identities along the anterior-posterior axis of animal embryos. Polycomb group (PcG) proteins form a cellular memory system that maintains the repressed state of homeotic gene expression. Conserved PcG proteins control multiple aspects of Arabidopsis development and maintain homeotic gene repression. In animals, PcG proteins repress their target genes by modifying histone tails through deacetylation and methylation, generating a PcG-specific histone code that recruits other chromatin remodeling proteins to establish a stable, heritable mechanism of epigenetic expression control. Plant PcG proteins might function through a similar biochemical mechanism owing to their conserved structural and functional relationship to animal PcG proteins.     

Exhaustion of the Frank-Starling mechanism in conscious dogs with heart failure induced by chronic coronary microembolization

The role of the Frank-Starling mechanism in the regulation of cardiac systolic function in the ischemic failing heart was examined in conscious dogs. Left ventricular (LV) dimension, pressure and systolic function were assessed using surgically implanted instrumentations and non-invasive echocardiogram. Heart failure was induced by daily intra-coronary injections of microspheres for 3-4 weeks via implanted coronary catheters. Chronic coronary embolization resulted in a progressive dilation of the left ventricle (12+/-3%), increase in LV end-diastolic pressure (118+/-19%), depression of LV dP/dt(max) (-19+/-4%), fractional shortening (-36+/-7%), and cardiac work (-60+/-9%), and development of heart failure, while the LV contractile response to dobutamine was depressed. A brief inferior vena caval occlusion in dogs with heart failure decreased LV preload to match the levels attained in their control state and caused a further reduction of LV dP/dt(max), fractional shortening, stroke work and cardiac work. Moreover, in response to acute volume loading, the change in the LV end-diastolic dimension-pressure (DeltaLVEDD-DeltaLVEDP) curve in the failing heart became steeper and shifted significantly to the left, while the increases in LV stroke work and cardiac work were blunted. Thus, our results suggest that the Frank-Starling mechanism is exhausted in heart failure and unable to further respond to increasing volume while it plays an important compensatory role in adaptation to LV dysfunction in heart failure.     

Thylakoid polypeptide composition and light-independent phosphorylation of the chlorophyll a,b-protein in Prochloron, a prokaryote exhibiting oxygenic photosynthesis

Thylakoids of the prokaryote Prochloron, present as a symbiont in ascidians isolated from the Red Sea at Eilat (Israel), showed polypeptide electrophoretic patterns comparable to those of thylakoids from eukaryotic oxygen-evolving organisms. Low temperature, fluorescence spectroscopy of Prochloron, having a chlorophyll a/b ratio of 3.8–5, and frozen in situ, demonstrated the presence of Photosystem II chlorophyll-protein complex emitting at 686 and 696 nm, as well as the emission band of Photosystem I at 720 nm which was so far not observed in Prochloron species. The latter emission was absent, if the cells or thylakoids were isolated prior to freezing. Energy transfer from chlorophyll b to chlorophyll a could be demonstrated to occur in vivo. The chlorophyll a,b-protein complex of Photosystem II, isolated by non-denaturing polyacrylamide gel electrophoresis, contained one major polypeptide of 34 kDa. The polypeptide was phosphorylated in vitro by a membrane-bound protein kinase which was not stimulated by light. A light-independent protein kinase activity was also found in isolated thylakoids of another prokaryote, the cyanophyte Fremyella diplosiphon. State I–State II transition could not be demonstrated in Prochloron by measurements of modulated fluorescence intensity in situ. We suggest that the presence of a light-independent thylakoid protein kinase of Prochloron, collected in the Red Sea at not less than 30 m depth, might be the result of an evolutionary process whereby this organism has adapted to an environment in which light, absorbed preferentially by Photosystem II, prevails.     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.      

Photosystem I reaction centers from maize bundle-sheath and mesophyll chloroplasts lack subunit III

Photosystem I reaction centers were isolated from mesophyll and bundle-sheath chloroplasts of the C4 maize plant. Both preparations were found to be free of chlorophyll b and to have the same spectral properties and chlorophyll/P700 ratio as photosystem I reaction centers isolated from C3 plants. Photosystem I reaction centers from both mesophyll and bundle sheath were found to consist of six subunits with apparent molecular masses of about 70 kDa, 20 kDa, 17 kDa, 16 kDa, 10 kDa and 8 kDa, corresponding to photosystem I reaction center subunits I, II, IV, V, VI and VII of spinach, as tested by their immunological cross-reactivity with antibody raised against the respective spinach subunits. No cross-reactivity was found with antibodies raised against subunit III of spinach, either in whole thylakoids or purified reaction centers of both bundle-sheath and mesophyll chloroplasts. It is concluded that photosystem I reaction centers of bundle-sheath and mesophyll thylakoids of maize are identical and lack the polypeptide corresponding to subunit III present in all C3 plants so far tested.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : V. A Radioautographic Study of Membrane Growth in a Mutant of Chlamydomonas reinhardi y-1

The development of photosynthetic lamellae during greening of dark-grown Chlamydomonas y-1 cells was investigated by radioautography. Acetate-³H was used as a marker for membrane lipids. In short pulse-labeling experiments, about 50–60% of the radioactivity incorporated was found in the lipid fraction and about 25–50% in starch granules present in the chloroplast of these algae. The relative specificity of acetate-³H used as a marker for membranes was artificially increased through quantitative removal of the starch granules from fixed cells by amylase treatment. Analysis of turnover coefficients of different membrane constituents and of the contribution of turnover and net synthesis to the total label incorporated in pulse experiments indicated that the incorporation of acetate into specific lipids was mainly due to net synthesis. The distribution of radioactivity in the different lipid constituents at the end of a short pulse and after 30- and 60-min chases indicated that transacylation is minimal and may be disregarded as a possible cause of randomization of the label. Statistical analysis of radioautographic grain distribution and measurements of different structural parameters indicate that (a) the chloroplast volume and surface remain constant during the process, whereas the growth of the photosynthetic lamellae parallels the increase in chlorophyll; (b) the lamellae do not develop from the chloroplast envelope or from the tubular system of the pyrenoid; (c) all the lamellae grow by incorporation of new material within preexisting structures; (d) different types of lamellae grow at different rates. The pyrenoid tubular system develops faster than the thylakoids, and single thylakoids develop about twice as fast as those which are paired or fused to grana. It is concluded that growth of the membranes occurs by a mechanism of random intussusception of molecular complexes within different types of preexisting membranes.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : IV. Lipid and Pigment Changes during Synthesis of Chloroplast Membranes in a Mutant of Chlamydomonas reinhardi y-1

The glycolipid, phospholipid, pigment, and fatty acid content in whole y-1 cells during the greening process have been investigated. The time course of their changes indicates that phosphatidyl glycerol and glycolipids are the main lipids synthesized specifically during illumination of dark-grown cells, concomitant with an increase in the polyunsaturated C18:2 and C18:3 fatty acids. The pigment complex of light-grown cells consists mainly of chlorophylls a and b, lutein, β-carotene, violaxanthin, and neoxanthin. During the greening process, chlorophylls a and b are synthesized in constant proportions (ratio a/b equals 2.6), β-carotene and violaxanthin do not change significantly, and lutein and neoxanthin increase. The molar ratios of the different lipids and pigment to total chlorophyll during greening has been calculated. It was found that during the initial phase of greening when chlorophyll is synthesized at increasing rates, the molar ratios of various lipids and pigments to chlorophyll decrease and tend to become constant when chlorophyll and membrane synthesis proceed at constant rates. The implication of these findings with respect to the concept of membrane assembly through a spontaneous single step process is discussed     

DYNAMIC CHANGES IN THE ULTRASTRUCTURE OF THE ACINAR CELL OF THE RAT PAROTID GLAND DURING THE SECRETORY CYCLE

Synchronization of the secretory cycle in vivo was obtained by injecting isoprenaline as an inducer of secretion. A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found. At the ultrastructural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge of the content. Each zymogen granule membrane, once connected with the lumen, acted as a lumen membrane. Fusion was thus sequential and resulted in a dramatic enlargement of the lumen space. During the entire process the passage between the lumen and the intercellular space remained blocked by the tight junctions, as shown by their impenetrability to ferritin. Reduction of the lumen size following enzyme discharge seemed to be achieved by withdrawal of lumen membrane in the form of small smooth vesicles which appeared mostly in the apical part of the cell. At the same time, the cell retracted towards the lumen, the whole process being completed within 2 hr from onset of secretion. Disappearance of the smooth vesicle followed, concomitant with formation of many condensing vacuoles and appearance of mature zymogen granules. The fate of the zymogen granule membrane, including its fusion with the lumen membrane, resorption in the form of small smooth vesicles, and its eventual reutilization mediated by the Golgi system, is discussed.