Photocontrol of anthocyanin formation in turnip seedlings

Summary The substitution of far-red for the first six hours of a prolonged irradiation with red light resulted in a large increase in anthocyanin yield, which was greater than the combined yields from far-red and red when the two treatments were given separately. When intermittent far-red irradiation was followed by a single short exposure to red, a considerable amount of anthocyanin was formed, although each treatment given separately had little effect. Four hours continuous far-red alone yielded some anthocyanin and also resulted in a further large increase in the effect of a short red treatment; this terminal red effect was fully reversible by a subsequent brief exposure to far-red. It is concluded that at least two photochemical reactions are involved in the responses to red and far-red, the first leading to the formation of substrate(s) used in the second reaction.When red light preceded exposure to the far-red/red irradiation sequence, the far-red enhancement effect was almost entirely lost and the anthocyanin yield approached that in red light. The effect of the red pre-irradiation treatment is attributed to destruction of phytochrome and it is suggested that phytochrome is the only pigment mediating anthocyanin synthesis in red and far-red. A possible interpretation is that the high-energy reaction in far-red and the low energy red/far-red reversible reaction are mediated by different forms of phytochrome.The substitution of blue for the first six hours of a prolonged irradiation with red light also resulted in a synergistic increase in anthocyanin yield; the enhancement effect of blue light was, however, not prevented by prior exposure to red. It is concluded that phytochrome is not the only pigment mediating the reactions occurring in blue light. The synergism between blue and red suggests that the high-energy reaction in blue light may lead to the production of substrates for phytochrome action.

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Zusammenfassung Die Substitution der ersten 6 Std einer Hellrot-Dauerbestrahlung durch Dunkelrot führte zu einem starken Anstieg im Anthocyangehalt, der höher war als die Summe aus Dunkelrot und Hellrot, wenn beide Bestrahlungen getrennt gegeben wurden. Folgte auf intermittierende Dunkelrot-Bestrahlung eine einmalige Dosis Hellrot, bildete sich eine beträchtliche Menge Anthocyan, obwohl jede Bestrahlung für sich kaum wirksam war. 4 Std Dauerdunkelrot induzierten bereits meßbare Anthocyanbildung, die durch kurze Hellrot-Bestrahlung weiter gesteigert werden konnte; der Effekt dieser terminalen Dosis Hellrot konnte durch nachfolgende kurze Dunkelrot-Bestrahlung wieder rückgängig gemacht werden. Daraus wird geschlossen, daß wenigstens zwei photochemische Reaktionen bei Bestrahlung mit Hellrot und Dunkelrot ablaufen, wobei die erste Substrat(e) für die zweite produziert.Wurde vor einer Dunkelrot-Hellrot-Sequenz mit Hellrot bestrahlt, ging die fördernde Wirkung von Dunkelrot fast vollständig verloren und der Anthocyangehalt entsprach annähernd dem in Hellrot. Der Effekt der Hellrot-Vorbestrahlung wird auf die Destruktion von Phytochrom zurückgeführt und es wird vermutet, daß Phytochrom das einzige Pigment ist, das bei der Anthocyansynthese in Hellrot und Dunkelrot beteiligt ist. Eine mögliche Interpretation wäre, daß die Hochenergiereaktion in Dunkelrot und die Hellrot-Dunkelrot reversible Niederenergiereaktion durch verschiedene Formen von Phytochrom vermittelt werden.Die Substitution der ersten 6 Std einer Dauerbelichtung mit Hellrot durch Blau ergab ebenfalls eine synergistische Zunahme im Anthocyangehalt. Der fördernde Effekt von Blaulicht konnte jedoch durch Vorbestrahlung mit Hellrot nicht verhindert werden. Daraus wird geschlossen, daß Phytochrom nicht das einzige Pigment sein kann, das die Reaktionen in Blaulicht vermittelt. Der Synergismus zwischen Blau und Hellrot läßt vermuten, daß die Hochenergiereaktion in Blau zur Produktion von Substrat führt, mit dem Phytochrom reagieren kann.

     

Effects of Burning and Grazing on a Coastal California Grassland

We tested the effects of fall burning and protection from livestock grazing as management to enhance native grasses on a coastal grassland in central California. Plants from the Mediterranean, introduced beginning in the late 1700s, have invaded and now dominate most of California's grasslands. Coastal grasslands are generally less degraded than those inland and have higher potential for restoration and conservation. Productivity of the experimental plots varied annually and declined over the course of the study because of rainfall patterns. Foliar cover of the native Danthonia californica (California oatgrass) increased more under grazing than grazing exclusion and did not respond to burning. Two other natives, Nassella pulchra (purple needlegrass) and Nassella lepida (foothill needlegrass), responded variably to treatments. The response of N. pulchra differed from that reported on more inland sites in California. Restoring these grasslands is complicated by differing responses of target species to protection from grazing and burning. The current practice of managing to enhance single species of native plants (e.g., N. pulchra) may be detrimental to other equally important native species.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

CYP704B1 Is a Long-Chain Fatty Acid ω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis

Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.The biopolymer sporopollenin is the major component of the outer walls in pollen and spores (exines). It is highly resistant to nonoxidative physical, chemical, and biological treatments and is insoluble in both aqueous and organic solvents. While the stability and resistance of sporopollenin account for the preservation of ancient pollen grains for millions of years with nearly full retention of morphology (Doyle and Hickey, 1976; Friis et al., 2001), these same qualities make it extremely difficult to study the chemical structure of sporopollenin. Thus, although the first studies on the composition of sporopollenin were reported in 1928 (Zetzsche and Huggler, 1928), the exact structure of sporopollenin remains unresolved. At present, it is thought that sporopollenin is a complex polymer primarily made of a mixture of fatty acids and phenolic compounds (Guilford et al., 1988; Wiermann et al., 2001).Fatty acids were first implicated as sporopollenin components when ozonolysis of Lycopodium clavatum and Pinus sylvestris exine yielded significant amounts of straight- and branched-chain monocarboxylic acids, characteristic fatty acid breakdown products (Shaw and Yeadon, 1966). More recently, improved purification and degradation techniques coupled with analytical methods, such as solid-state ¹³C-NMR spectroscopy, Fourier transform infrared spectroscopy, and ¹H-NMR, have shown that sporopollenin is made up of polyhydroxylated unbranched aliphatic units and also contains small amounts of oxygenated aromatic rings and phenylpropanoids (Guilford et al., 1988; Ahlers et al., 1999; Domínguez et al., 1999; Bubert et al., 2002). Biochemical studies using thiocarbamate herbicide inhibition of the chain-elongating steps in the synthesis of long-chain fatty acids and radioactive tracer experiments provided further evidence that lipid metabolism is involved in the biosynthesis of sporopollenin (Wilwesmeier and Wiermann, 1995; Meuter-Gerhards et al., 1999).Relatively little is known about the genetic network that determines sporopollenin synthesis. However, several Arabidopsis (Arabidopsis thaliana) genes implicated in exine biosynthesis encode proteins with sequence homology to enzymes that are involved in fatty acid metabolism. Mutations in MALE STERILITY2 (MS2) eliminate exine and affect a protein with sequence similarity to fatty acyl reductases; the predicted inability of ms2 plants to reduce pollen wall fatty acids to the corresponding alcohols suggests that this reaction is a key step in sporopollenin synthesis (Aarts et al., 1997). The FACELESS POLLEN1 (FLP1) gene, whose loss causes the flp1 exine defect, encodes a protein similar to those involved in wax synthesis (Ariizumi et al., 2003). The no exine formation1 (nef1) mutant accumulates reduced levels of lipids, and the NEF1 protein was suggested to be involved in either lipid transport or the maintenance of plastid membrane integrity, including those plastids in the secretory tapetum of anthers, where many of the sporopollenin components are synthesized (Ariizumi et al., 2004). The dex2 mutant has mutations in the evolutionarily conserved anther-specific cytochrome P450, CYP703A2 (Morant et al., 2007), which catalyzes in-chain hydroxylation of saturated medium-chain fatty acids, with lauric acid (C12:0) as a preferred substrate (Morant et al., 2007). A recently described gene, ACOS5, encodes a fatty acyl-CoA synthetase that has in vitro preference for medium-chain fatty acids (de Azevedo Souza et al., 2009). Mutations in all of these genes compromise exine formation.Here, we describe an evolutionarily conserved cytochrome P450, CYP704B1, and demonstrate that this gene is essential for exine biosynthesis and plays a role different from that of CYP703A2. Heterologously expressed CYP704B1 catalyzed ω-hydroxylation of several saturated and unsaturated C14-C18 fatty acids. These results suggest the possibility that ω-hydroxylated fatty acids produced by CYP704B1, together with in-chain hydroxylated lauric acids provided by the action of CYP703A2, may serve as key monomeric aliphatic building blocks in sporopollenin formation. Analyses of the genetic relationships between CYP704B1, MS2, and CYP703A2 suggest that all three genes are involved in the same pathway within the sporopollenin biosynthesis framework.     

The extracellular lipase EXL4 is required for efficient hydration of Arabidopsis pollen

Pollination in species with dry stigmas begins with the hydration of desiccated pollen grains on the stigma, a highly regulated process involving the proteins and lipids of the pollen coat and stigma cuticle. Self-incompatible species of the Brassicaceae block pollen hydration, and while the early signaling steps of the self-incompatibility response are well studied, the precise mechanisms controlling pollen hydration are poorly understood. Both lipids and proteins are important for hydration; loss of pollen coat lipids and proteins results in defective or delayed hydration on the stigma surface. Here, we examine the role of the pollen coat protein extracellular lipase 4 (EXL4), in the initial steps of pollination, namely hydration on the stigma. We identify a mutant allele, exl4-1, that shows a reduced rate of pollen hydration. exl4-1 pollen is normal with respect to pollen morphology and the downstream steps in pollination, including pollen tube germination, growth, and fertilization of ovules. However, owing to the delay in hydration, exl4-1 pollen is at a disadvantage when competed with wild-type pollen. EXL4 also functions in combination with GRP17 to promote the initiation of hydration. EXL4 is similar to GDSL lipases, and we show that it functions in hydrolyzing ester bonds. We report a previously unknown function for EXL4, an abundant pollen coat protein, in promoting pollen hydration on the stigma. Our results indicate that changes in lipid composition at the pollen–stigma interface, possibly mediated by EXLs, are required for efficient pollination in species with dry stigmas.     

Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization,core and urea optimization and in vivo efficacy

Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.     

Is Visual Selective Attention in Deaf Individuals Enhanced or Deficient? The Case of the Useful Field of View

Background

Early deafness leads to enhanced attention in the visual periphery. Yet, whether this enhancement confers advantages in everyday life remains unknown, as deaf individuals have been shown to be more distracted by irrelevant information in the periphery than their hearing peers. Here, we show that, in a complex attentional task, a performance advantage results for deaf individuals.

Methodology/Principal Findings

We employed the Useful Field of View (UFOV) which requires central target identification concurrent with peripheral target localization in the presence of distractors – a divided, selective attention task. First, the comparison of deaf and hearing adults with or without sign language skills establishes that deafness and not sign language use drives UFOV enhancement. Second, UFOV performance was enhanced in deaf children, but only after 11 years of age.

Conclusions/Significance

This work demonstrates that, following early auditory deprivation, visual attention resources toward the periphery slowly get augmented to eventually result in a clear behavioral advantage by pre-adolescence on a selective visual attention task.