Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.Prenylation is an important derivatization of plant aromatics, contributing to the chemical diversification of phenolic secondary metabolites in plants due to differences in prenylation positions, prenyl chain lengths, and further modifications of prenyl chains. To date, about 1,000 prenylated aromatic compounds have been isolated as biologically active substances from various plant species, including many medicinal plants.Coumarins (α-benzopyrones) are a large group of plant secondary metabolites. Many biologically active coumarins are prenylated, with the prenyl residue enhancing the biological activities of the aromatic core compound. For example, imperatorin (dimethylallylated xanthotoxol), a strong inhibitor of a Manduca sexta midgut cytochrome P450, has 100-fold greater activity than the nonprenylated coumarin compound, suggesting that prenylation is involved in chemoprevention against biotic stress in plants (Neal and Wu, 1994). Prenylated compounds are also beneficial for human health. For example, geranylation of umbelliferone at the OH position to form auraptene results in a 25-fold enhancement of the inhibition of Epstein Barr virus activity, a test used to screen antitumor compounds (Murakami et al., 1997). Moreover, in tuberculosis, 8-geranyloxypsoralen was reported to decrease the growth rate of Mycobacterium smegmatis (Adams et al., 2006).There are many reports on the detection of prenyltransferase (PT) activities for coumarins in various plant species. For example, umbelliferone-dimethylallyltransferase activities were reported in cultured parsley (Petroselinum crispum) cells, Ruta graveolens, and Ammi majus, and plastidial localization of the enzyme activity is also reported (Ellis and Brown, 1974; Dhillon and Brown, 1976; Tietjen and Matern, 1983; Hamerski and Matern, 1988; Hamerski et al., 1990). In addition, bergaptol 5-O-geranyltransferase activity, which yields bergamottin, a major coumarin derivative, was characterized using the microsomal fraction of lemon (Citrus limon) peel flavedo, the outer part of the lemon fruit (Frérot and Decorzant, 2004; Munakata et al., 2012). In the lemon flavedo, 8-geranyltransferase activity for umbelliferone was also detected (Munakata et al., 2012). To date, only one gene encoding these enzymes has been described; this gene, which encodes a parsley PT (PcPT), was very recently isolated (Karamat et al., 2014).The first flavonoid-specific PT identified was naringenin 8-dimethylallyltransferase (SfN8DT1) from a leguminous medicinal plant, Sophora flavescens (Sasaki et al., 2008). Since then, genes encoding various flavonoid PTs have been identified in Leguminosae (Akashi et al., 2009; Sasaki et al., 2011; Shen et al., 2012). Although other prenylated aromatic compounds, including coumarins, xanthons, phenylpropanoids, and phloroglucinols, have been isolated from many plant species, no gene encoding a PT for those aromatics has been isolated, except for the gene encoding a phloroglucinol-specific enzyme (HlPT1) from hops (Humulus lupulus) and a the recently isolated coumarin dimethylallyltransferase from parsley (Tsurumaru et al., 2010, 2012; Karamat et al., 2014). These isolated plant aromatic PTs show strong preference for dimethylallyl diphosphate (DMAPP) as the prenyl donor substrate, although in nature, many geranylated phenolics and less farnesylated phenolics have been described. This raises questions about the enzymes and reaction mechanisms involved in the synthesis of these phenolic compounds, such as substrate specificity and prenylation sites. Better understanding of these reactions requires the identification of PTs with other enzymatic activities. It is also necessary to identify PTs producing prenylated phenolics in nonleguminosaeous plants. Four different tracks should be explored to identify enzymes that (1) recognize nonflavonoid substrates, e.g. coumarins, phenylpropanoids, and xanthons, (2) are specific for longer chain prenyl diphosphates such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), (3) are from nonlegume origins, and (4) catalyze O-prenylation.Citrus species, including lemons, contain large quantities of geranylated coumarins. We therefore isolated a complementary DNA (cDNA) encoding a PT from lemon peel, identifying the novel PT-encoding gene ClPT1. Phylogenetic analysis showed that this enzyme shares homologies with homogentisate PTs involved in vitamin E and plastoquinone biosynthesis but is located in a new clade. We provide evidence showing that this unique enzyme is highly specific for GPP as a prenyl donor and coumarin as a prenyl acceptor. We also show that the gene product is targeted to plastid in plant cells.     

Differential proteomics reveals multiple components in retrogradely transported axoplasm after nerve injury

Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of approximately 4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components.     

Temporal mating isolation driven by a behavioral gene in Drosophila

Speciation is the evolutionary process in which new barriers to gene exchange are created. These barriers may be physical, leading to spatial separation of subpopulations and resulting in allopatric speciation, or they may be temporal, giving rise to allochronic speciation, and may include the time of day or the time of year when mating takes place. Drosophila melanogaster and D. pseudoobscura show different temporal patterns of circadian locomotor activity that are determined by the circadian clock gene period (per). Genes that control aspects of behavior that might be relevant to courtship and mating, such as locomotor patterns, become obvious candidates for involvement in the speciation process. However, evidence for the role of individual genes in the mechanism of mate choice has proved elusive. We have used transgenic flies carrying the natural per genes from these two Drosophila species to reveal that per has the potential to provide the permissive conditions for speciation, by affecting mate choice through a mechanism involving the species-specific timing of mating behavior.     

Red foliage color reliably indicates low host quality and increased metabolic load for development of an herbivorous insect

Plant chemical defense and coevolved detoxification mechanisms in specialized herbivorous insects are fundamental in determining many insect–plant interactions. For example, Brassicale plants protect themselves from herbivory by producing glucosinolates, but these secondary metabolites are effectively detoxified by larvae of Pierid butterflies. Nevertheless, not all Brassicales are equally preferred by these specialist herbivores. Female Pieris butterflies avoid laying eggs on anthocyanin-rich red foliage, suggesting red color is a visual cue affecting oviposition behavior. In this study, we reared P. brassicae larvae on green and red cabbage leaves, to determine whether foliage color reliably indicates host plant quality. We did not find a difference in survival rates or maximal larval body mass in the two food treatments. However, larvae feeding on red cabbage leaves exhibited significantly lower growth rates and longer durations of larval development. Interestingly, this longer development was coupled with a higher consumption rate of dry food matter. The lower ratio of body mass gain to food consumption in larvae feeding on red cabbage leaves was coupled with significantly higher (ca. 10 %) larval metabolic rates. This suggests that development on red foliage may incur an increased metabolic load associated with detoxification of secondary plant metabolites. Energy and oxygen allocation to detoxification could come at the expense of growth and thus compromise larval fitness as a result of extended development. From an evolutionary perspective, red foliage color may serve as an honest defensive cue, as it reliably indicates the plant’s low quality as a substrate for larval development.     

Transition from Anaerobic to Aerobic Growth Conditions for the Sulfate-Reducing Bacterium Desulfovibrio oxyclinae Results in Flocculation

A chemostat culture of the sulfate-reducing bacterium Desulfovibrio oxyclinae isolated from the oxic layer of a hypersaline cyanobacterial mat was grown anaerobically and then subjected to gassing with 1% oxygen, both at a dilution rate of 0.05 h⁻¹. The sulfate reduction rate under anaerobic conditions was 370 nmol of SO₄²⁻ mg of protein⁻¹ min⁻¹. At the onset of aerobic gassing, sulfate reduction decreased by 40%, although viable cell numbers did not decrease. After 42 h, the sulfate reduction rate returned to the level observed in the anaerobic culture. At this stage the growth yield increased by 180% compared to the anaerobic culture to 4.4 g of protein per mol of sulfate reduced. Protein content per cell increased at the same time by 40%. The oxygen consumption rate per milligram of protein measured in washed cell suspensions increased by 80%, and the thiosulfate reduction rate of the same samples increased by 29% with lactate as the electron donor. These findings indicated possible oxygen-dependent enhancement of growth. After 140 h of growth under oxygen flux, formation of cell aggregates 0.1 to 3 mm in diameter was observed. Micrometer-sized aggregates were found to form earlier, during the first hours of exposure to oxygen. The respiration rate of D. oxyclinae was sufficient to create anoxia inside clumps larger than 3 μm, while the levels of dissolved oxygen in the growth vessel were 0.7 ± 0.5 μM. Aggregation of sulfate-reducing bacteria was observed within a Microcoleus chthonoplastes-dominated layer of a cyanobacterial mat under daily exposure to oxygen concentrations of up to 900 μM. Desulfonema-like sulfate-reducing bacteria were also common in this environment along with other nonaggregated sulfate-reducing bacteria. Two-dimensional mapping of sulfate reduction showed heterogeneity of sulfate reduction activity in this oxic zone.