A seed-specific regulator of triterpene saponin biosynthesis in Medicago truncatula

Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16α hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula. In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins

CYP712K4 catalyzes the C-29 oxidation of friedelin in the Maytenus ilicifolia quinone methide triterpenoid biosynthesis pathway

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids

Point Sources from a Spitzer IRAC Survey of the Galactic Center

We have obtained Spitzer/IRAC observations of the central 2.0 x 1.4 degrees (~280 x 200 pc) of the Galaxy at 3.6-8.0 microns. A point source catalog of 1,065,565 objects is presented. The catalog includes magnitudes for the point sources at 3.6, 4.5, 5.8, and 8.0 microns, as well as JHK photometry from 2MASS. The point source catalog is confusion limited with average limits of 12.4, 12.1, 11.7, and 11.2 magnitudes for [3.6], [4.5], [5.8], and [8.0], respectively. We find that the confusion limits are spatially variable because of stellar surface density, background surface brightness level, and extinction variations across the survey region. The overall distribution of point source density with Galactic latitude and longitude is essentially constant, but structure does appear when sources of different magnitude ranges are selected. Bright stars show a steep decreasing gradient with Galactic latitude, and a slow decreasing gradient with Galactic longitude, with a peak at the position of the Galactic center. From IRAC color-magnitude and color-color diagrams, we conclude that most of the point sources in our catalog have IRAC magnitudes and colors characteristic of red giant and AGB stars.Comment: 44 pages, 13 figures, ApJS in pres

Measuring Dust Production in the Small Magellanic Cloud Core-Collapse Supernova Remnant 1E 0102.2-7219

We present mid-infrared spectral mapping observations of the core-collapse supernova remnant 1E 0102.2-7219 in the Small Magellanic Cloud using the InfraRed Spectrograph (IRS) on the Spitzer Space Telescope. The remnant shows emission from fine structure transitions of neon and oxygen as well as continuum emission from dust. Comparison of the mid-IR dust emission with observations at x-ray, radio and optical wavelengths shows that the dust is associated with the supernova ejecta and is thus newly formed in the remnant. The spectrum of the newly formed dust is well reproduced by a model that includes 3x10^-3 solar masses of amorphous carbon dust at 70 K and 2x10^-5 solar masses of Mg2SiO4 (forsterite) at 145 K. Our observations place a lower limit on the amount of dust in the remnant since we are not sensitive to the cold dust in the unshocked ejecta. We compare our results to observations of other core-collapse supernovae and remnants, particularly Cas A where very similar spectral mapping observations have been carried out. We observe a a factor of ~10 less dust in E 0102 than seen in Cas A, although the amounts of amorphous carbon and forsterite are comparable.Comment: submitted to Ap

Formation and Evolution of Dust in Type IIb Supernova with Application to the Cassiopeia A Supernova Remnant

We investigate the formation of dust grains in the ejecta of a SN IIb and their evolution in the shocked gas in the SNR by considering the uniform and power-law density structures for the CSM. Based on these calculations, we also simulate the time evolution of thermal emission from the shock-heated dust in the SNR and compare the results with the observations of Cas A SNR. We find that the total mass of dust formed in the SN IIb is as large as 0.167 M_sun but the average radius of dust is smaller than 0.01 mum and is significantly different from those in SNe II-P with the massive H-envelope. In the explosion with the small-mass H-envelope, the expanding He core undergoes little deceleration, so that the gas density in the He core is too low for large-sized grains to form. In addition, the low-mass H-envelope of the SN IIb leads to the early arrival of the reverse shock at the dust-forming region. If the CSM is more or less spherical, therefore, the newly formed grains would be completely destroyed in the relatively dense shocked gas for the CSM density of n_H > 0.1 cm^{-3}. However, the actual CSM is likely to be non-spherical, so that a part of grains could be ejected into the ISM without being shocked. We demonstrate that the time evolution of the SED by thermal dust emission is sensitive to the ambient gas density and structure that affects the passage of the reverse shock into the ejecta. Thus, the SED evolution well reflects the evolution of dust through erosion by sputtering and stochastic heating. For Cas A, we consider the CSM produced by the steady mass loss of ~8x10^{-5} M_sun/yr during the supergiant phase. Then we find the infrared SED of Cas A is reasonably reproduced by thermal emission from the newly formed dust of 0.08 M_sun, which consists of 0.008 M_sun shocked warm dust and 0.072 M_sun unshocked cold dust.Comment: 50 pages, 17 figures, 3 tables. Accepted for publication in Ap

Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins

Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. Results: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of -amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2-hydroxyoleanolic acid, hederagenin and total saponin levels. Conclusions: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis

The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids

HST/NICMOS Paschen-alpha Survey of the Galactic Center: Overview

We have recently carried out the first wide-field hydrogen Paschen-alpha line imaging survey of the Galactic Center (GC), using the NICMOS instrument aboard the Hubble Space Telescope. The survey maps out a region of 2253 pc^2 around the central supermassive black hole (Sgr A*) in the 1.87 and 1.90 Micron narrow bands with a spatial resolution of 0.01 pc at a distance of 8 kpc. Here we present an overview of the observations, data reduction, preliminary results, and potential scientific implications, as well as a description of the rationale and design of the survey. We have produced mosaic maps of the Paschen-alpha line and continuum emission, giving an unprecedentedly high resolution and high sensitivity panoramic view of stars and photo-ionized gas in the nuclear environment of the Galaxy. We detect a significant number of previously undetected stars with Paschen-alpha in emission. They are most likely massive stars with strong winds, as confirmed by our initial follow-up spectroscopic observations. About half of the newly detected massive stars are found outside the known clusters (Arches, Quintuplet, and Central). Many previously known diffuse thermal features are now resolved into arrays of intriguingly fine linear filaments indicating a profound role of magnetic fields in sculpting the gas. The bright spiral-like Paschen-alpha emission around Sgr A* is seen to be well confined within the known dusty torus. In the directions roughly perpendicular to it, we further detect faint, diffuse Paschen-alpha emission features, which, like earlier radio images, suggest an outflow from the structure. In addition, we detect various compact Paschen-alpha nebulae, probably tracing the accretion and/or ejection of stars at various evolutionary stages.Comment: accepted for publication in MNRAS; a version of higher resolution images may be found at http://www.astro.umass.edu/~wqd/papers/hst/paper1.pd

The role of experiments in understanding fishery-induced evolution

Evidence of fishery-induced evolution has been accumulating rapidly from various avenues of investigation. Here we review the knowledge gained from experimental approaches. The strength of experiments is in their ability to disentangle genetic from environmental differences. Common garden experiments have provided direct evidence of adaptive divergence in the wild and therefore the evolvability of various traits that influence production in numerous species. Most of these cases involve countergradient variation in physiological, life history, and behavioral traits. Selection experiments have provided examples of rapid life history evolution and, more importantly, that fishery-induced selection pressures cause simultaneous divergence of not one but a cluster of genetically and phenotypically correlated traits that include physiology, behavior, reproduction, and other life history characters. The drawbacks of experiments are uncertainties in the scale-up from small, simple environments to larger and more complex systems; the concern that taxons with short life cycles used for experimental research are atypical of those of harvested species; and the difficulty of adequately simulating selection due to fishing. Despite these limitations, experiments have contributed greatly to our understanding of fishery-induced evolution on both empirical and theoretical levels. Future advances will depend on integrating knowledge from experiments with those from modeling, field studies, and molecular genetic approaches