The tannosome is an organelle forming condensed tannins in the chlorophyllous organs of Tracheophyta

Background and Aims

Condensed tannins (also called proanthocyanidins) are widespread polymers of catechins and are essential for the defence mechanisms of vascular plants (Tracheophyta). A large body of evidence argues for the synthesis of monomeric epicatechin on the cytosolic face of the endoplasmic reticulum and its transport to the vacuole, although the site of its polymerization into tannins remains to be elucidated. The aim of the study was to re-examine the cellular frame of tannin polymerization in various representatives of the Tracheophyta.

Methods

Light microscopy epifluorescence, confocal microscopy, transmission electron microscopy (TEM), chemical analysis of tannins following cell fractionation, and immunocytochemistry were used as independent methods on tannin-rich samples from various organs from Cycadophyta, Ginkgophyta, Equisetophyta, Pteridophyta, Coniferophyta and Magnoliophyta. Tissues were fixed in a caffeine–glutaraldehyde mixture and examined by TEM. Other fresh samples were incubated with primary antibodies against proteins from both chloroplastic envelopes and a thylakoidal chlorophyll-carrying protein; they were also incubated with gelatin–Oregon Green, a fluorescent marker of condensed tannins. Coupled spectral analyses of chlorophyll and tannins were carried out by confocal microscopy on fresh tissues and tannin-rich accretions obtained through cell fractionation; chemical analyses of tannins and chlorophylls were also performed on the accretions.

Key Results and Conclusions

The presence of the three different chloroplast membranes inside vacuolar accretions that constitute the typical form of tannin storage in vascular plants was established in fresh tissues as well as in purified organelles, using several independent methods. Tannins are polymerized in a new chloroplast-derived organelle, the tannosome. These are formed by pearling of the thylakoids into 30 nm spheres, which are then encapsulated in a tannosome shuttle formed by budding from the chloroplast and bound by a membrane resulting from the fusion of both chloroplast envelopes. The shuttle conveys numerous tannosomes through the cytoplasm towards the vacuole in which it is then incorporated by invagination of the tonoplast. Finally, shuttles bound by a portion of tonoplast aggregate into tannin accretions which are stored in the vacuole. Polymerization of tannins occurs inside the tannosome regardless of the compartment being crossed. A complete sequence of events apparently valid in all studied Tracheophyta is described.     

Two Binding Modes of Netropsin are Involved in the Complex Formation with Poly(dA-dT)·Poly(dA-dT) and other Alternating DNA Duplex Polymers

Abstract

Using CD measurements we show that the interaction of netropsin to poly(dA-dT)·poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA·dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA)·poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT)·poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice

Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss‐of‐function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild‐type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss‐of‐function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo–endosperm size control, the genetic network of the related genes should be elucidated.     

Parasitoid developmental mortality in the field: patterns, causes and consequences for sex ratio and virginity

1. Sex ratio theory predicts that developmental mortality can affect sex ratio optima under Local Mate Competition and also lead to 'virgin' broods containing only females with no sibling-mating opportunities on maturity. 2. Estimates of developmental mortality and its sex ratio effects have been laboratory based, and both models and laboratory studies have treated mortality as a phenomenon without identifying its biological causes. 3. We contribute a large set of field data on Metaphycus luteolus Timberlake (Hymenoptera: Encyrtidae), an endoparasitoid of soft scale insects (Hemiptera: Coccidae), which has sex allocation conditional on host quality and female-biased brood sex ratios. Developmental mortality within broods can be both assessed and attributed to distinct causes, including encapsulation by the host and larval-larval competition. 4. Thirty per cent of M. luteolus offspring die during development with 65% of this mortality because of encapsulation and 28% because of larval competition. The distributions of mortality overall and for each cause of mortality separately were overdispersed. 5. The probability of an individual being encapsulated increased with clutch size, while the probability of being killed by a brood mate declined with increasing clutch size and with increasing per capita availability of resources. 6. The sexual compositions of broods at emergence were influenced by both the degree and the type of mortality operating. At higher levels of mortality, single sex broods were more common and sex ratios were less precise. Overall, virginity was more prevalent than predicted and was more greatly affected by the occurrence of competition than by other sources of mortality, almost certainly because competition tended to eliminate males. 7. The reproductive and developmental biology of M. luteolus appears to be influenced by a complex interplay of maternal clutch size and sex allocation strategies, offspring-offspring developmental interactions, host defence mechanisms and postemergence mating behaviour. Despite the great sophistication of sex ratio theory, it has not yet evolved to the point where it is capable of considering all of these influences simultaneously.     

Gene genealogies reveal differentiation at sex pheromone olfactory receptor loci in pheromone strains of the European corn borer, Ostrinia nubilalis

Males of the E and Z strains of the European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae) are attracted to different blends of the same pheromone components. The difference in male behavioral response is controlled by the sex-linked locus Resp. The two types of males have identical neuroanatomy but their physiological specificity is reversed, suggesting that variation at the periphery results in behavioral change. Differences in the olfactory receptors (ORs) could explain the strain-specific antennal response and blend preference. Gene genealogies can provide insights into the processes involved in speciation and allow delineation of genome regions that contribute to reproductive barriers. We used intronic DNA sequences from five OR-encoding genes to investigate whether they exhibit fixed differences between strains and therefore might contribute to reproductive isolation. Although two genealogies revealed shared polymorphism, molecular polymorphism at three genes revealed nearly fixed differences between strains. These three OR genes map to the sex chromosome, but our data indicate that the distance between Resp and the ORs is >20 cM, making it unlikely that variation in pheromone-sensitive OR genes is directly responsible for the difference in behavioral response. However, differences in male antennal response may have their origin in the selection of strain-specific alleles.     

Interactions of β-lactoglobulin with serotonin and arachidonyl serotonin

β‐Lactoglobulin (β‐LG) is a lipocalin, which is the major whey protein of cow's milk and the milk of other mammals. However, it is absent from human milk. The biological function of β‐LG is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. β‐LG has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5‐hydroxytryptamine, 5‐HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA‐5HT), with β‐LG was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with β‐LG forming equimolar complexes. The binding constant for the serotonin/β‐LG interaction is between 10⁵ and 10⁶ M⁻¹, whereas for the AA‐5HT/β‐LG complex it is between 10⁴ and 10⁵ M⁻¹ as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25% EtOH). The interactions between serotonin/β‐LG and AA‐5HT/β‐LG may compete with self‐association (micellization) of both the ligand and the protein. According to far‐ and near‐UV CD results, these ligands have no apparent influence on β‐LG secondary structure, however they partially destabilize its tertiary structure. Their binding by β‐LG may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk‐fed animals. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 871–880, 2011.     

Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson's disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C-terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC-COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra-molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra-molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC-COR tandem of LRRK2, showing that Y1699 is positioned at the intra-molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra-molecular ROC : COR interface, strengthens the intra-molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC-COR tandem domain resulting in decreased GTPase activity.     

Effect of spinal level and loading conditions on the production of vertebral burst fractures in a porcine model

Vertebral burst fractures are commonly studied with experimental animal models. There is however a lack of consensus as to what parameters are important to create an unstable burst fracture with a significant canal encroachment on such model. This study aims to assess the effect of the loading rate, flexion angle, spinal level, and their interactions on the production of a vertebral thoracolumbar burst fracture on a porcine model. Sixteen functional spinal units composed of three vertebrae were harvested from mature Yucatan minipigs. Two loading rates (0.01 and 500 mm/s), two flexion angles (0° and 15°), and two spinal levels (T11-T13 and T14-L2) were studied, following a full factorial experimental plan with one repetition. Compression was applied to each functional unit to create a vertebral fracture. The load-to-failure, loss of compressive stiffness, final canal encroachment, and fracture type were used as criteria to evaluate the resulting fracture. All specimens compressed without flexion resulted in burst fractures. Half of the specimens compressed with the 15° flexion angle resulted in compression fractures. Specimens positioned without flexion lost more of their compressive stiffness and had more significant canal encroachment. Fractured units compressed with a higher loading rate resulted in a greater loss of compressive stiffness. The spinal level had no significant effect on the resulting fractures. The main parameters which affect the resulting fracture are the loading rate and the flexion angle. A higher loading rate and the absence of flexion favors the production of burst fractures with a greater canal encroachment.