Elucidating the Role of Transport Processes in Leaf Glucosinolate Distribution

In Arabidopsis (Arabidopsis thaliana), a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long-distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root-to-shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.Feeding behavior of herbivorous insects and distribution of defense compounds in plants have been suggested to be a result of an arms race between plants and insects that has spanned millions of years (Ehrlich and Raven, 1964). Whether insects adapted first to plants or the other way around is an ongoing debate in this research field (Schoonhoven et al., 2005; Ali and Agrawal, 2012). Leaf margin accumulation of defense compounds has been demonstrated in various plant species (Gutterman and Chauser-Volfson, 2000; Chauser-Volfson et al., 2002; Kester et al., 2002; Cooney et al., 2012). In the model plant Arabidopsis (Arabidopsis thaliana), higher concentration of glucosinolates, which constitute a major part of the chemical defense system in this plant (Kliebenstein et al., 2001a; Halkier and Gershenzon, 2006), was found at the leaf midrib and margins compared with the leaf lamina (Shroff et al., 2008; Sønderby et al., 2010). This nonuniform leaf distribution of glucosinolates appeared to explain the feeding pattern of a generalist herbivore (Helicoverpa armigera), as it avoided feeding at the leaf margin and midrib (Shroff et al., 2008). A similar feeding pattern on Arabidopsis was observed for a different generalist herbivore, Spodoptera littoralis (Schweizer et al., 2013). Interestingly, S. littoralis was shown to favor feeding from Arabidopsis leaf margins in glucosinolate-deficient mutants (Schweizer et al., 2013), which could indicate an inherent preference for margin feeding and that Arabidopsis adapted to such behavior by accumulating defense compounds here. A damaged leaf margin may be more critical for leaf stability than damage to inner leaf parts (Shroff et al., 2008), further motivating protection of this tissue. The margin-feeding preference of S. littoralis might be explained by better nutritional value of the leaf margin cells (Schweizer et al., 2013), which has been shown to consist of specialized elongated cell files (Koroleva et al., 2010; Nakata and Okada, 2013).Other distribution patterns have been reported for glucosinolates in an Arabidopsis leaf. A study investigating spatiotemporal metabolic shifts during senescence in Arabidopsis reported that fully expanded mature leaves exhibited a glucosinolate gradient from base to tip, with highest level of glucosinolates at the leaf base (Watanabe et al., 2013). In contrast to the horizontal plane, less has been reported on distribution of glucosinolates in the vertical plane of a leaf. A localization study of cyanogenic glucosides, defense molecules related to glucosinolates (Halkier and Gershenzon, 2006), determined that these compounds primarily were located in the epidermis of sorghum (Sorghum bicolor; Kojima et al., 1979). Whereas epidermis-derived trichomes in Arabidopsis were recently demonstrated to contain glucosinolates and to express glucosinolate biosynthetic genes (Frerigmann et al., 2012), no studies have investigated glucosinolates in the epidermal cell layer.Based on promoter-GUS studies, biosynthesis of glucosinolates in leaves of Arabidopsis has been associated with primarily major and minor veins in leaves and silique walls (Mikkelsen et al., 2000; Reintanz et al., 2001; Tantikanjana et al., 2001; Chen et al., 2003; Grubb et al., 2004; Schuster et al., 2006; Gigolashvili et al., 2007; Li et al., 2011; Redovniković et al., 2012). The discrepancy between vasculature-associated glucosinolate biosynthesis and margin accumulation of glucosinolates suggests that transport processes must be involved in establishing the distribution pattern of glucosinolates within a leaf.Plant transport systems include the apoplastic xylem, the symplastic phloem, and plasmodesmata. Xylem transport is mainly driven by an upward pull generated by transpiration from aerial plant organs, thereby directing transport to sites of rapid evaporation (such as leaf margins; Sattelmacher, 2001). Phloem flow is facilitated by an osmosis-regulated hydrostatic pressure difference between source and sink tissue, primarily generated by Suc bulk flow (Lucas et al., 2013). Plasmodesmata are intercellular channels that establish symplasmic pathways between neighboring cells, and most cell types in a plant are symplastically connected via plasmodesmata (Roberts and Oparka, 2003). Translocation of small molecules in these channels is driven by diffusion and is regulated developmentally as well as spatially to form symplastically connected domains (Roberts and Oparka, 2003; Christensen et al., 2009). To what extent any of these transport processes are involved in establishing specific distribution patterns of glucosinolates within leaves is not known.Recently, two plasma membrane-localized, glucosinolate-specific importers, GLUCOSINOLATE TRANSPORTER1 (GTR1) and GTR2, were identified in Arabidopsis (Nour-Eldin et al., 2012). In leaf, their expression patterns were shown to be in leaf veins (GTR1 and GTR2) and surrounding mesophyll cells (GTR1; Nour-Eldin et al., 2012). Absence of aliphatic and indole glucosinolates in seeds of the gtr1gtr2 double knockout (dKO) mutant (gtr1gtr2 dKO) demonstrated that these transporters are essential for long-distance glucosinolate transport to the seeds and indicates a role in phloem loading (Nour-Eldin et al., 2012). Another study investigating long-distance transport of glucosinolates in the 3-week-old wild type and gtr1gtr2 dKO indicated that GTR1 and GTR2 were involved in bidirectional transport of aliphatic glucosinolates between root and shoot via both phloem and xylem pathways (Andersen et al., 2013). The authors suggested a role for GTR1 and GTR2 in the retention of long-chained aliphatic glucosinolates in roots by removing the compounds from the xylem (Andersen et al., 2013).Identification of the glucosinolate transporters GTR1 and GTR2 has provided a molecular tool to investigate the role of transport processes in establishing leaf glucosinolate distribution. In this study, we have performed a detailed spatial investigation of the distribution of an exogenously fed glucosinolate (sinigrin) and endogenous glucosinolates within mature wild-type and gtr1gtr2 dKO Arabidopsis leaves, achieved by collecting and analyzing leaf parts at the horizontal (y axis: petiole, base, and tip; x axis: midrib, lamina, and margin) as well as at the vertical leaf plane (z axis: abaxial epidermis). Furthermore, we analyze wild-type and gtr1gtr2 dKO root xylem sap and leaf apoplastic fluids for glucosinolates. Based on our results, we propose a model where GTR1 and GTR2 import glucosinolates from the apoplast to the symplast and where the glucosinolate distribution pattern within an Arabidopsis leaf is established via symplasmic movement of glucosinolates through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in peripheral cell layers.     

AID Enzymatic Activity Is Inversely Proportional to the Size of Cytosine C5 Orbital Cloud

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl > hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.     

AID is essential for immunoglobulin V gene conversion in a cultured B cell line

Following productive V gene rearrangement, the functional immunoglobulin genes in the B lymphocytes of man and mouse are subjected to two further types of genetic modification. Class-switch recombination, a region-specific but largely nonhomologous recombination process, leads to a change in constant region of the expressed antibody. Somatic hypermutation introduces multiple single nucleotide substitutions in and around the rearranged V gene segments and underpins affinity maturation. However, in chicken and rabbits (but not man or mouse), an additional mechanism, gene conversion, is a major contributor to V gene diversification. It has been demonstrated recently that both switch recombination and hypermutation are ablated in mice and humans lacking AID, a B cell-specific protein of unknown molecular activity. Here we show that disruption of AID in the DT40 chicken B cell lymphoma leads to a failure to perform immunoglobulin V gene conversion. Thus, AID is required for all three immunoglobulin gene modification programs (gene conversion, hypermutation, and switch recombination) and acts in the initiation or execution of these processes rather than in bringing the B cell to an appropriate stage of differentiation.     

Insulins with built-in glucose sensors for glucose responsive insulin release.

Derivatization of insulin with phenylboronic acids is described, thereby equipping insulin with novel glucose sensing ability. It is furthermore demonstrated that such insulins are useful in glucose‐responsive polymer‐based release systems. The preferred phenylboronic acids are sulfonamide derivatives, which, contrary to naïve boronic acids, ensure glucose binding at physiological pH, and simultaneously operate as handles for insulin derivatization at LysB29. The glucose affinities of the novel insulins were evaluated by glucose titration in a competitive assay with alizarin. The affinities were in the range 15–31 mM (K_d), which match physiological glucose fluctuations. The dose‐responsive glucose‐mediated release of the novel insulins was demonstrated using glucamine‐derived polyethylene glycol polyacrylamide (PEGA) as a model, and it was shown that Zn(II) hexamer formulation of the boronated insulins resulted in steeper glucose sensitivity relative to monomeric insulin formulation. Notably, two of the boronated insulins displayed enhanced insulin receptor affinity relative to native insulin (113%–122%) which is unusual for insulin LysB29 derivatives. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Distribution of the alphaGal- and the non-alphaGal T-antigens in the pig kidney: potential targets for rejection in pig-to-man xenotransplantation

Carbohydrate antigens, present on pig vascular endothelial cells, seem to be the prime agents responsible for graft rejection, and although genetically modified animals that express less amounts of carbohydrate antigen are available, it is still useful to decide the localization of the reactive xenoantigens in organs contemplated for xenotransplantation. Here we compare the distribution in pig kidney of antigens important in xenograft destruction, namely the Galalpha1-3Gal (alphaGal) glycans, with the localization of the T-antigen (Galbeta1-3GalNAc). The alpha-galactose-specific lectin Griffonia simplicifolia isolectin 1B4 was used to detect the Galalpha1-3Gal glycans, whereas Arachis hypogaea (PNA) lectin and a monoclonal antibody (3C9) detected T-antigen. In addition, two vascular markers (anti-caveolin-1 and anti-von Willebrand factor) served to identify vascular structures of the kidney. Both conventional fluorescence and confocal microscopy were used to distinguish lectin and immunohistochemical staining. On the basis of fluorescence signals, the results indicate that the carbohydrate antigens are heterogeneously distributed in the pig kidney. alphaGal epitopes were sparse in the capillary loops forming the glomeruli and in the capillaries surrounding the convoluted tubules, but showed stronger staining in capillaries surrounding the limbs of Henle. In addition, the brush border and basement membranes of the convoluted tubules strongly reacted with the GS1-B4-lectin. Finally, the Galalpha1-3Gal glycans were also present on epithelial cells of the large collecting tubules. Regarding the T-antigen, PNA and 3C9 reacted with different glomerular cells, whereas both reacted strongly with the endothelial cells lining the large kidney vessels. Human serum incubation of pig kidney sections, in which the alphaGal epitopes were blocked by unconjugated GS1-B4, showed staining of the same vascular structures as were obtained after incubation with the T-antigen-detecting agents. The study thus proves a complex spatial distribution of carbohydrate antigens relevant for xenotransplantation of pig kidney.     

Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA

Background  

Serology is often used for the diagnosis of Mycoplasma pneumoniae. It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M. pneumoniae. The two proteins, P116 and P1, are found to be immunogenic. By using these in ELISA it is possible to identify an immune response against M. pneumoniae in serum samples.     

Statistical power and measurement allocation in ergonomic intervention studies assessing upper trapezius EMG amplitude. A case study of assembly work.

The present study aimed at exploring the statistical power of ergonomic intervention studies using electromyography (EMG) from the upper trapezius muscle. Data from a previous study of cyclic assembly work were reanalyzed with respect to exposure variability between subjects, between days, and within days. On basis of this information, the precision and power of different data collection strategies were explored. A sampling strategy comprising four registrations of about two min each (i.e. two work cycles) for one day per subject resulted in coefficients of variation between subjects on the 10-, 50-, and 90-APDF-percentiles of 0.44, 0.31, and 0.29, respectively. The corresponding necessary numbers of subjects in a study aiming at detecting a 20% exposure difference between two independent groups of equal size were 154, 78, and 68, respectively (p< or = 0.05, power 0.80). Multiple measurement days per subject would improve power, but only to a marginal extent beyond 4 days of recording. Increasing the number of recordings per day would have minor effects. Bootstrap resampling of the data set revealed that estimates of variability and power were associated with considerable uncertainty. The present results in combination with an overview of other occupational studies showed that common-size investigations using trapezius EMG percentiles are at great risk of suffering from insufficient statistical power, even if the expected intervention effect is substantial. The paper suggests a procedure of how to retrieve and use exposure variability information as an aid when studies are planned, and how to allocate measurements efficiently.     

Synthesis and structural determination of stereoisomers of muscarinic ligands of the (3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycloalkane type

Methods for the synthesis of each of the four stereoisomers of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane ( 10, 11, 12 , and 13 ) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane ( 18, 19, 20 , and 21 ), and the two stereoisomers of 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane ( 27 and 28 ) were developed. The relative configuration of the compounds was determined on the basis of previously described ¹H NOE experiments, and the absolute configuration of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octanes ( 10, 11, 12 , and 13 ) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane ( 27 and 28 ) was determined by single crystal X-ray crystallography. Optical purity was determined by capillary electrophoresis (CE) using chiral selectors as trimethyl-β-cyclodextrin and heparin dissolved in the running buffer. All the 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycles had low nanomolar affinity for muscarinic receptors as determined by displacement of radiolabelled oxotremorine-M (³H-Oxo-M) and pirenzepine (³H-Pz) from cortical rat brain homogenates. The binding assay discriminated between diastereomers, but only a minor degree of enantioselectivity was observed in the binding assays. Chirality 9:739–749, 1997. © 1997 Wiley-Liss, Inc.     

Wind Conditions and Pollen Deposition in a Mixed Deciduous Forest

Pollen deposition in a mixed deciduous forest in South Jutland, Denmark, was studied by means of pollen analyses (Andersen, 1970) and pollen collectors. In this connection it was important to know the wind conditions outside and inside the forest. Wind velocities and wind directions in the flowering seasons were measured at 2 stations in a 3-year period. High wind velocities prevail above the forest in spring and summer. At such wind velocities the difference in loss of large and small pollen grains by filtration in the tree tops is reduced. The winds inside the forest tend to turn into the direction of easiest flow. The velocities are about 20% of the outside wind before and 12–15% after the leafing. Single pollen grains released from the trees will be carried wide distances before deposition in the spring, and it is suggested that the tree pollen falls to the ground rather in aggregates or included in rain-drops.