Development of polymorphic microsatellite loci in Nothapodytes nimmoniana, a medicinally important tree from the Western Ghats, India

Nothapodytes nimmoniana is a medicinally important tree species that occur in the Western Ghats, a megadiversity hotspot in southern India. Inner stem bark of the tree contains an important anti-cancer alkaloid, camptothecin for which the natural population of the tree is heavily harvested. In this paper, we report the isolation and characterization of eight polymorphic microsatellite loci using enrichment hybridization protocol. Analysis of 36 individuals representing two populations revealed three to 12 alleles per locus. Observed heterozygosity ranged from 0.21 to 0.94 for the two populations. None of the loci tested showed linkage disequilibrium. These markers are invaluable for evaluating the genetic structure and assessing the genetic impacts of harvesting of N. nimmoniana in the Western Ghats to formulate strategies for conservation of the species.     

Phytochrome B Promotes Branching in Arabidopsis by Suppressing Auxin Signaling

Many plants respond to competition signals generated by neighbors by evoking the shade avoidance syndrome, including increased main stem elongation and reduced branching. Vegetation-induced reduction in the red light:far-red light ratio provides a competition signal sensed by phytochromes. Plants deficient in phytochrome B (phyB) exhibit a constitutive shade avoidance syndrome including reduced branching. Because auxin in the polar auxin transport stream (PATS) inhibits axillary bud outgrowth, its role in regulating the phyB branching phenotype was tested. Removing the main shoot PATS auxin source by decapitation or chemically inhibiting the PATS strongly stimulated branching in Arabidopsis (Arabidopsis thaliana) deficient in phyB, but had a modest effect in the wild type. Whereas indole-3-acetic acid (IAA) levels were elevated in young phyB seedlings, there was less IAA in mature stems compared with the wild type. A split plate assay of bud outgrowth kinetics indicated that low auxin levels inhibited phyB buds more than the wild type. Because the auxin response could be a result of either the auxin signaling status or the bud’s ability to export auxin into the main shoot PATS, both parameters were assessed. Main shoots of phyB had less absolute auxin transport capacity compared with the wild type, but equal or greater capacity when based on the relative amounts of native IAA in the stems. Thus, auxin transport capacity was unlikely to restrict branching. Both shoots of young phyB seedlings and mature stem segments showed elevated expression of auxin-responsive genes and expression was further increased by auxin treatment, suggesting that phyB suppresses auxin signaling to promote branching.The development of shoot branches is a multistep process with many potential points of regulation. After the formation of an axillary meristem in the leaf axil, an axillary bud may form through the generation of leaves and other tissues. The axillary bud may grow out to form a branch, or may remain dormant or semidormant for an indefinite period of time (Bennett and Leyser, 2006). In Arabidopsis (Arabidopsis thaliana), the position of the bud in the rosette is a strong determinant of its fate, with upper buds displaying greater outgrowth potential than lower buds. In fact, the varying potential of buds at different positions is maintained even in buds that are activated to form branches, with the upper buds growing out first and most robustly, and lower buds growing out after a time lag and with less vigor (Hempel and Feldman, 1994; Finlayson et al., 2010).The disparate fate of buds at different rosette positions is mediated, at least in part, by the process of correlative inhibition, whereby remote parts of the plant inhibit the outgrowth of the buds (Cline, 1997). Correlative inhibition is typically associated with the bud-inhibiting effects of auxin sourced in the shoot apex and transported basipetally in the polar auxin transport stream (PATS). Auxin in the PATS does not enter the bud and thus must act indirectly; however, the exact mechanism by which auxin inhibits bud outgrowth is not well understood, despite many years of intensive study (Waldie et al., 2010; Domagalska and Leyser, 2011). Evidence supports divergent models by which auxin may regulate branching. One model contends that the PATS modulates a bud outgrowth inhibiting second messenger (Brewer et al., 2009). Another model postulates a mechanism whereby competition between the main shoot and the axillary bud for auxin export in the PATS regulates bud activity (Bennett et al., 2006; Prusinkiewicz et al., 2009; Balla et al., 2011).In addition to intrinsic developmental programming, branching is also modulated by environmental signals, including competition signals generated by neighboring plants. The red light:far-red light ratio (R:FR) is an established competition signal that is modified (reduced) by neighboring plants and sensed by the phytochrome family of photoreceptors. A low R:FR evokes the shade avoidance syndrome with plants displaying, among other phenotypes, enhanced shoot elongation and reduced branching (Smith, 1995; Ballaré, 1999; Franklin and Whitelam, 2005; Casal, 2012). Phytochrome B (phyB) is the major sensor contributing to R:FR responses, and loss of phyB function results in a plant that displays a phenotype similar to constitutive shade avoidance. It should be noted that actual shade avoidance is mediated by additional phytochromes and that the complete absence of functional phyB in the loss-of-function mutant may also result in a phenotype that does not exactly mirror shade avoidance. Loss of phyB function leads to reduced branching and altered expression of genes associated with hormone pathways and bud development in the axillary buds (Kebrom et al., 2006; Finlayson et al., 2010; Kebrom et al., 2010; Su et al., 2011). In Arabidopsis, phyB deficiency differentially affects the outgrowth of buds from specific positions in the rosette and thus demonstrates an important function in the regulation of correlative inhibition (Finlayson et al., 2010; Su et al., 2011), a process known to be influenced by auxin. Many aspects of auxin signaling are dependent on AUXIN RESISTANT1 (AXR1), which participates in activating the Skip-Cullin-F-box auxin signaling module (del Pozo et al., 2002). Reduced auxin signaling resulting from AXR1 deficiency enabled phyB-deficient plants to branch profusely and reduced correlative inhibition, thus establishing auxin signaling downstream of phyB action (Finlayson et al., 2010). Although a link between auxin signaling and phyB regulation of branching was demonstrated, the details of the interaction were not discovered.The relationship between auxin and shade avoidance responses has been investigated in some detail. Auxin signaling was implicated in shade avoidance responses mediated by ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 in young Arabidopsis seedlings (Steindler et al., 1999). Rapid changes in leaf development resulting from canopy shade were also shown to involve TRANSPORT INHIBITOR RESPONSE1-dependent auxin signaling (Carabelli et al., 2007). A link between auxin abundance and the response to the R:FR was demonstrated in Arabidopsis deficient for the TRP AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) auxin biosynthetic enzyme (Tao et al., 2008). Young wild-type seedlings respond to a decreased R:FR by increasing indole-3-acetic acid (IAA) biosynthesis, accumulating IAA, increasing hypocotyl and petiole elongation, and increasing leaf elevation. However, these responses are reduced in plants deficient in TAA1. Subsequent studies confirmed the importance of auxin in responses to the R:FR (Pierik et al., 2009; Kozuka et al., 2010; Keller et al., 2011), and also identified the auxin transporter PIN-FORMED3 as essential for hypocotyl elongation responses in young seedlings (Keuskamp et al., 2010). In addition to the roles of auxin abundance and transport in the process, auxin sensitivity has also been implicated in shade avoidance. Several auxin signaling genes are direct targets of the phytochrome signaling component PHYTOCHROME INTERACTING FACTOR5 (PIF5), and these genes are misregulated in Arabidopsis deficient in either PHYTOCHROME INTERACTING FACTOR4 (PIF4) or PIF5 (Hornitschek et al., 2012; Sun et al., 2013). Auxin-responsive hypocotyl elongation and auxin-induced gene expression were also reduced in young seedlings of the pif4pif5 double mutant (Hornitschek et al., 2012), which show defects in shade avoidance responses (Lorrain et al., 2008).Although some aspects of the regulation of branching are now understood, there are still many gaps in our knowledge of the process, especially as related to the regulation of branching by light signals. Because auxin is known to play a major role in regulating branch development, and because recent studies have implicated auxin in general shade avoidance responses and specifically in the regulation of branching by phyB, the hypothesis that auxin homeostasis, transport, and/or signaling may contribute to the hypobranching phenotype of phyB-deficient plants was generated and tested, using a variety of physiological and molecular approaches.     

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.     

Characterization of an infection structure-specific gene from the rust fungus Uromyces appendiculatus

Uredospores of the plant pathogen, Uromyces appendiculatus, infect leaves of the bean plant, Phaseolus vulgaris, through stomata. Physical stimuli provided by the stomate induce differentiation of the germ tube to form a series of infection structures involved in host colonization. Contact between the uredospores and the oil-collodion membranes induces formation of infection structures in the absence of the host. This report describes the characterization of a Uromyces gene, INF24, that is induced by the physical stimulus of an oil-collodion membrane. INF24 contains a 450-bp open reading frame which encodes a 16.4-kDa polypeptide. The N terminus of the INF24-encoded protein, and the C terminus of human single-stranded DNA-binding protein are both glycine-rich and share homology.