Immunodetection of protein glycoforms encoded by two independent genes of the self-incompatibility multigene family of brassica

Glycoprotein products of two highly homologous Brassica S gene family members were studied: SLSG (S locus-specific glycoprotein), product of an SLG gene at the S locus, and SLR1 (S locus-related) protein, product of the SLR1 gene, a gene unlinked to the S locus. A polyclonal antibody directed against a trpE-SLR1 fusion protein facilitated study of the SLR1 protein. SLR1 protein was detected in a number of crucifer species. No variation in the level of this protein was found between self-compatible and self-incompatible plants. Both SLSG and SLR1 protein occurred as glycoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Each glycoform had several charge forms, indicated by elution patterns from a high performance liquid chromatography cation exchange column and behavior on two-dimensional gels. Deglycosylation of both SLSG and SLR1 protein caused loss of the glycoforms, which apparently arose from differences in glycosylation. Consistent with their apparent similar post-translational processing, immunolocalization showed that SLR1 protein, like SLSG, accumulated in the stigma papillae cell walls. Thus, both SLSG and SLR1 protein are present at the site of pollen-stigma interaction.     

MASS FRAGMENTOGRAPHY OF PHENYLETHYLAMINE, m- AND p-TYRAMINE AND RELATED AMINES IN PLASMA, CEREBROSPINAL FLUID, URINE, AND BRAIN

—A mass fragmentographic method for the assay of phenylethylamine (PEA) and a number of related amines in several biological materials is described. The gas chromatographic column employed for this analysis is a 12ft 1/8 in. o.d. steel column packed with 0.5% OV₂₂+ 2% SE54 + 1% OV₂₁₀ coated on 80/100 mesh chromosorb W (HP). The mass spectral characteristics of these amines are illustrated, compared, and discussed. Of the various monoamines which could be measured, only PEA, m- and p-tyramine were detected in measurable quantities. Phenylethanolamine and p-octopamine were found in trace amounts in urine, plasma, cerebrosponal fluid, and rat brain. No diurnal variation in the urinary excretion of PEA, m- and p-tyramine was observed. Plasma concentration of PEA or p-tyramine did not significantly change 1 h after eating a breakfast. Furthermore, consuming 200 g of Cadbury milk chocolate containing about 1 mg of PEA, 0.1 mg of phenylethanolamine and 10 mg of p-tyramine did not significantly alter urine excretions of these three amines. In the brain, as has been reported by others, we found that PEA and p-tyramine are not evenly distributed and that the highest concentrations are found in the hypothalamus and caudate. From the results obtained we concluded that PEA, m- and p-tyramine are probably produced from endogenous sources and that the direct contribution of diet to their urine excretion is small.     

A superfamily of S locus-related sequences in Arabidopsis: diverse structures and expression patterns.

Six sequences that are closely related to the S gene family of the largely self-incompatible Brassica species have been identified in self-fertilizing Arabidopsis. The sequences define four genomic regions that map to chromosomes 1 and 3. Of the four functional genes identified, only the previously reported Arabidopsis AtS1 gene was expressed specifically in papillar cells and may function in pollination. The remaining three genes, including two novel genes designated ARK2 and ARK3, encode putative receptor-like serine/threonine protein kinases that are expressed predominantly in vegetative tissues. ARK2 promoter activity was detected exclusively in above-ground tissues, specifically in cotyledons, leaves, and sepals, in correlation with the maturation of these structures. ARK3 promoter activity was detected in roots as well as above-ground tissues but was limited to small groups of cells in the root-hypocotyl transition zone and at the base of lateral roots, axillary buds, and pedicels. The nonoverlapping patterns of expression of the ARK genes and the divergence of their sequences, particularly in their predicted extracellular domains, suggest that these genes perform nonredundant functions in specific aspects of development or growth of the plant body.     

S locus genes and the evolution of self-fertility in Arabidopsis thaliana

Loss of self-incompatibility (SI) in Arabidopsis thaliana was accompanied by inactivation of genes required for SI, including S-LOCUS RECEPTOR KINASE (SRK) and S-LOCUS CYSTEINE-RICH PROTEIN (SCR), coadapted genes that constitute the SI specificity-determining S haplotype. Arabidopsis accessions are polymorphic for PsiSRK and PsiSCR, but it is unknown if the species harbors structurally different S haplotypes, either representing relics of ancestral functional and structurally heteromorphic S haplotypes or resulting from decay concomitant with or subsequent to the switch to self-fertility. We cloned and sequenced the S haplotype from C24, in which self-fertility is due solely to S locus inactivation, and show that this haplotype was produced by interhaplotypic recombination. The highly divergent organization and sequence of the C24 and Columbia-0 (Col-0) S haplotypes demonstrate that the A. thaliana S locus underwent extensive structural remodeling in conjunction with a relaxation of selective pressures that once preserved the integrity and linkage of coadapted SRK and SCR alleles. Additional evidence for this process was obtained by assaying 70 accessions for the presence of C24- or Col-0-specific sequences. Furthermore, analysis of SRK and SCR polymorphisms in these accessions argues against the occurrence of a selective sweep of a particular allele of SCR, as previously proposed.     

Persistence of native and exotic plants 10 years after prairie reconstruction

Prairie reconstructions are a critical component of preservation of the imperiled tallgrass prairie ecosystem in the Midwestern United States. Sustainability of this endeavor depends on establishment of persistent cover of planted native species and resistance to noxious weeds. The goal of this study was to understand the influence of early reconstruction practices on long‐term outcomes. Twelve replicates of three planting methods (dormant‐season broadcast, growing‐season broadcast, and growing‐season drill) and three seed mix richness levels (10, 20, or 34 species), fully crossed in a completely randomized design were planted in 2005 on nine former agricultural fields located in Iowa and Minnesota. Cover by species was estimated in 2005–2007, 2010, and 2015. In 2015, cover of planted species, native nonplanted species, and exotic species were similar to those recorded in 2010. Cover of the noxious weed Cirsium arvense had also declined by an average of 49% without herbicide from a peak in 2007 to low stable levels from 2010 to 2015. Richness of planted forbs, on the other hand, were still increasing in high‐richness broadcast treatments (e.g. 17–59% increase 2010–1015 in Minnesota). Two results in 2015 are reasons for concern: cover of planted species is only slightly over 50% in both Minnesota and Iowa, though with forbs still increasing, this may improve; and the cool‐season exotic grasses Poa pratensis and Bromus inermis are increasing at both Minnesota and Iowa sites. Control of these invasive grasses will be necessary, but care will be needed to avoid negative impacts of control methods on natives.     

Reanalysis of the Rituximab in ANCA-Associated Vasculitis trial identifies granulocyte subsets as a novel early marker of successful treatment

IntroductionIn the present study, we sought to identify markers in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) that distinguish those achieving remission at 6 months following rituximab or cyclophosphamide treatment from those for whom treatment failed in the Rituximab in ANCA-Associated Vasculitis (RAVE) trial.MethodsClinical and flow cytometry data from the RAVE trial were downloaded from the Immunology Database and Analysis Portal and Immune Tolerance Network TrialShare public repositories. Flow cytometry data were analyzed using validated automated gating and joined with clinical data. Lymphocyte and granulocyte populations were measured in patients who achieved or failed to achieve remission.ResultsThere was no difference in lymphocyte subsets and treatment outcome with either treatment. We defined a Granularity Index (GI) that measures the difference between the percentage of hypergranular and hypogranular granulocytes. We found that rituximab-treated patients who achieved remission had a significantly higher GI at baseline than those who did not (p = 0.0085) and that this pattern was reversed in cyclophosphamide-treated patients (p = 0.037). We defined optimal cutoff values of the GI using the Youden index. Cyclophosphamide was superior to rituximab in inducing remission in patients with GI below −9.25 % (67 % vs. 30 %, respectively; p = 0.033), whereas rituximab was superior to cyclophosphamide for patients with GI greater than 47.6 % (83 % vs. 33 %, respectively; p = 0.0002).ConclusionsWe identified distinct subsets of granulocytes found at baseline in patients with AAV that predicted whether they were more likely to achieve remission with cyclophosphamide or rituximab. Profiling patients on the basis of the GI may lead to more successful trials and therapeutic courses in AAV.

Trial registration

ClinicalTrials.gov identifier (for original study from which data were obtained): {"type":"clinical-trial","attrs":{"text":"NCT00104299","term_id":"NCT00104299"}}NCT00104299. Date of registration: 24 February 2005.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0778-z) contains supplementary material, which is available to authorized users.     

Ligand-Mediated cis-Inhibition of Receptor Signaling in the Self-Incompatibility Response of the Brassicaceae

The inhibition of self-pollination in self-incompatible Brassicaceae is based on allele-specific trans-activation of the highly polymorphic S-locus receptor kinase (SRK), which is displayed at the surface of stigma epidermal cells, by its even more polymorphic pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. In an attempt to achieve constitutive activation of SRK and thus facilitate analysis of self-incompatibility (SI) signaling, we coexpressed an Arabidopsis lyrata SCR variant with its cognate SRK receptor in the stigma epidermal cells of Arabidopsis (Arabidopsis thaliana) plants belonging to the C24 accession, in which expression of SRK and SCR had been shown to exhibit a robust SI response. Contrary to expectation, however, coexpression of SRK and SCR was found to inhibit SRK-mediated signaling and to disrupt the SI response. This phenomenon, called cis-inhibition, is well documented in metazoans but has not as yet been reported for plant receptor kinases. We demonstrate that cis-inhibition of SRK, like its trans-activation, is based on allele-specific interaction between receptor and ligand. We also show that stigma-expressed SCR causes entrapment of its SRK receptor in the endoplasmic reticulum, thus disrupting the proper targeting of SRK to the plasma membrane, where the receptor would be available for productive interaction with its pollen coat-derived SCR ligand. Although based on an artificial cis-inhibition system, the results suggest novel strategies of pollination control for the generation of hybrid cultivars and large-scale seed production from hybrid plants in Brassicaceae seed crops and, more generally, for inhibiting cell surface receptor function and manipulating signaling pathways in plants.Ligand receptor signaling plays important roles in cell-cell communication between neighboring cells in a variety of developmental and physiological processes. This communication typically relies on the interaction of transmembrane receptors displayed on the surface of signal-receiving cells with their cognate ligands derived from signal-sending neighboring cells, which, in turn, leads to the activation of receptor-mediated signaling cascades that modify intracellular activities of the signal-receiving cell. Such is the case with communication between pollen grains and stigma epidermal cells, a process that has an important role in directing reproductive success and determining pollination modes (i.e. selfing or outcrossing) in the Brassicaceae. In this family, outcrossing is enforced by self-incompatibility (SI), a mechanism controlled by haplotypes of the S locus, by which the stigma epidermal cells of a plant recognize and reject self pollen grains (i.e. those derived from the same flower, the same plant, or plants expressing the same S-locus haplotype), thus preventing self-pollination, while allowing the growth of tubes from nonself pollen grains (i.e. those derived from plants expressing a different S-locus haplotype; Nasrallah and Nasrallah, 2014a). Inhibition of self pollen in the SI response is initiated by allele-specific interaction between two highly polymorphic proteins encoded at the S locus: the S-locus receptor kinase (SRK), which is localized at the plasma membrane of stigma epidermal cells (Stein et al., 1991, 1996), and its ligand, the S-locus cysteine-rich protein (SCR), which accumulates in the pollen coat and diffuses onto the stigma surface upon pollen-stigma contact (Schopfer et al., 1999; Takayama et al., 2000; Shiba et al., 2001). The interaction of the SRK extracellular domain, or S domain, with its cognate SCR ligand is thought to activate downstream signaling cascades in stigma epidermal cells, which lead to inhibition of pollen germination on the stigma surface and/or pollen tube penetration through the stigma epidermal cell wall. The SRK and SCR genes are the primary determinants of the transition between the outcrossing and selfing modes of mating in the Brassicaceae, as demonstrated by the observation that transformation of SRK and SCR gene pairs derived from self-incompatible Arabidopsis lyrata or Capsella grandiflora restored SI in several accessions of the normally self-fertile Arabidopsis (Arabidopsis thaliana; Nasrallah et al., 2002, 2004; Boggs et al., 2009).Tight regulation of the SI response is critical for ensuring reproductive success in self-incompatible plants. Activation of SRK signaling must be triggered only by pollen-derived cognate SCR ligand upon interaction of stigma epidermal cells with self pollen grains, because constitutive activation of SI signaling in stigma epidermal cells would result in inhibition of nonself as well as self pollen grains and would result in female sterility. This adverse outcome is averted by tight regulation of the SCR gene, which is expressed exclusively in the anthers of self-incompatible plants and whose protein products are localized exclusively in the pollen coat (Schopfer and Nasrallah, 2000; Shiba et al., 2001). For experimental studies of SI, however, constitutive activation of SRK-mediated signaling in stigma epidermal cells would be useful, as it might provide a convenient means of identifying components of the poorly understood SRK-mediated signaling pathway.A reaction that resembles a constitutive SI response, in which stigma epidermal cells inhibit both self and nonself pollen grains, has been obtained by manual application of purified recombinant SCR proteins produced in bacteria (Kachroo et al., 2001; Chookajorn et al., 2004) or synthetic SCR (Takayama et al., 2001) to stigmas that express their cognate SRK receptors. Unlike the highly localized activation induced by pollen-derived SCR at the site of pollen-stigma contact, treatment of the stigma surface with SCR protein can clearly cause global activation of SRK in most, if not all, epidermal cells of a stigma. However, treating stigmas in the numbers required for analysis of SRK signaling is extremely laborious, can damage stigmas, and produces inconsistent results. Therefore, a method that circumvents these problems would be advantageous. In metazoans, constitutive activation of receptor kinases has been shown to result not only from receptor mutations that cause constitutive kinase activity (Webster and Donoghue, 1996; Hirota et al., 1998) and mutations in signaling components that cause ligand-independent activation of downstream cascades (Wang et al., 2012, 2014; Roberts et al., 2013; Han, 2014), but also from ectopic expression of ligands within the same cells as their receptors, as occurs in several pathological conditions (Sporn and Roberts, 1985; Castellano et al., 2006; Krasagakis et al., 2011).Accordingly, an attempt was made to generate Arabidopsis plants having a stable constitutive stigma SI response by coexpressing an SRK variant and its cognate SCR in stigma epidermal cells, which should, in principle, constitutively activate the SI response in these cells. This report shows that, while pollen-derived SCR trans-activates the SRK-mediated SI response, stigma-expressed SCR inhibits the activity of its cognate SRK by causing entrapment of the receptor in the endoplasmic reticulum (ER). This phenomenon is similar in its outcome to the ligand-mediated cis-inhibition phenomenon that had previously been observed in metazoans for some signaling systems that use transmembrane proteins as ligands (Yaron and Sprinzak, 2012) but had not been described for plant receptor-like kinases. The results suggest novel strategies for control of receptor-like kinase activity and manipulation of signaling pathways in plants and for pollination control in hybrid breeding programs and seed production from hybrid plants in the Brassicaceae.