Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.     

Purification of the 22000- and 20000-mol.wt. forms of human somatotropin and characterization of their binding to liver and mammary binding sites.

Quantitative data concerning the binding of 22000-mol.wt. human somatotropin and its 20000-mol.wt. variant are described using pregnant-rabbit liver and mammary-gland receptors. The purification and the complete chemical characterization of both human somatotropin and its 20000-mol.wt. variant is also presented. Contamination of the 20000-mol.wt.-variant preparation by 22000-mol.wt. hormone was found to be 0.5% by weight as measured in radioimmunoassay using monoclonal antibody. Labelling of human somatotropin and its 20000-mol.wt. variant using the Iodogen method is described as well as the characterization of the binding to pregnant-rabbit liver and mammary-gland receptor preparations. The maximum binding capacity of the 125I-labelled human somatotropin was between 50 and 60% to liver particulate receptor, whereas that of the 20000-mol.wt. variant was 30%. The specificity of binding of both forms to rabbit hepatic and mammary-gland receptor was found to be similar for both proteins in the same system. The affinity constants and capacity were respectively 0.7 X 10(10)M-1 and 815 fmol/mg of protein for human somatotropin and 0.6 X 10(10)M-1 and 1.250 fmol/mg of protein for the 20000-mol.wt. variant. These data suggest that both proteins behave as partial agonists to the receptors studied.     

Coronin 1B supports RhoA signaling at cell-cell junctions through Myosin II

Non-muscle myosin II (NMII) motor proteins are responsible for generating contractile forces inside eukaryotic cells. There is also a growing interest in the capacity for these motor proteins to influence cell signaling through scaffolding, especially in the context of RhoA GTPase signaling. We previously showed that NMIIA accumulation and stability within specific regions of the cell cortex, such as the zonula adherens (ZA), allows the formation of a stable RhoA signaling zone. Now we demonstrate a key role for Coronin 1B in maintaining this junctional pool of NMIIA, as depletion of Coronin 1B significantly compromised myosin accumulation and stability at junctions. The loss of junctional NMIIA, upon Coronin 1B knockdown, perturbed RhoA signaling due to enhanced junctional recruitment of the RhoA antagonist, p190B Rho GAP. This effect was blocked by the expression of phosphomimetic MRLC-DD, thus reinforcing the central role of NMII in regulating RhoA signaling.     

Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.     

Identification of QTLs associated with agronomic performance under nitrogen-deficient conditions using chromosome segment substitution lines of a wild rice relative, <Emphasis Type="Italic">Oryza rufipogon</Emphasis>

Improved root system architecture can enhance agronomic performance by increasing water and nitrogen (N) acquisition efficiency. However, little is known about interaction between root system architecture and agronomic performance under field environments. To gain a better understanding about the genetic basis of these relationships, we evaluated a set of chromosome segment substitution lines (CSSLs) derived from crosses between a tropical japonica rice cultivar ‘Curinga’ and a wild species Oryza rufipogon accession IRGC105491. Root system architectural traits were investigated using the CSSLs at 40 days old seedlings using the root basket method under hydroponic conditions, and agronomic performances were also tested under field conditions with different N treatments. Agronomic performances were computed as the ratio of a trait value under low to high N treatments, including grain yield and biomass yield as nitrogen-deficiency tolerance (NDT) traits. Root architecture and NDT trait QTLs were mapped using 238 SNP marker loci. A total of 13 QTLs for root system architectural, NDT and morpho-physiological traits were identified on chromosomes 1, 3, 4, 5, 7, 8, 9, 10 and 12. Interestingly, a QTL for deeper root number was identified the region of SNP markers between id1012330 and id1021697 on chromosome 1 under hydroponic conditions overlapped with a QTL for NDT trait of relative grain yield (qRGY1). These results suggest that deeper root trait is helpful to maintain grain yield under nitrogen-deficient conditions. The QTL associated root architecture could potentially be used in future rice-breeding efforts to increase agronomic performance under nitrogen-deficient conditions.     

Ring‐opening of azetidiniums by nucleophiles. Synthesis of polysubstituted linear amines

This microreview focuses on the nucleophilic ring‐opening of azetidiniums presenting various substitution patterns at C2, C3, and C4. In most cases, the nucleophilic ring‐opening occurred in a stereoselective and regioselective fashion producing functionalized linear amines. Experimental selectivities associated with Density Functional Theory (DFT) calculations have allowed a better understanding of the parameters governing the regioselectivities.