The Arabidopsis leucine-rich repeat receptor kinase BIR3 negatively regulates BAK1 receptor complex formation and stabilizes BAK1

BAK1 is a co-receptor and positive regulator of multiple ligand-binding leucine-rich-repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana. BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand-binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand-binding receptors to prevent BAK1 receptor complex formation

Exploring natural genetic variation in tomato sucrose synthases on the basis of increased kinetic properties

Sucrose synthase (SuSy) is one key enzyme directly hydrolyzing sucrose to supply substrates for plant metabolism, and is considered to be a biomarker for plant sink strength. Improvement in plant sink strength could lead to enhanced plant growth and yield. Cultivated tomatoes are known to have a narrow genetic diversity, which hampers further breeding for novel and improved traits in new cultivars. In this study, we observed limited genetic variation in SuSy1, SuSy3 and SuSy4 in 53 accessions of cultivated tomato and landraces, but identified a wealth of genetic diversity in 32 accessions of related wild species. The variation in the deduced amino acid sequences was grouped into 23, 22, and 17 distinct haplotypes for SuSy1/3/4, respectively. Strikingly, all known substrate binding sites were highly conserved, as well as most of the phosphorylation sites except in SuSy1. Two SuSy1 and three SuSy3 protein variants were heterologously expressed to study the effect of the amino acid changes on enzyme kinetic properties, i.e. maximal sucrose hydrolyzing capacity (Vmax), affinity for sucrose (Km), and catalytic efficiency (Vmax/Km) at 25°C and 16°C. SuSy1-haplotype#3 containing phosphorylation site Ser-16 did not have an improvement in the kinetic properties compared to the reference SuSy1-haplotype#1 containing Arg-16. Meanwhile SuSy3-haplotype#9 from a wild accession, containing four amino acid changes S53A, S106I, E727D and K741E, showed an increase in Vmax/Km at 16°C compared to the reference SuSy3-haplotype#1. This study demonstrates that SuSy kinetic properties can be enhanced by exploiting natural variation, and the potential of this enzyme to improve sucrose metabolism and eventually sink strength in planta.</p

Модель интерпретации литературного текста

The article discloses the main text categories and defines their interweaving with the most important stylistics’ aspects, forming a detailed literary text interpretation model. The main categories of a literary text, which will be analyzed in the article, are: conceptuality, discreteness, implicitness, integration, modality and personality/impersonality of the author’s image. And as the most colorful semasiological expressive means, the figures of qualification (metonymy, metaphor and irony), opposition (antithesis and oxymoron) and some figures of inequality (pun and zeugma) will be described below.Стаття розкриває основні текстові категорії і визначає їх переплетіння з найважливішими стилістичними аспектами, формируючи модель детальної інтерпретації літературного тексту. Основні текстові категорії, що будуть проаналізовані в даній статті, наступні: концептуальність, дискретність, імпліцитність, інтеграція, модальність й індивідуальність чи безособистість наявності автора. І, як найяскравіші семасіологічні засоби експресії, фігури якісності (метонімія, метафора, іронія), фігури опозиції (оксюморон й антитеза) та деякі фігури нерівності (зевгма і каламбур) будуть описані нижче.Статья раскрывает основные текстовые категории и рассматривает их переплетение с важнейшими стилистическими аспектами, формируя модель детальной интерпретации литературного текста. Основные текстовые категории, которые проанализированы в данной статье, следующие: концептуальность, дискретность, имплицитность, интеграция, модальность и индивидуальность или безличность присутствия автора

Structural and functional mechanisms of Desulfovibrio vulgaris cytochrome c3 revealed by site specific mutagenesis

The structural basis for the redox properties of the tetrahaem Desulfovibrio vulgaris cytochrome c3, has been investigated by site specific mutagenesis. Thermodynamic and structural characterisation of mutated cytochromes revealed the functional role of Lys45 and Thr24 in the wild type protein. Mutation of Lys45, located in the vicinity of the propionates of haem I, by the uncharged residues Thr and Gin, allowed to conclude that Lys45 controls the pKa of such propionate groups and confirms the involvement of this residue in the redox-Bohr effect ~. More recently, the replacement of Thr24, located in the vicinity ofhaem III, by Val showed how crucial Thr24 is for the stabilisation of the oxidised form of the protein. This single mutation increases the microscopic redox potential ofhaem III by 106 mV. This mutation also destabilizes the concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild type protein. Therefore, Thr24 is responsible for the balance of the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer 2

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential

Flavodoxin II from Azotobacter vinelandii is a “long-chain” flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel β-sheet flanked on either side by α-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 310 helix preceding the start of the α3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular