До питання визначення змістовної та видової характеристики категорій «інтеграція» та «правова інтеграція» у доктрині конституційного права

Розглядаються теоретико-методологічні, онтологічні, праксіологічні та аксіологічні аспекти визначення змістовної характеристики категорій «інтеграція» та «правової інтеграції». Окремо приділяється увага значенню даних категорій для розвитку сучасної доктрини конституційного права, що формується в умовах міждержавної інтеграції.Рассматриваются теоретико-методологические, онтологические, праксиологические и аксиологические аспекты определения содержательной характеристики категорий «интеграция» и «правовая интеграции». Отдельно уделяется внимание значению данных категорий для развития современной доктрины конституционного права, которая формируется в условиях межгосударственной интеграции.The article considers the theoretical, the methodological, the praxiological, the ontological and the axiological issues of defining meaningful description of such categories as «integration» and legal integration. It separately pays attention to the meaning of such categories for the developing of modern constitutional law doctrine, which is formed in conditions of interstate integration

Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation

Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema etal., 2004, Plant Mol. Biol. 54: 853-863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the β-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 8

Sucrose-mediated translational stalling involves a conserved ribosomal pocket

Within eukaryotes, 20-50% of the mRNAs contain short open reading frames (uORFs) located upstream of the main ORF. A significant fraction of these uORFs encode conserved peptides (CPuORFs) that regulate translation in response to specific metabolites. A well-studied example includes uORF2 of the plant growth inhibiting transcription factor bZIP11. Elevated intracellular sucrose levels lead to ribosome stalling at the stop codon of uORF2, thus reducing bZIP11 protein synthesis. Similar examples can be found in bacteria and animals, e.g. on the bacterial TnaC and human CDH1-NPN* ORFs that both induce stalling at the stop codon when in the presence of tryptophan and the drug-like molecule PF846, respectively. In this study, we affinity-purified in vitro translated sucrose-stalled wheat ribosomes translating bZIP11-uORF2 and determined the ribosomes’ structures using cryo-electron microscopy. This revealed density inside a pocket in the ribosomal exit tunnel of the plant Triticum aestivum, that colocalizes with the binding locations of tryptophan and PF846 in E. coli and humans, respectively. We suggest this density corresponds to sucrose. Tryptophan and PF846 mode-of-action was previously proposed to inhibit release factor binding or function. Mutation of the uORF2 stop codon shows that its presence is crucial for sucrose-induced stalling, suggesting that the stalling only manifests during termination and not elongation. Moreover, the structural similarities with tryptophan-induced stalled ribosomes near the peptidyl transferase center indicates that an analogous mechanism of inhibition of release factor function is likely. Our findings suggest a conserved mechanistic framework across different organisms, wherein specific molecules interact with the nascent peptide and ribosome to modulate protein synthesis

Expression patterns within the Arabidopsis C/S1 bZIP transcription factor network: availability of heterodimerization partners controls gene expression during stress response and development

Members of the Arabidopsis group C/S1 basic leucine zipper (bZIP) transcription factor (TF) network are proposed to implement transcriptional reprogramming of plant growth in response to energy deprivation and environmental stresses. The four group C and five group S1 members form specific heterodimers and are, therefore, considered to cooperate functionally. For example, the interplay of C/S1 bZIP TFs in regulating seed maturation genes was analyzed by expression studies and target gene regulation in both protoplasts and transgenic plants. The abundance of the heterodimerization partners significantly affects target gene transcription. Therefore, a detailed analysis of the developmental and stress related expression patterns was performed by comparing promoter: GUS and transcription data. The idea that the C/S1 network plays a role in the allocation of nutrients is supported by the defined and partially overlapping expression patterns in sink leaves, seeds and anthers. Accordingly, metabolic signals strongly affect bZIP expression on the transcriptional and/or post-transcriptional level. Sucrose induced repression of translation (SIRT) was demonstrated for all group S1 bZIPs. In particular, transcription of group S1 genes strongly responds to various abiotic stresses, such as salt (AtbZIP1) or cold (AtbZIP44). In summary, heterodimerization and expression data provide a basic framework to further determine the functional impact of the C/S1 network in regulating the plant energy balance and nutrient allocation

HISTONE DEACETYLASE 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion

Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis

Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis

Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought