Active BR signalling adjusts the subcellular localisation of BES1/HSP90 complex formation.

Heat shock proteins 90 (HSP90) are essential and play critical roles in the adaptation of organisms to diverse stimuli. In plants, HSP90 are involved in auxin, jasmonate and brassinosteroid (BR) signalling pathways. The BR-promoted activation of the BES1 transcription factor regulates BR-responsive genes. Using genetic, physiological, fluorescence live cell imaging, molecular and biochemical approaches, such as phenotypic analysis, co-immunoprecipitation assay, yeast-two hybrid and Bimolecular fluorescence complementation (BiFC), we studied complex formation between BES1 and HSP90 under control conditions and active BR signalling. Further, we determined the effect of the pharmacological inhibition of HSP90 ATPase activity on hypocotyl elongation of bes1-D mutant. We determined that HSP90 interact with BES1 in the nucleus and in the cytoplasm. During active BR signalling, nuclear complexes were absent while cytoplasmic HSP90/BES1 complexes were prominent. Our results showed that the hypocotyl length of bes1-D mutants was highly reduced when HSP90 was challenged by the geldanamycin (GDA) inhibitor of the ATPase activity of HSP90. Active BR signalling could not rescue the GDA effect on the hypocotyl elongation of bes1-D. Our results reveal that the constitutively active BES1 in the bes1-D mutant is hypersensitive to GDA. The interaction of HSP90 with BES1 argues that HSP90 facilitate the nuclear metastable conformation of BES1 to regulate BR-dependent gene expression, and our data show that HSP90 assist in the compartmentalised cycle of BES1 during active BR signalling.

The role of Arabidopsis thaliana RASD1 gene in ABA-dependent abiotic stress response.

Abiotic stress is one of the key parameters affecting plant productivity. Drought and soil salinity, in particular, challenge plants to activate various response mechanisms to withstand these adverse growth conditions. While the molecular events that take place are complex and to a large extent unclear, the plant hormone abscisic acid (ABA) is considered a major player in mediating the adaptation of plants to stress. Here we report the identification of an ABA-insensitive mutant from Arabidopsis thaliana. A combination of molecular, genetic and physiology approaches were implemented, to characterise the AtRASD1 locus (RESPONSIVENESS TO ABA SALT AND DROUGHT 1) and to investigate its role in plant development. RASD1 is expressed predominantly in the vascular system of A. thaliana and encodes a peptide of unknown function with no similarity to any known sequence to date. The protein is localised in the nucleus and the cytoplasm, and RASD1-impaired plants are drought-intolerant and insensitive to exogenous ABA and NaCl during germination and root growth. Our data indicate that RASD1 is involved in ABA-dependent signal transduction pathways and therefore in enabling plants to activate response mechanisms related to seed germination and abiotic stress.

Self-reported autonomic symptoms in Parkinson's disease: properties of the SCOPA-AUT scale.

BACKGROUND AND AIM: Autonomic symptoms in Parkinson's disease (PD) are very common and contribute to the severity of patient's disability. We evaluated the occurrence of autonomic symptoms in Greek patients with PD utilizing the Scales for Outcomes in Parkinson's Disease-Autonomic questionnaire (SCOPA-AUT), a specific 23-item self-completed questionnaire for the assessment of autonomic dysfunction in patients with PD. SUBJECTS AND METHODS: One hundred and sixty-one PD patients and forty matched controls were enrolled in the study. Clinical assessment was performed with the Hoehn and Yahr scale. Patients completed a demographic questionnaire, the Non-Motor Symptoms Questionnaire (NMSQuest), the Parkinson's Disease Questionnaire (PDQ-39) and the SCOPA-AUT scale which was properly translated into Greek and validated for the study. RESULTS: SCOPA-AUT scale showed a good reliability profile and  correlated well with other measures for non-motor symptoms and health-related quality measures in PD patients. PD patients scored higher than controls in the total SCOPA -AUT score (mean score 11.9 versus 6.4). Patients reported problems in many items of the SCOPA-AUT, but the most common autonomic symptoms emerged in the Urinary and the Gastrointestinal domains. Especially sialorrhea, constipation, straining for defecation, incontinence and nocturia differentiated patients from controls. Furthermore, mean total SCOPA-AUT score correlated with duration and severity of the disease. CONCLUSION: Autonomic symptoms in PD are too important to remain undetected. By incorporating into everyday practice the use of suitable and reliable questionnaires, physicians will be able to adequately detect and manage these symptoms. Hippokratia 2016, 20(2):115-120.

Evaluation of non-motor symptoms in Parkinson's Disease: An underestimated necessity.

BACKGROUND AND AIM: Νon-motor symptoms in Parkinson's disease (PD) are very common and contribute to the severity of patient's disability. We evaluated the frequency of nonmotor symptoms in patients with PD and we explored the influence of disease characteristics on the presence of these symptoms. PATIENTS AND METHODS: One hundred sixty six patients and sixty six matched controls were enrolled in the study. The Non-Motor Symptoms Questionnaire (NMSQuest), a 30-item self-completed questionnaire, was used for the evaluation of nonmotor symptoms. RESULTS: Non-motor symptoms were more common in PD patients than controls. Mean ± SD NMSQuest score was 6.76 ± 4.22 in PD patients and 5.44 ± 4.45 in controls (p=0.035). The more common non-motor symptoms in PD patients were urinary urgency (54.3%), nocturia (51.8%), constipation (45.7%) and sadness (42.1%). There was a correlation between NMSQuest score and severity of the disease. CONCLUSION: Non-motor symptoms in PD are too important to remain undetected. By incorporating into every day practice the use of suitable, reliable questionnaires, we will be able to facilitate detection and management of these symptoms.

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system.

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Genomic organization of hsp90 gene family in Arabidopsis.

We have isolated six members of the hsp90 gene family from Arabidopsis thaliana. Three genes designated hsp81.2, 81.3 and 81.4 are clustered within a 15 kb genomic region while two of these are 1.5 kb apart in a head-to-head orientation. The deduced amino acid sequence shows that the members can be divided into two types. The hsp81.1, 81.2, 81.3 and 81.4 genes comprise the cytosolic hsp90 type having few introns. However, the hsp88.1 and 89.1 genes comprising the organelle type are composed of 18 or 19 introns. Sequence comparison showed there is high homology among the cytosolic members while there is less homology among the organelle members. The expression of the hsp90 genes and mRNA accumulation in plants and calli is very low at control temperatures and is strongly induced by heat-shock. Arsenite stress strongly stimulates the expression of this gene family.

The beta-tubulin genes of Drosophila auraria are arranged in a cluster.

When the beta 1-, beta 2- and beta 3-tubulin-specific DNAs from Drosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes of Drosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a lambda EMBL3 genomic library of D. auraria, and they all contain beta-tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.