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It has been possible to use the yeast artificial chromosome-based physical map of chromosome 4 to construct a sequence-ready map of cosmid and bacterial artificial chromosome clones covering a 1.9-megabase (Mb) contiguous region of the plant Arabidopsis thaliana. A region between markers COP9 and G3845 on the long arm of chromosome 4 from the ecotype Columbia was chosen for sequencing. It was found that there is a consistently high gene density over an extended contiguous region, with 389 predicted genes in 1.87 Mb.

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 (RESPONSIVENESSTOABASALTANDDROUGHT1) 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. [ABSTRACT FROM AUTHOR]

Background and aim: Non-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. [ABSTRACT FROM AUTHOR]

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana , such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 ( aprf1 ) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Polyamine oxidases (PAOs) have been correlated with numerous physiological and developmental processes, as well as responses to biotic and abiotic stress conditions. Their transcriptional regulation is driven by signals generated by various developmental and environmental cues, including phytohormones. However, the inductive mechanism(s) of the corresponding genes remains elusive. Out of the five previously characterized Arabidopsis PAO genes, none of their regulatory sequences have been analyzed to date. In this study, a GUS reporter-aided promoter deletion approach was used to investigate the transcriptional regulation of AtPAO3 during normal growth and development as well as under various inductive environments. AtPAO3 contains an upstream open reading frame (uORF) and a short inter-cistronic sequence, while the integrity of both appears to be crucial for the proper regulation of gene expression. The full-length promoter contains several cis -acting elements that regulate the tissue-specific expression of AtPAO3 during normal growth and development. Furthermore, a number of TFBS that are involved in gene induction under various abiotic stress conditions display an additive effect on gene expression. Taken together, our data indicate that the transcription of AtPAO3 is regulated by multiple environmental factors, which probably work alongside hormonal signals and shed light on the fine-tuning mechanisms of PAO regulation.

Circuitries of signaling pathways integrate distinct hormonal and environmental signals, and influence development in plants. While a crosstalk between brassinosteroid (BR) and gibberellin (GA) signaling pathways has recently been established, little is known about other components engaged in the integration of the two pathways. Here, we provide supporting evidence for the role of HSP90 (HEAT SHOCK PROTEIN 90) in regulating the interplay of the GA and BR signaling pathways to control hypocotyl elongation of etiolated seedlings in Arabidopsis. Both pharmacological and genetic depletion of HSP90 alter the expression of GA biosynthesis and catabolism genes. Major components of the GA pathway, like RGA (REPRESSOR of ga1-3 ) and GAI (GA-INSENSITIVE) DELLA proteins, have been identified as physically interacting with HSP90. Interestingly, GA-promoted DELLA degradation depends on the ATPase activity of HSP90, and inhibition of HSP90 function stabilizes the DELLA/BZR1 (BRASSINAZOLE-RESISTANT 1) complex, modifying the expression of downstream transcriptional targets. Our results collectively reveal that HSP90, through physical interactions with DELLA proteins and BZR1, modulates DELLA abundance and regulates the expression of BZR1-dependent transcriptional targets to promote plant growth.

The activation of BRASSINOSTEROID INSENSITIVE1 (BRI1) and its association with the BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) are key steps for the initiation of the BR signaling cascade mediating hypocotyl elongation. Heat shock protein 90 (HSP90) is crucial in the regulation of signaling processes and the activation of hormonal receptors. We report that HSP90 is required for the maintenance of the BRI1 receptor at the plasma membrane (PM) and its association with the BAK1 co-receptor during BL-ligand stimulation. HSP90 mediates BR perception and signal transduction through physical interactions with BRI1 and BAK1, while chaperone depletion resulted in lower levels of BRI1 and BAK1 receptors at the PM and affected the spatial partitioning and organization of BRI1/BAK1 heterocomplexes at the PM. The BRI1/BAK1 interaction relies on the HSP90-dependent activation of the kinase domain of BRI1 which leads to the confinement of the spatial dynamics of the membrane resident BRI1 and the attenuation of the downstream signaling. This is evident by the impaired activation and transcriptional activity of BRI1 EMS SUPPRESSOR 1 (BES1) upon HSP90 depletion. Our findings provide conclusive evidence that further expands the commitment of HSP90 in BR signaling through the HSP90-mediated activation of BRI1 in the control of the BR signaling cascade in plants.

WD40-repeat-containing proteins (WDRs) are highly abundant in all eukaryotes. Several have been implicated as subunits of multi-protein CRL E3 ligase complexes that regulate ubiquitination mediated protein degradation and thus various cellular and developmental processes. Impairment of the WDR protein ULCS1 from Arabidopsis causes pleiotropic phenotypes during plant development, including reduced lignification, anther indehiscence, and sterility. Here we show that RNAi-mediated downregulation of ULCS1 results in a fast-growing phenotype during vegetative development. Due to accelerated growth, ulcs1i mutants reach their vegetative to reproductive transition point earlier than WT plants. However, their comparable germination rate and their similar number of secondary branches and rosette leaves at bolting indicate that ulcs1i is not an early flowering time mutant. GUS staining of progeny, obtained from crosses between ulcs1i and CYCB1::GUS plants, revealed an increased number of mitotic cell divisions in the root meristems of ulcs1i compared to WT. Immunolabeling of homogalacturonans (HGAs) epitopes showed significant fluorescent signal differences at the cell walls and the mucilage of the seeds between ulcs1i and WT. Furthermore, we demonstrate that ULCS1 interacts with the UBA-like protein in a yeast two-hybrid assay, suggesting a direct or indirect physical coupling of these proteins in Arabidopsis.

Auxin homeostasis and signaling affect a broad range of developmental processes in plants. The interplay between HSP90 and auxin signaling is channeled through the chaperoning capacity of the HSP90 on the TIR1 auxin receptor. The sophisticated buffering capacity of the HSP90 system through the interaction with diverse signaling protein components drastically shapes genetic circuitries regulating various developmental aspects. However, the elegant networking capacity of HSP90 in the global regulation of auxin response and homeostasis has not been appreciated. Arabidopsis hsp90 mutants were screened for gravity response. Phenotypic analysis of root meristems and cotyledon veins was performed. PIN1 localization in hsp90 mutants was determined. Our results showed that HSP90 affected the asymmetrical distribution of PIN1 in plasma membranes and influenced its expression in prompt cell niches. Depletion of HSP90 distorted polar distribution of auxin, as the acropetal auxin transport was highly affected, leading to impaired root gravitropism and lateral root formation. The essential role of the HSP90 in auxin homeostasis was profoundly evident from early development, as HSP90 depletion affected embryo development and the pattern formation of veins in cotyledons. Our data suggest that the HSP90-mediated distribution of PIN1 modulates auxin distribution and thereby auxin signaling to properly promote plant development., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

The objective of this work is to present a methodology for the selection of nanoparticles such as liposomes to be used as acoustic probes for the detection of very low concentrations of DNA. Liposomes, applied in the past as mass amplifiers and detected through frequency measurement, are employed in the current work as probes for energy-dissipation enhancement. Because the dissipation signal is related to the structure of the sensed nanoentity, a systematic investigation of the geometrical features of the liposome/DNA complex was carried out. We introduce the parameter of dissipation capacity by which several sizes of liposome and DNA structures were compared with respect to their ability to dissipate acoustic energy at the level of a single molecule/particle. Optimized 200 nm liposomes anchored to a dsDNA chain led to an improvement of the limit of detection (LoD) by 3 orders of magnitude when compared to direct DNA detection, with the new LoD being 1.2 fmol (or 26 fg/μL or 2 pM). Dissipation monitoring was also shown to be 8 times more sensitive than the corresponding frequency response. The high versatility of this new methodology is demonstrated in the detection of genetic biomarkers down to 1-2 target copies in real samples such as blood. This study offers new prospects in acoustic detection with potential use in real-world diagnostics.

Stomatal ontogenesis, patterning, and function are hallmarks of environmental plant adaptation, especially to conditions limiting plant growth, such as elevated temperatures and reduced water availability. The specification and distribution of a stomatal cell lineage and its terminal differentiation into guard cells require a master regulatory protein phosphorylation cascade involving the YODA mitogen-activated protein kinase kinase kinase. YODA signaling results in the activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MPK3 and MPK6), which regulate transcription factors, including SPEECHLESS (SPCH). Here, we report that acute heat stress affects the phosphorylation and deactivation of SPCH and modulates stomatal density. By using complementary molecular, genetic, biochemical, and cell biology approaches, we provide solid evidence that HEAT SHOCK PROTEINS 90 (HSP90s) play a crucial role in transducing heat-stress response through the YODA cascade. Genetic studies revealed that YODA and HSP90.1 are epistatic, and they likely function linearly in the same developmental pathway regulating stomata formation. HSP90s interact with YODA, affect its cellular polarization, and modulate the phosphorylation of downstream targets, such as MPK6 and SPCH, under both normal and heat-stress conditions. Thus, HSP90-mediated specification and differentiation of the stomatal cell lineage couples stomatal development to environmental cues, providing an adaptive heat stress response mechanism in plants., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

The chaperones, heat shock proteins (HSPs), stabilize proteins to minimize proteotoxic stress, especially during heat stress (HS) and polyamine (PA) oxidases (PAOs) participate in the modulation of the cellular homeostasis of PAs and reactive oxygen species (ROS). An interesting interaction of HSP90s and PAOs was revealed in Arabidopsis thaliana by using the pLFY:HSP90RNAi line against the four AtHSP90 genes encoding cytosolic proteins, the T-DNA Athsp90-1 and Athsp90-4 insertional mutants, the Atpao3 mutant and pharmacological inhibitors of HSP90s and PAOs. Silencing of all cytosolic HSP90 genes resulted in several-fold higher levels of soluble spermidine (S-Spd), acetylated Spd (N 8 -acetyl-Spd) and acetylated spermine (N 1 -acetyl-Spm) in the transgenic Arabidopsis thaliana leaves. Heat shock induced increase of soluble-PAs (S-PAs) and soluble hydrolyzed-PAs (SH-PAs), especially of SH-Spm, and more importantly of acetylated Spd and Spm. The silencing of HSP90 genes or pharmacological inhibition of the HSP90 proteins by the specific inhibitor radicicol, under HS stimulatory conditions, resulted in a further increase of PA titers, N 8 -acetyl-Spd and N 1 -acetyl-Spm, and also stimulated the expression of PAO genes. The increased PA titers and PAO enzymatic activity resulted in a profound increase of PAO-derived hydrogen peroxide (H 2 O 2 ) levels, which was terminated by the addition of the PAO-specific inhibitor guazatine. Interestingly, the loss-of-function Atpao3 mutant exhibited increased mRNA levels of selected AtHSP90 genes. Taken together, the results herein reveal a novel function of HSP90 and suggest that HSP90s and PAOs cross-talk to orchestrate PA acetylation, oxidation, and PA/H 2 O 2 homeostasis.

Armadillo (ARM) repeat containing proteins constitute a large family in plants and are involved in diverse cellular functions, like signal transduction, proliferation and differentiation. In animals, ARM repeat proteins have been implicated in cancer development. In this study, we aimed in characterizing the VPNB1 gene from Arabidopsis thaliana and its role in plant development, by implementing a number of genetic and molecular approaches. AtVPNB1 encodes for an ARM repeat protein of unknown function, exclusively expressed in the cambium as well as in the differentiating xylem and phloem cells of the vascular system. Subcellular localization experiments showed that VPNB is confined in nucleoplasmic speckle-like structures unrelated to cajal bodies. Transgenic VPNB-impaired plants exhibit a slower growing phenotype and a non-canonical pattern of xylem tissue. On the contrary, VPNB overexpression lines display an inverted phenotype of increased growth, accompanied by an increased deposition of phloem and xylem cell layers. In line with the above data, qPCR analysis revealed a deregulation of several key master genes of secondary wall biosynthesis, underlining the involvement of VPNB1 in the regulation and differentiation of the root and shoot vascular tissue., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The ability to derive information on the conformation of surface attached biomolecules by using simple techniques such as biosensors is currently considered of great importance in the fields of surface science and nanotechnology. Here we present a nanoshape sensitive biosensor where a simple mathematical expression is used to relate acoustic measurements to the geometrical features of a surface-attached biomolecule. The underlying scientific principle is that the acoustic ratio (ΔD/ΔF) is a measure of the hydrodynamic volume of the attached entity, mathematically expressed by its intrinsic viscosity [η]. A methodology is presented in order to produce surfaces with discretely bound biomolecules where their native conformation is maintained. Using DNA anchors we attached a spherical protein (streptavidin) and a rod-shaped DNA (47bp) to a quartz crystal microbalance (QCM) device in a suspended way and predicted correctly through acoustic measurements their conformation, i.e., shape and length. The methodology can be widely applied to draw conclusions on the conformation of any biomolecule or nanoentity upon specific binding on the surface of an acoustic wave device.

Arabidopsis thaliana flowering time mutants revealed the function of numerous genes that regulate the transition from vegetative to reproductive growth. Analyses of their loci have shown that many of them act as chromatin modifiers. In this study, a combination of molecular and genetic approaches have been implemented, to characterize the function of APRF1 (ANTHESIS POMOTING FACTOR 1) gene in A. thaliana and to investigate its role in plant development. APRF1 encodes for a low molecular weight nuclear WDR protein which displays functional homology to the Swd2 protein, an essential subunit of the yeast histone methylation COMPASS complex. Compared to WT plants, total loss-of-function aprf1 mutants exhibited shoot apical meristem (SAM) alterations and increased growth rates. However, the vegetative phase of aprf1 plants was prolonged and bolting was delayed, indicating an impairment in flowering under long days (LD). On the contrary, overexpression of APRF1 accelerates flowering. Consistent with the late flowering phenotype, the molecular data confirmed that FLC and SOC1 expression were significantly altered in the aprf1 mutants. Our data suggest that APRF1 acts upstream of FLC and promotes flowering under LD., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Highly interactive signaling processes constitute a set of parameters intertwining in a continuum mode to shape body formation and development. A sophisticated gene network is required to integrate environmental and endogenous cues in order to modulate flowering. However, the molecular mechanisms that coordinate the circuitries of flowering genes remain unclear. Here using complemented experimental approaches, we uncover the decisive and essential role of HEAT SHOCK PROTEIN 90 (HSP90) in restraining developmental noise to an acceptable limit. Localized depletion of HSP90 mRNAs in the shoot apex resulted in low penetrance of vegetative-to-reproductive phase transition and completely abolished flower formation. Extreme variation in expression of flowering genes was also observed in HSP90 mRNA-depleted transformed plants. Transient heat-shock treatments moderately increased HSP90 mRNA levels and rescued flower arrest. The offspring had a low, nevertheless noticeable failure to promote transition from vegetative into the reproductive phase and showed flower morphological heterogeneity. In floral tissues a moderate variation in HSP90 transcript levels and in the expression of flowering genes was detected. Key flowering proteins comprised clientele of the molecular chaperone demonstrating that the HSP90 is essential during vegetative-to-reproductive phase transition and flower development. Our results uncover that HSP90 consolidates a molecular scaffold able to arrange and organize flowering gene network and protein circuitry, and effectively counterbalance the extent to which developmental noise perturbs phenotypic traits., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

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.