Arabidopsis SWI/SNF chromatin remodeling complex binds both promoters and terminators to regulate gene expression

ATP-dependent chromatin remodeling complexes are important regulators of gene expression in Eukaryotes. In plants, SWI/SNF-type complexes have been shown critical for transcriptional control of key developmental processes, growth and stress responses. To gain insight into mechanisms underlying these roles, we performed whole genome mapping of the SWI/SNF catalytic subunit BRM in Arabidopsis thaliana, combined with transcript profiling experiments. Our data showthatBRM occupies thousands of sites in Arabidopsis genome, most of which located within or close to genes. Among identified direct BRM transcriptional targets almost equal numbers were up- and downregulated upon BRM depletion, suggesting that BRM can act as both activator and repressor of gene expression. Interestingly, in addition to genes showing canonical pattern of BRM enrichment near transcription start site, many other genes showed a transcription termination sitecentred BRM occupancy profile. We found that BRMbound 3� gene regions have promoter-like features, including presence of TATA boxes and high H3K4me3 levels, and possess high antisense transcriptional activity which is subjected to both activation and repression by SWI/SNF complex. Our data suggest that binding to gene terminators and controlling transcription of non-coding RNAs is another way through which SWI/SNF complex regulates expression of its targets

Polyprenols Are Synthesized by a Plastidial cis-Prenyltransferase and Influence Photosynthetic Performance

Plants accumulate a family of hydrophobic polymers known as polyprenols, yet how they are synthesized, where they reside in the cell, and what role they serve is largely unknown. Using Arabidopsis thaliana as a model, we present evidence for the involvement of a plastidial cis-prenyltransferase (AtCPT7) in polyprenol synthesis. Gene inactivation and RNAi-mediated knockdown of AtCPT7 eliminated leaf polyprenols, while its overexpression increased their content. Complementation tests in the polyprenol-deficient yeast Δrer2 mutant and enzyme assays with recombinant AtCPT7 confirmed that the enzyme synthesizes polyprenols of ~55 carbons in length using geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate as substrates. Immunodetection and in vivo localization of AtCPT7 fluorescent protein fusions showed that AtCPT7 resides in the stroma of mesophyll chloroplasts. The enzymatic products of AtCPT7 accumulate in thylakoid membranes, and in their absence, thylakoids adopt an increasingly “fluid membrane” state. Chlorophyll fluorescence measurements from the leaves of polyprenol-deficient plants revealed impaired photosystem II operating efficiency, and their thylakoids exhibited a decreased rate of electron transport. These results establish that (1) plastidial AtCPT7 extends the length of GGPP to;55 carbons, which then accumulate in thylakoid membranes; and (2) these polyprenols influence photosynthetic performance through their modulation of thylakoid membrane dynamics

Spatial Nano-Morphology of the Prolamellar Body in Etiolated Arabidopsis thaliana Plants With Disturbed Pigment and Polyprenol Composition

The prolamellar body (PLB) is a periodic bicontinuous membrane structure based on tubular tetrahedral units. PLBs are present in plant etioplasts and, upon illumination, directly transform into the lamellar thylakoid networks within chloroplasts. Efficient tubular-lamellar rearrangement and later formation of the photosynthetically active thylakoid membranes are crucial steps in the development of plant autotrophy. PLB membranes are mainly composed of galactolipids, carotenoids, and protochlorophyllide (Pchlide), the chlorophyll precursor, bound in a complex with NADPH and Pchlide oxidoreductase. Although the PLB structure has been studied for over 50 years, the direct role of particular membrane components in the formation of the PLB paracrystalline network remains elusive. Moreover, despite the numerous literature data regarding the PLB geometry, their reliable comparative analysis is complicated due to variable experimental conditions. Therefore, we performed comprehensive ultrastructural and low-temperature fluorescence analysis of wild type Arabidopsis thaliana (Arabidopsis) seedlings grown in different conditions typical for studies on etiolated seedlings. We established that the addition of sucrose to the growing media significantly affected the size and compactness of the PLB. The etiolation period was also an important factor influencing the PLB structural parameters and the ratio of free to complex-bound Pchlide. Thus, a reliable PLB structural and spectral analysis requires particular attention to the applied experimental conditions. We investigated the influence of the pigment and polyprenol components of the etioplast membranes on the formation of the PLB spatial structure. The PLB 3D structure in several Arabidopsis mutants (ccr1-1, lut5-1, szl1-1npq1-2, aba1-6, pif1, cpt7) with disturbed levels of particular pigments and polyprenols using electron tomography technique was studied. We found that the PLB nano-morphology was mainly affected in the pif1 and aba1-6 mutants. An increased level of Pchlide (pif1) resulted in the substantial shift of the structural balance between outer and inner PLB water channels and overall PLB compactness compared to wild type plants. The decrease in the relative content of β-branch xanthophylls in aba1-6 plants was manifested by local disturbances in the paracrystalline structure of the PLB network. Therefore, proper levels of particular etioplast pigments are essential for the formation of stable and regular PLB structure

Medium-chain-length polyprenol (C45–C55) formation in chloroplasts of Arabidopsis is brassinosteroid-dependent

Brassinosteroids are important plant hormones influencing, among other processes, chloroplast development, the electron transport chain during light reactions of photosynthesis, and the Calvin-Benson cycle. Medium-chainlength polyprenols built of 9–11 isoprenoid units (C45–C55 carbons) are a class of isoprenoid compounds present in abundance in thylakoid membranes. They are synthetized in chloroplast by CPT7 gene from Calvin cycle derived precursors on MEP methylerythritol 4-phosphate) isoprenoid biosynthesis pathway. C45–C55 polyprenols affect thylakoid membrane ultra-structure and hence influence photosynthetic apparatus performance in plants such as Arabidopsis and tomato. So far nothing is known about the hormonal or environmental regulation of CPT7 gene expression. The aim of our study was to find out if medium-chain-length polyprenol biosynthesis in plants may be regulated by hormonal cues.We found that the CPT7 gene in Arabidopsis has a BZR1 binding element (brassinosteroid dependent) in its promoter. Brassinosteroid signaling mutants in Arabidopsis accumulate a lower amount of medium-chain-length C45–C55 polyprenols than control plants. At the same time carotenoid and chlorophyll content is increased, and the amount of PsbD1A protein coming from photosystem II does not undergo a significant change. On contrary, treatment of WT plants with epi-brassinolide increases C45–C55 polyprenols content. We also report decreased transcription of MEP enzymes (besides C45–C55 polyprenols, precursors of numerous isoprenoids, e.g. phytol, carotenoids are derived from this pathway) and genes encoding biosynthesis of medium-chain-length polyprenol enzymes in brassinosteroid perception mutant bri1-116. Taken together, we document that brassinosteroids affect biosynthetic pathway of C45–C55 polyprenols

The effects of implementing a point-of-care electronic template to prompt routine anxiety and depression screening in patients consulting for osteoarthritis (the Primary Care Osteoarthritis Trial): A cluster randomised trial in primary care

Background This study aimed to evaluate whether prompting general practitioners (GPs) to routinely assess and manage anxiety and depression in patients consulting with osteoarthritis (OA) improves pain outcomes. Methods and findings We conducted a cluster randomised controlled trial involving 45 English general practices. In intervention practices, patients aged ≥45 y consulting with OA received point-of-care anxiety and depression screening by the GP, prompted by an automated electronic template comprising five questions (a two-item Patient Health Questionnaire–2 for depression, a two-item Generalized Anxiety Disorder–2 questionnaire for anxiety, and a question about current pain intensity [0–10 numerical rating scale]). The template signposted GPs to follow National Institute for Health and Care Excellence clinical guidelines for anxiety, depression, and OA and was supported by a brief training package. The template in control practices prompted GPs to ask the pain intensity question only. The primary outcome was patient-reported current pain intensity post-consultation and at 3-, 6-, and 12-mo follow-up. Secondary outcomes included pain-related disability, anxiety, depression, and general health. During the trial period, 7,279 patients aged ≥45 y consulted with a relevant OA-related code, and 4,240 patients were deemed potentially eligible by participating GPs. Templates were completed for 2,042 patients (1,339 [31.6%] in the control arm and 703 [23.1%] in the intervention arm). Of these 2,042 patients, 1,412 returned questionnaires (501 [71.3%] from 20 intervention practices, 911 [68.0%] from 24 control practices). Follow-up rates were similar in both arms, totalling 1,093 (77.4%) at 3 mo, 1,064 (75.4%) at 6 mo, and 1,017 (72.0%) at 12 mo. For the primary endpoint, multilevel modelling yielded significantly higher average pain intensity across follow-up to 12 mo in the intervention group than the control group (adjusted mean difference 0.31; 95% CI 0.04, 0.59). Secondary outcomes were consistent with the primary outcome measure in reflecting better outcomes as a whole for the control group than the intervention group. Anxiety and depression scores did not reduce following the intervention. The main limitations of this study are two potential sources of bias: an imbalance in cluster size (mean practice size 7,397 [intervention] versus 5,850 [control]) and a difference in the proportion of patients for whom the GP deactivated the template (33.6% [intervention] versus 27.8% [control]). Conclusions In this study, we observed no beneficial effect on pain outcomes of prompting GPs to routinely screen for and manage comorbid anxiety and depression in patients presenting with symptoms due to OA, with those in the intervention group reporting statistically significantly higher average pain scores over the four follow-up time points than those in the control group. Trial registration ISRCTN registry ISRCTN4072198

HSFA1 proteins mediate heat-induced accumulation of CPT7-derived polyprenols affecting thylakoid organization

AbstractPolyprenols are ubiquitous isoprenoid compounds that accumulate in large quantities in plant photosynthetic tissues. While our knowledge of polyprenol biochemistry is constantly expanding, the regulation of their biosynthesis as well as the molecular basis of their cellular action are still poorly understood. In Arabidopsis, the polyprenols Pren-9, −10 and −11, synthesized by cis-prenyltransferase 7 (CPT7), are localized in plastidial membranes and affect the photosynthetic performance of chloroplasts.In this report we present evidence that plastidial polyprenols are among the major constituents of thylakoid membranes. Disturbances in polyprenol level, caused by alterations in CPT7 expression, change chloroplast ultrastructure, affect aggregation of LHCII complexes and modulate non-photochemical quenching (NPQ). Moreover, we show that Arabidopsis responds to high temperature by upregulating expression of CPT7 and increasing the accumulation of CPT7-derived polyprenols. These heat-induced changes in polyprenol biosynthesis are mediated by Heat Shock Transcription Factors of the HSFA1 family, the master regulators of heat stress response. Collectively, results presented in this report bring us closer to understanding the mechanisms by which polyprenols affect plant physiology and provide an additional link between chloroplast biology and plant responses to changing environmental conditions.One sentence summaryHeat Shock Transcription Factors induce biosynthesis of polyprenols - isoprenoid compounds that affect the organization and function of chloroplasts.</jats:sec

Transcriptional profile of <i>brm-1</i> overlaps with that of <i>ga1-3</i>.

<p>(A), Overlap between differentially regulated genes in mutants <i>brm-1</i>, <i>ga1-3</i> and <i>ga1-3/brm-1</i> identified in microarray data, shown by a Venn diagram. (B), Genes up- and down-regulated in all three mutants, shown by a heat map. The color scale represents normalized expression levels. (C), 94% of the genes commonly mis-expressed in all three mutants show expression changes in a similar direction. Green – genes mis-regulated only in <i>brm-1</i>; blue – genes mis-regulated in <i>brm-1</i>, <i>ga1-3</i> and <i>ga1-3/brm-1</i>; orange – genes mis-regulated in a similar direction in all three mutants; gray – genes mis-regulated in an opposite direction in one of the mutants.</p