16 research outputs found
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 ÎČ-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
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
3ïżœ 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
Medium-chain-length polyprenol (C45âC55) 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 9â11 isoprenoid units (C45âC55 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. C45âC55 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 C45âC55 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
C45âC55 polyprenols content. We also report decreased transcription of MEP enzymes (besides C45âC55 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 C45âC55 polyprenols
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 Îrer2 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 âfluid membraneâ 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
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 â„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 Questionnaireâ2 for depression, a two-item Generalized Anxiety Disorderâ2 questionnaire for anxiety, and a question about current pain intensity [0â10 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 â„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
Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes
Arabidopsis SWI/SNF chromatin remodeling complex binds both promoters and terminators to regulate gene expression
BRM acts through distinct mechanisms to regulate GA-mediated responses.
<p>(A), Germination of the <i>brm-1</i> mutant on 10 ”M PAC is rescued by the <i>triple della</i> mutation. The progeny of <i>brm-1/BRM</i> plants were analyzed 10 days after sowing. (B), Phenotypes of 3-week-old plants grown on 2.5 ”M PAC. The <i>brm-1/3xdella</i> line shows an intermediate growth phenotype. Barâ=â5 mm. (C), RT-qPCR analysis of relative transcript levels of the <i>OFP16, EXP5, CYS2</i> and <i>LTP2</i> genes in 18-d-old wild type, <i>brm-1</i>, <i>ga1-3</i>, <i>ga1-3/brm-1</i>, <i>ga1-3/3xdella</i> and <i>ga1-3/brm-1/3xdella</i> lines. Transcript levels in the wild type were set to 1. Data are the means ± s.d. of 3 biological replicates. (D), Model of the role of BRM in regulating the expression of GA-responsive genes. BRM positively regulates the <i>GA3ox1</i> and <i>SCL3</i> genes involved in GA biosynthesis and signaling, and probably through this influences the expression of many GA-responsive genes in the opposite manner to DELLA repressors. In addition, BRM seems to act on a subset of GA-responsive genes independently of DELLA repressors. Also in this case, the effect exerted by BRM is typically in the opposite direction to that of DELLAs and is observed both for genes up- and down-regulated by the SWI/SNF complex (blue and red lines, respectively).</p
Concentration of gibberellins in wild type and mutant lines.
<p>The values are ng/g dry weight (s.e.). They are the means of three biological replicates except for the GA<sub>12</sub> and GA<sub>9</sub> measurements in <i>ga1-3</i>, for which 2 replicates were used. n.d. â not determined.</p
GA responses of the <i>brm-1</i> mutant.
<p>(A, B), Elongation of <i>brm-1</i> hypocotyls and roots in response to 1 ”M GA<sub>4</sub>. Plants were grown on Âœ MS medium for 8 days under long-days conditions in the presence or absence of 1 ”M GA<sub>4</sub>. GA application caused considerable elongation of the hypocotyls, but had little effect on <i>brm-1</i> root growth. Barâ=â5 mm. (B), Hypocotyl length of plants grown as in A. Presented data are the means of 12 measurements ± s.d. (C), Flowering of <i>brm-1</i> plants in response to exogenous gibberellins. Plants were grown in soil under short-day conditions and treated with 10 ”M GA<sub>3</sub>. At least 15 plants of each line/condition were scored. Data are the means ± s.d. Asterisks indicate significant differences from the wild type plants (p<0.01).</p