Arabidopsis Plastoglobule Proteome with ABC1 atypical kinase mutants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
TISSUE(S): Plant Cell, Leaf
SUBMITTER: 
William Comstock
LAB HEAD: Klaas van Wijk
PROVIDER: PXD017380 | Pride | 2020-02-06
REPOSITORIES: Pride
Similar Datasets
Project description:Chloroplasts and chromoplasts of tomato fruits contain lipid droplets known as plastoglobules (PG). Recent studies have shown that PG act as a metabolic sink to store triacylglycerol (TAG), carotenoids, vitamin E, and K as well as other lipophile compounds. In addition, it has been shown that proteins associated with PG contribute to prenyl lipid metabolic pathways and homeostasis. Proteome studies in Arabidopsis and bell pepper, have demonstrated that PG contains a specific proteome containing around 30 proteins. So far, the tomato PG proteome particularly that of red fruit (chromoplast) has not yet been established. We have investigated the chromoplast PG proteome and metabolome and identified 17 new candidate proteins of chromoplast PG with known PG proteins. Interestingly, most of the carotenoid biosynthetic pathway enzymes such as PSY‐1, PDS, ZDS, CRTISO, and β‐LYC are enriched in chromoplast PG compared with chloroplast PG. In this study, we propose a model of chromoplast PG acting as a carotenoid biosynthesis platform.
2023-03-11 | PXD035763 | Pride
Project description:Protein complexes separation of isolated plastoglobules (PGs) from Arabidopsis thaliana (Col-0 ecotype). PGs were isolated from the plants when they were 4 weeks old under long day photoperiod ( during the morning in dark adapted plants,16 hours of light and 8 darkness photoperiod, LED light of 120 uE intensity). The PGs were solubilized with 1.1 % n-Dodecyl--D-Maltoside and separated in a Blue-Native gel. The gel lanes were excised and further separated in a denaturing second dimension by SDS-PAGE. The resulting gel was visualized by silver staining and the protein spots excised for in-gel trypsin digestion. Samples from the 2D gel were subjeted to shotgun proteomics. The protein samples were measured on an LTQ Orbitrap (Thermo Fisher Scientific Inc, Waltham Massachusetts, USA) coupled to a Rheos Allegro liquid chromatograph (Flux Instruments GmbH, Klaus Kaiser Hochstr. 48, 4002 Basel, CH).
2021-04-13 | PXD019765 | Pride
Project description:Proteome comparison of isolated plastoglobules (PGs) from Arabidopsis thaliana (Col-0 ecotype) under unstress (UNS) and high light (HL) stress conditions. For both conditions, PGs were isolated from three independent set of plants each (i.e. three for UNS and three for HL). PGs were isolated from the plants when they were 4 weeks old under long day photoperiod (16 hours of light and 8 darkness). The light stress was applied to the designated set of plants 5 days prior the completion of the 4 weeks by shifting the light intensity from 120 uE to 500 E. The PGs were solubilized with 1.1 % n-Dodecyl--D-Maltoside and separated in a Blue-Native gel. After the electrophoretic separation, gel lanes were cut and further divided into 10 independent slices corresponding to different molecular weights. These gel slices were subjeted to in-gel digestion with trypsin and the resulting peptides labeled with TMT reagents. Labeled peptides were mixed 1:1 and purified by solid phase extraction using c18 StageTips. Eluted peptides were sprayed into a ThermoScientific Q-Exactive HF-X mass spectrometer. Survey scans were taken in the Orbi trap (120000 resolution, determined at m/z 200) and the top 20 ions in each survey scan are then subjected to automatic higher energy collision induced dissociation (HCD) with fragment spectra acquired at 45000 resolution. Additionally, PG samples that were not separated in a Blue-native gel were trypsin digested and subjected for mass-spectrometry analysis as previously described.
2021-04-13 | PXD019735 | Pride
Project description:We studied the function of PG localized protease, PGM48 both in vitro and in vivo during natural senescence in Arabidopsis thaliana.Our results show that PGM48 is a metalloprotease and a senescence inducible protein. In vitro assay by recombinant protein revealed a proteolytic activity. Quantitative proteome analysis of PGs from senescing rosettes of overexpression plants lines showed a significantly reduced level of CAROTENOID CLEAVAGE ENZYME 4 ( CCD4) protein but 2-3 fold higher level of phytyl-esterase (PES1) compared to wild type; the opposite results were found in RNAi lines. Yeast-2-hybrid analysis and in vitro pull-down experiments showed that PGM48 interacts with the kinase ABC1K3, PES1 and CCD4, but not with chlorophyll catabolic enzymes. We propose that PGM48 acts as a positive regulator of leaf senescence.
2019-04-09 | PXD003684 | Pride
Project description:Analysis of the subunits composition of the thylakoids protein complexes in Picea abies (Norway spruce) by means of two-dimensional large-pore Blue-Native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D lpBN/SDS-PAGE) and in-gel tryptic digestion of single spots.
2019-04-02 | PXD010071 | Pride
Project description:Physcomitrella patens is a model plant representing the group of mosses, which diverged from vascular plant ancestors early after land colonization. To investigate the unique characteristic of the composition and post-translational regulation of Physcomitrella patens photosynthetic apparatus, we have separated the thylakoids protein complexes in their subunits composition by means of two-dimensional large-pore Blue-Native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D lpBN/SDS-PAGE), followed by protein in-gel tryptic digestion of single spots. To identify the kinase responsible for phosphorylation of the major thylakoid protein complex photosystem II, a mutant line lacking the ortholog of kinase STN8 in the model angiosperm Arabidopsis thaliana has been generated and the identified thylakoid protein phosphorylation pattern have been compared with the WT moss.
2019-05-08 | PXD012501 | Pride
Project description:Light is the driving force of photosynthesis. However, excessive light can be harmful. Photoinhibition, or lightinduced photodamage, is one of the key processes limiting photosynthesis. When the absorbed light exceeds the amount that can be dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). A welldefined mechanism that protects the photosynthetic apparatus from photoinhibition has been described in the model green alga Chlamydomonas reinhardtii and plants. Chlorella ohadii is a green microalga, isolated from biological desert soil crusts, that thrives under extreme high light (HL) in which other organisms do not survive. Here, we show that this alga evolved unique protection mechanisms distinct from those of C. reinhardtii and plants. When grown under extreme HL, significant structural changes were noted in the C. ohadii thylakoids, including a drastic reduction in the antennae and the formation of stripped core PSII, lacking its outer and inner antennae. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, the stressrelated light harvesting complex (LHCSR), PSII protein phosphorylation and statetransitions are entirely absent or were barely detected in C. ohadii. A carotenoid biosynthesis related protein (CBR) is largely accumulated in the LHCII remains of HL cells and in the absence of psbS and LHCSR can function in sensing the HL and protecting from PI. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extremelight intensities where other photosynthetic organisms fail to survive.
2023-05-08 | PXD038704 | Pride
Project description:Coping of evergreen conifers of boreal forests with freezing temperatures on bright winter days puts the photosynthetic machinery in great risk of oxidative damage. To survive harsh winter conditions, conifers have evolved a unique but poorly characterised photoprotection mechanism, a sustained form of non-photochemical quenching (sustained NPQ). Here we focused on functional properties and underlying molecular mechanisms related to the development of sustained NPQ in Norway spruce (Picea abies). Data was collected during four consecutive years (2016-19) from trees growing in sun and shade habitats. When day temperatures dropped below -4°C, specific N-terminally triply phosphorylated LHCB1 isoform (3p-LHCII) and phosphorylated PSBS (p-PSBS) were detected in the thylakoid membrane. Development of sustained NPQ coincided with the highest level of 3p-LHCII and p-PSBS, occurring after prolonged combination of bright winter days and temperature close to -10°C. Artificial induction of both the sustained NPQ and recovery from naturally induced sustained NPQ provided information on differential dynamics and light-dependence of 3p-LHCII and p-PSBS accumulation and dephosphorylation as essential prerequisites of sustained NPQ. Data obtained collectively suggest three novel components related to sustained NPQ in spruce. (i) Freezing temperatures induce 3p-LHCII accumulation independently of light, which is suggested to initiate de-stacking of appressed thylakoid membranes due to increased electrostatic repulsion of adjacent membranes. (ii) p-PSBS accumulation is both light- and temperature-dependent and closely linked to the initiation of sustained NPQ, which (iii) in concert with PSII photoinhibition is likely to trigger sustained NPQ in spruce.
2020-07-27 | PXD018941 | Pride
Project description:The Uncharacterized Protein Family 0016 (UPF0016) is conserved throughout evolution. In humans, the member TMEM165 is a putative Golgi localized Ca2+ and/or Mn2+ transporter. Genes encoding UPF0016 proteins are also present in many bacteria, including the cyanobacteria Synechocystis (MNX/SynPAM71), where it has been described as Mn2+ exporter. In Arabidopsis, two members of this family are embedded in the chloroplast membranes; the CMT1 protein is localized in the inner envelope membrane and the PAM71 protein is localized in the thylakoid membrane, and both cooperate to transport Mn2+ to the oxygen evolving complex of photosystem II. We used the pam71 mutant of Arabidopsis as a platform to perform intra- and inter-species complementation assays. The N-terminus of PAM71 was fused in front of TMEM165, CMT1 and MNX to direct them into chloroplast membranes. We performed LC-MS/MS experiments to assess their sub-plastidial localization.
2021-04-20 | PXD022763 | Pride
Project description:Chloroplast glutamyl endopeptidase (CGEP), a stromal S9D peptidase, was found to be a member of the peptidase network that governs chloroplast proteostasis. Using the PICS technique, CGEP was found to degrade proteins smaller than 25 kDa, cleaving the C-terminal of glutamate. Comparative proteomics showed that the null mutant (cgep) altered protein accumulation levels in starch metabolism, stromal protein folding machinery, and ribosomal proteins.
2020-01-21 | PXD017189 | Pride