Dataset Information

Deciphering thylakoid microdomains using a mass spectrometry-based approach

ABSTRACT: In photosynthetic eukaryotes, photosynthesis occurs in the chloroplast, and more specifically in the thylakoid membranes. The thylakoids consist of two main domains: the grana, which are piles of membranes gathered together by stacking forces, and the stroma-lamellae, which are unstacked thylakoids connecting the grana. The major photosynthetic complexes are unevenly distributed, within these compartments, due to steric and electrostatic constraints and to their role in the photosynthetic process. To gain information on the sub-thylakoid distribution of these proteins (i.e. in the grana and in the stroma-lamellae) we combined experimental approaches to perform a complete survey of the protein composition of these thylakoid sub-compartments using i) thylakoid membrane fractionations to improve the dynamic range detection of minor thylakoid proteins and ii) a quantitative proteomic approach coupled to a dedicated data analysis pipeline and manual annotation to differentiate genuine grana and stroma-lamellae proteins from possible contaminants. Besides confirming previous information on the localization of photosynthetic proteins, these experiments provide some unexpected results, and allow revisiting the composition of the thylakoid micro-domains.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Hesse Anne-Marie

LAB HEAD: Myriam Ferro

PROVIDER: PXD000546 | Pride | 2015-09-23

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_31943.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_31943.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_31943.xml.gz	Xml
	Samples_and_datafile_names.xlsx	Xlsx
	VELOS10439.raw	Raw

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Publications

Deciphering thylakoid sub-compartments using a mass spectrometry-based approach.

Tomizioli Martino M Lazar Cosmin C Brugière Sabine S Burger Thomas T Salvi Daniel D Gatto Laurent L Moyet Lucas L Breckels Lisa M LM Hesse Anne-Marie AM Lilley Kathryn S KS Seigneurin-Berny Daphné D Finazzi Giovanni G Rolland Norbert N Ferro Myriam M

Molecular & cellular proteomics : MCP 20140528 8

Photosynthesis has shaped atmospheric and ocean chemistries and probably changed the climate as well, as oxygen is released from water as part of the photosynthetic process. In photosynthetic eukaryotes, this process occurs in the chloroplast, an organelle containing the most abundant biological membrane, the thylakoids. The thylakoids of plants and some green algae are structurally inhomogeneous, consisting of two main domains: the grana, which are piles of membranes gathered by stacking forces ...[more]

PMID: 24872594

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Project description:The regulation of the photosynthetic apparatus in higher plants is highly connected to the organization of the thylakoid membrane into appressed and non-appressed regions. The two photosystems (I and II) are the spatially strongest segregated protein complexes by this thylakoid heterogenous organization, with PSII predominantly present in the appressed membranes (grana), while PSI is confined to the stroma-exposed non-appressed thylakoid membranes (lamellae). Stacking of thylakoid membranes in grana is highly dynamic and influenced by the levels of PSII and LHCII protein phosphorylation which, in turn, are dependent on changes in light intensity and quality and thus, ultimately, on the redox state of the electron transfer chain. Dynamics of this lateral heterogeneity controls the spillover of excitation energy from PSII to PSI and optimizes the photochemistry in constantly fluctuating light conditions. Recently, a family of membrane-integral thylakoid proteins named CURVATURE THYLAKOID 1 (CURT1) have been proposed as new key components in shaping both the width and the number of layers per granum in response to changes in light intensity. CURT1B, in particular, has been known for a long time as a relatively abundant phosphorylated and acetylated protein, but so far the role and dynamics of these post-translational modifications (PTMs) have not been revealed. To this end, we have quantified by means of targeted proteomics the amount of CURT1 proteins and the level of CURT1B PTMs after short-term fluctuating light treatments in wild type Arabidopsis thaliana and the knock-out mutants of the kinases STN7 and STN8 and of the phosphatase TAP38. The CURT1B protein was first localized to a specific curvature domain largely depleted of the chlorophyll-protein complexes. Moreover, we have found that CURT1B is either phosphorylated or acetylated in the N-terminus, but never both. While the level of acetylation is never affected by the light treatments, the phosphorylation increases in light conditions that lead to sudden increase in PSII core protein phosphorylation, and these dynamics are largely abolished in stn8 plants but not in stn7 or tap38. Intriguingly, the phosphorylation dynamics as well as the level of the other CURT1 proteins, are instead highly affected in psb33 plants, which lack LHCII phosphorylation dynamics, and in the psal plants, which show constitutively phosphorylated LHCII. These results provide important information for assessing the relationships between PSII-LHCII phosphorylation, CURT1B phosphorylation and the dynamics of the appressed thylakoids, which in turn allow optimal photosynthesis and provide photoprotection under fluctuations in light intensity.

Project description:During Zea mays (maize) C4 differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C3 plants and produce the ATP and NADPH that drive metabolic activities. Regulation of this differentiation process is poorly understood but involves expression and coordination of nuclear and plastid genomes. Here, we identify a recessive allele of the maize Hcf136 homologue that in Arabidopsis thaliana functions as a PSII stability or assembly factor located in the thylakoid lumen. Proteome analysis of the thylakoids and electron microscopy reveal that Zm hcf136 lacks PSII complexes and grana thylakoids in M chloroplasts, consistent with the previously defined Arabidopsis function. Interestingly, hcf136 is also defective in processing the full-length psbB-psbT-psbH-petB-petD polycistron specifically in M chloroplasts. To determine whether the loss of PSII in M cells affects C4 differentiation, we performed cell-type specific transcript analysis of hcf136 and wild-type seedlings. The results indicate that M and BS cells respond uniquely to the loss of PSII, with little overlap in gene expression changes between data sets. These results are discussed in the context of signals that may drive differential gene expression in C4 photosynthesis. To explore the disruption of PSII activity on gene expression, transcript profiles from separated M and BS cells were examined using two-label microarray analysis. Total RNA was isolated from the second leaves of mutant and wild-type silbings. Six biological replicates were used to compare wild-type and mutant transcript profiles in separate M and BS experiments. To maximize biological replication, different seedling pools were used for each of the 12 hybridizations. Microarray experiments and analyses were performed using the Genisphere MPX900 kit and the Maize Array Consortium oligonucleotide platform (GPL5439; GPL5440). Feature intensity values were log-transformed and corrected for local background signal, and a LOWESS procedure (Dudoit et al., 2002) was used to normalize between channels. Features with either low or saturating signal intensity were discarded from further analysis. High expression filtering was less stringent to avoid elimination of previously characterized, high abundance, C4 cell-specific transcripts. After filtering, features that were not assigned an MZ number by the Maize Array Consortium were discarded from further analysis. The moderated t-test (Smyth, 2004) using the R package limma was applied to identify differentially expressed genes. The p-values for each test (gene) were converted to q-values for false discovery rate analysis as described by Storey et al. (2004). To avoid confounding treatment effects associated with direct comparisons of M and BS transcriptomes (Sawers et al., 2007), comparisons were only made using the same cell type across the hcf136 and wild-type sibling genotypes. Bundle sheath (BS) Samples: GSM245063-GSM245164 Mesophyll (M) Samples: GSM245165 - GSM245206