Dataset Information

Thylakoids of Picea abies - The unique photosynthetic apparatus of Pinaceae - Analysis of photosynthetic complexes in Norway spruce (Piceaabies)

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Picea Abies

TISSUE(S): Photosynthetic Cell, Leaf

SUBMITTER: Steffen Grebe

LAB HEAD: Eva-Mari Aro

PROVIDER: PXD010071 | Pride | 2019-04-02

REPOSITORIES: Pride

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Publications

The unique photosynthetic apparatus of Pinaceae: analysis of photosynthetic complexes in Picea abies.

Grebe Steffen S Trotta Andrea A Bajwa Azfar A AA Suorsa Marjaana M Gollan Peter J PJ Jansson Stefan S Tikkanen Mikko M Aro Eva-Mari EM

Journal of experimental botany 20190601 12

Pinaceae are the predominant photosynthetic species in boreal forests, but so far no detailed description of the protein components of the photosynthetic apparatus of these gymnosperms has been available. In this study we report a detailed characterization of the thylakoid photosynthetic machinery of Norway spruce (Picea abies (L.) Karst). We first customized a spruce thylakoid protein database from translated transcript sequences combined with existing protein sequences derived from gene models ...[more]

PMID: 30938447

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Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.

Project description:Purpose: The plant hormone abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. RNA-seq was employed to obtain differential expression data in response to ABA, dehydration and mannitol treatments in wild-type tissue. This was compared against the targeted gene knockout line PpanrKO. The ANR gene encodes a novel ABA regulator identified from ABA non-responsive (anr) mutant screens. Methods: RNA-seq on the Illumina HiSeq platform and mapping to the V3.0 genome and transcriptome assemblies using tophat2 enabled gene abundance data to be generated for differential gene expression analysis with edgeR in the Trinity package. Results: We observe a coordinated up-regulation of ca. 900 genes, with extensive overlaps between hormone and stress treatments as well as ca. 900 down-regulated genes. Of the up-regulated genes, those encoding LEA proteins, membrane channel proteins and transporters and oxidative-stress-associated genes comprised a significant fraction while those encoding growth-associated cell wall enzymes (e.g. expansins) and components of secondary metabolism (notably enzymes in the phenylpropanoid pathway) were prominent in the down-regulated genes. The ANR gene, a modular MAP3 kinase comprising an N-terminal PAS domain, a central EDR domain and a C-terminal MAPKKK-like domain unique to basal plant lineages, is found to play a central role in these responses with mutants failing to accumulate the wide spectrum of dehydration tolerance-associated gene products characteristic of the wild-type response and do not acquire ABA-dependent desiccation tolerance. Conclusions: Our study adds considerable data to the stress responses in bryophytes helping understand the mechanisms behind vegetative dehydration tolerance; an important trait for crop species. We also confirmed the vital role that PpANR plays in these responses in the moss Physcomitrella patens likely representing an ancestral mechanism for ABA-mediated dehydration tolerance vital in the conquest of land. RNA from 3 biological replicas was bulked per library. 8 libraries were generated from two genotypes, wild-type and PpanrkO, which included 3 treatments and a control sample for each.

Dataset Information

Thylakoids of Picea abies - The unique photosynthetic apparatus of Pinaceae - Analysis of photosynthetic complexes in Norway spruce (Piceaabies)

Publications

The unique photosynthetic apparatus of Pinaceae: analysis of photosynthetic complexes in Picea abies.

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