Subcellular Proteomics as a Unified Approach of Experimental Localizations and Computed Prediction Data for Arabidopsis and Crop Plants.

In eukaryotic organisms, subcellular protein location is critical in defining protein function and understanding sub-functionalization of gene families. Some proteins have defined locations, whereas others have low specificity targeting and complex accumulation patterns. There is no single approach that can be considered entirely adequate for defining the in vivo location of all proteins.

Establishment of Photosynthesis through Chloroplast Development Is Controlled by Two Distinct Regulatory Phases.

Chloroplasts develop from undifferentiated proplastids present in meristematic tissue. Thus, chloroplast biogenesis is closely connected to leaf development, which restricts our ability to study the process of chloroplast biogenesis per se. As a consequence, we know relatively little about the regulatory mechanisms behind the establishment of the photosynthetic reactions and how the activities of the two genomes involved are coordinated during chloroplast development.

A MEM1-like motif directs mesophyll cell-specific expression of the gene encoding the C4 carbonic anhydrase in Flaveria.

The first two reactions of C photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F.

SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations.

The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories.

AEF1/MPR25 is implicated in RNA editing of plastid atpF and mitochondrial nad5, and also promotes atpF splicing in Arabidopsis and rice.

RNA editing is an essential mechanism that modifies target cytidines to uridine in both mitochondrial and plastid mRNA. Target sites are recognized by pentatricopeptide repeat (PPR) proteins. Using bioinformatics predictions based on the code describing sequence recognition by PPR proteins, we have identified an Arabidopsis editing factor required for editing of atpF in plastids.

Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue.

Background: An important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment.

Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family.

Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions.

SUBAcon: a consensus algorithm for unifying the subcellular localization data of the Arabidopsis proteome.

Motivation: Knowing the subcellular location of proteins is critical for understanding their function and developing accurate networks representing eukaryotic biological processes. Many computational tools have been developed to predict proteome-wide subcellular location, and abundant experimental data from green fluorescent protein (GFP) tagging or mass spectrometry (MS) are available in the model plant, Arabidopsis. None of these approaches is error-free, and thus, results are often contradictory.

The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I.

Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain.

Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants.

Fluorescent protein (FP) tagging approaches are widely used to determine the subcellular location of plant proteins. Here we give a brief overview of FP approaches, highlight potential technical problems, and discuss what to consider when designing FP/protein fusion constructs and performing transformation assays. We analyze published FP tagging data sets along with data from proteomics studies collated in SUBA3, a subcellular location database for Arabidopsis proteins, and assess the reliability of these data sets by comparing them.

The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing.

RNA editing in plants is an essential post-transcriptional process that modifies the genetic information encoded in organelle genomes. Forward and reverse genetics approaches have revealed the prevalent role of pentatricopeptide repeat (PPR) proteins in editing in both plastids and mitochondria, confirming the shared origin of this process in both organelles. The E domain at or near the C terminus of these proteins has been shown to be essential for editing, and is presumed to recruit the enzyme that deaminates the target cytidine residue.

SUBA3: a database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis.

The subcellular location database for Arabidopsis proteins (SUBA3, http://suba.plantenergy.uwa.

The SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LHCB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis in Arabidopsis seedlings.

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons.

In silico methods for identifying organellar and suborganellar targeting peptides in Arabidopsis chloroplast proteins and for predicting the topology of membrane proteins.

Numerous experimental and in silico approaches have been developed for attempting to identify the -subcellular localisation of proteins. Approximately 2,000-4,000 proteins are thought to be targeted to plastids in plants, but a complete and unambiguous catalogue has yet to be drawn up. This article reviews the various prediction methods that identify plastid targeting sequences, and those that can help estimate location and topology within the plastid or plastid membranes.

Insights into the composition and assembly of the membrane arm of plant complex I through analysis of subcomplexes in Arabidopsis mutant lines.

NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.

The pentatricopeptide repeat protein OTP87 is essential for RNA editing of nad7 and atp1 transcripts in Arabidopsis mitochondria.

In plant organelles, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in RNA of both mitochondria and plastids, altering the information encoded by the gene. The cytidine to be edited is determined by a cis-element surrounding the editing site that is specifically recognized and bound by a trans-acting factor. All the trans-acting editing factors identified so far in plant organelles are members of a large protein family, the pentatricopeptide repeat (PPR) proteins.

OTP70 is a pentatricopeptide repeat protein of the E subgroup involved in splicing of the plastid transcript rpoC1.

Over 20 proteins of the pentatricopeptide repeat (PPR) family have been demonstrated to be involved in RNA editing in plant mitochondria and chloroplasts. All of these editing factors contain a so-called 'E' domain that has been shown to be essential for editing to occur. The presumption has been that this domain recruits the (unknown) editing enzyme to the RNA.

MASCP Gator: an aggregation portal for the visualization of Arabidopsis proteomics data.

Proteomics has become a critical tool in the functional understanding of plant processes at the molecular level. Proteomics-based studies have also contributed to the ever-expanding array of data in modern biology, with many generating Web portals and online resources that contain incrementally expanding and updated information. Many of these resources reflect specialist research areas with significant and novel information that is not currently captured by centralized repositories.

A study of new Arabidopsis chloroplast RNA editing mutants reveals general features of editing factors and their target sites.

RNA editing in higher plant organelles results in the conversion of specific cytidine residues to uridine residues in RNA. The recognition of a specific target C site by the editing machinery involves trans-acting factors that bind to the RNA upstream of the C to be edited. In the last few years, analysis of mutants affected in chloroplast biogenesis has identified several pentatricopeptide repeat (PPR) proteins from the PLS subfamily that are essential for the editing of particular RNA transcripts.

The pentatricopeptide repeat protein OTP82 is required for RNA editing of plastid ndhB and ndhG transcripts.

Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the pentatricopeptide repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase.

Remodeled respiration in ndufs4 with low phosphorylation efficiency suppresses Arabidopsis germination and growth and alters control of metabolism at night.

Respiratory oxidative phosphorylation is a cornerstone of cellular metabolism in aerobic multicellular organisms. The efficiency of this process is generally assumed to be maximized, but the presence of dynamically regulated nonphosphorylating bypasses implies that plants can alter phosphorylation efficiency and can benefit from lowered energy generation during respiration under certain conditions. We characterized an Arabidopsis (Arabidopsis thaliana) mutant, ndufs4 (for NADH dehydrogenase [ubiquinone] fragment S subunit 4), lacking complex I of the respiratory chain, which has constitutively lowered phosphorylation efficiency.

Loss of the transit peptide and an increase in gene expression of an ancestral chloroplastic carbonic anhydrase were instrumental in the evolution of the cytosolic C4 carbonic anhydrase in Flaveria.

C(4) photosynthesis has evolved multiple times from ancestral C(3) species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) and is involved in both C(3) and C(4) photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C(3) and C(4) plants. To understand the molecular changes underlying the evolution of the C(4) pathway, three cDNAs encoding distinct beta-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C(3) plant Flaveria pringlei.

The Flaveria bidentis beta-carbonic anhydrase gene family encodes cytosolic and chloroplastic isoforms demonstrating distinct organ-specific expression patterns.

Carbonic anhydrase (CA) catalyzes the interconversion of CO(2) and bicarbonate, the forms of inorganic carbon used by the primary carboxylating enzymes of C(3) and C(4) plants, respectively. Multiple forms of CA are found in both photosynthetic subtypes; however, the number of isoforms and the location and function of each have not been elucidated for any single plant species. Genomic Southern analyses showed that the C(4) dicotyledon Flaveria bidentis 'Kuntze' contains a small gene family encoding beta-CA and cDNAs encoding three distinct beta-CAs, named CA1, CA2, and CA3, were isolated.