Explore the vast range of titles available.

Plasmodesmata permit solutes to move between cells nonspecifically and without having to cross a membrane. This symplastic connectivity, while straightforward to observe using fluorescent tracers, has proven difficult to quantify. We use fluorescence recovery after photobleaching, combined with a mathematical model of symplastic diffusion, to assay plasmodesmata-mediated permeability in the Arabidopsis (Arabidopsis thaliana) root meristem in wild-type and transgenic lines, and under selected chemical treatments. The permeability measured for the wild type is nearly 10-times greater than previously reported. Plamodesmal permeability remains constant in seedlings treated with auxin (30 nM indoleacetic acid for 2 and 24 h; 100 nM indoleacetic acid for 2 h); however, permeability is diminished in two lines previously reported ro have impaired plasmodesmal function as well as in wild-type seedlings treated for 24 h with 0.6 mM tryptophan. Moreover, plasmodesmal permeability is strongly altered by applied hydrogen peroxide within 2 h of treatment, being approximately doubled at a low concentration (0.6 mM) and nearly eliminated at a higher one (6 mM). These results reveal that the plasmodesmata in the root meristem carry a substantial flux of small molecules and that this flux is subject to rapid regulation.

Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.

The ability of the plant hormone auxin to enter a cell is critical to auxin transport and signaling. Auxin can cross the cell membrane by diffusion or via auxin‐specific influx carriers. There is little knowledge of the magnitudes of these fluxes in plants. Radiolabeled auxin uptake was measured in protoplasts isolated from roots of Arabidopsis thaliana. This was done for the wild‐type, under treatments with additional unlabeled auxin to saturate the influx carriers, and for the influx carrier mutant auxin resistant 1 (aux1). We also used flow cytometry to quantify the relative abundance of cells expressing AUX1‐YFP in the assayed population. At pH 5.7, the majority of auxin influx into protoplasts – 75% – was mediated by the influx carrier AUX1. An additional 20% was mediated by other saturable carriers. The diffusive influx of auxin was essentially negligible at pH 5.7. The influx of auxin mediated by AUX1, expressed as a membrane permeability, was 1.5 ± 0.3 μm s⁻¹. This value is comparable in magnitude to estimates of efflux permeability. Thus, auxin‐transporting tissues can sustain relatively high auxin efflux and yet not become depleted of auxin.

Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.

cpSRP54 (for chloroplast SIGNAL RECOGNITION PARTICLE54) is involved in cotranslational and posttranslational sorting of thylakoid proteins. The Arabidopsis (Arabidopsis thaliana) cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western-blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecY/E translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2 plants. To further understand the impact of cpSRP54 deletion, a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves using stable isotope labeling (isobaric stable isotope labeling and isotopecoded affinity tags) and two-dimensional gels. This showed that cpSRP54 deletion led to a change in light-harvesting complex composition, an increase of PsbS, and a decreased photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. The Calvin cycle was down-regulated in mature leaves to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 clpc1 double mutant. This mutant was seedling lethal under autotrophic conditions but could be partially rescued under heterotrophic conditions.

Plasmodesmata permit solutes to move between cells nonspecifically and without having to cross a membrane. This symplastic connectivity, while straightforward to observe using fluorescent tracers, has proven difficult to quantify. We use fluorescence recovery after photobleaching, combined with a mathematical model of symplastic diffusion, to assay plasmodesmata-mediated permeability in the Arabidopsis (Arabidopsis thaliana) root meristem in wild-type and transgenic lines, and under selected chemical treatments. The permeability measured for the wild type is nearly 10-times greater than previously reported. Plamodesmal permeability remains constant in seedlings treated with auxin (30 mM indoleacetic acid for 2 and 24 h; 100 nm indoleacetic acid for 2 h); however, permeability is diminished in two lines previously reported to have impaired plasmodesmal function as well as in wild-type seedlings treated for 24 h with 0.6 mM tryptophan. Moreover, plasmodesmal permeability is strongly altered by applied hydrogen peroxide within 2 h of treatment, being approximately doubled at a low concentration (0.6 mM) and nearly eliminated at a higher one (6 mM). These results reveal that the plasmodesmata in the root meristem carry a substantial flux of small molecules and that this flux is subject to rapid regulation.

cpSRP54 (for chloroplast SIGNAL RECOGNITION PARTICLE54) is involved in cotranslational and posttranslational sorting of thylakoid proteins. The Arabidopsis (Arabidopsis thaliana) cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western-blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecY/E translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2 plants. To further understand the impact of cpSRP54 deletion, a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves using stable isotope labeling (isobaric stable isotope labeling and isotope-coded affinity tags) and two-dimensional gels. This showed that cpSRP54 deletion led to a change in light-harvesting complex composition, an increase of PsbS, and a decreased photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. The Calvin cycle was down-regulated in mature leaves to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 clpc1 double mutant. This mutant was seedling lethal under autotrophic conditions but could be partially rescued under heterotrophic conditions.

Plastids are essential organelles of prokaryotic origin that are present in almost every plant cell. 11-14% of the nuclear genes of Arabidopsis thaliana are predicted to encode for plastid-localized proteins. The development of plastids into specific types, such as chloroplasts, is not only dependent on the coordinated expression of nuclear- and plastid-encoded genes, but also on efficient and reliable protein targeting, assembly and proteolysis. Chloroplasts play a central role in leaf growth, and characterization of the chloroplast proteome and its dynamics is needed to understand chloroplast function. A large-scale and comprehensive analysis of the A. thaliana chloroplast proteome identified more than 1300 proteins. This resulted also in a better definition of the N-terminal targeting signals of nuclear-encoded chloroplast proteins, and determined N-terminal modifications that may affect protein stability and function. The abundance of 48 stromal proteins involved in proteome homeostasis (including aminopeptidases, deformylases, chaperones) is highlighted. Chloroplast SRP54 is involved in co- and post-translational sorting of thylakoid proteins. A quantitative proteomics analysis determined that deletion of cpSRP54 resulted in specific changes in the composition of the thylakoid photosynthetic apparatus, in particular light harvesting proteins and the photosystems. The Calvin cycle and photorespiratory pathway were up-regulated in young leaves and the Calvin cycle was subsequently down-regulated in mature leaves. Up-regulation of thylakoid proteases and stromal chaperones, including ClpC, was observed in young leaves. The stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. The significance of ClpC up regulation was confirmed through the generation of an ffc x clpc1 double mutant. The Trigger Factor (TIG) protein in prokaryotes is a unique peptidyl prolyl cis/trans isomerase (PPIase) ribosome associated chaperone that prevents miss-folding of nascent chains. TIG and SRP54 compete for the interaction with nascent chains at the ribosome. An A. thaliana null mutant (tig-1) of chloroplast localized TIG has no phenotype suggesting functional redundancy to other biogenesis components, such as chaperones. To test the functional relationship between cpSRP54 and TIG in A. thaliana, double mutants in ffc x tig-1 were generated. These show a suppression of the ffc phenotype, suggesting the participation of AtTIG in biogenesis of chloroplast-encoded proteins, and providing new insight in the primary cause of the ffc phenotype. Plastid proteases and chaperones play a key role in chloroplast biogenesis and protein homeostasis. The Clp protease system consists of a tetradecameric core protease complex of ClpP and ClpR proteins. Substrates are unfolded and delivered into this core complex via interacting ClpC,D chaperones, with ClpS possibly stimulating selection of N-end rule substrates. Double mutants in clps/clpc1 and clps/clpd were generated and resulting phenotypes are discussed. The presence of an ssrA substrate tagging and recognition system in the chloroplast is explored.