Mutants defective in PGE and Clp protease activity are resistant to plastidial isoprenoid inhibitors.

<p>Resistance to FSM was estimated by quantifying seedling establishment (SE, number of plants producing true leaves) and chlorophyll levels (CHL) in plants germinated and grown in the presence of 30 μM FSM relative to those obtained with no inhibitor (100%). Similarly, NFZ resistance was calculated based on chlorophyll levels in media with 35 nM NFZ. Data correspond to the mean and SEM values of n≥3 independent experiments and asterisks mark statistically significant differences (<i>t</i> test: p<0.05) relative to WT samples.</p

Both Hsp70 chaperone and Clp protease plastidial systems are required for protection against oxidative stress

<p>Environmental stress conditions such as high light, extreme temperatures, salinity or drought trigger oxidative stress and eventually protein misfolding in plants. In chloroplasts, chaperone systems refold proteins after stress, while proteases degrade misfolded and aggregated proteins that cannot be refolded. We observed that reduced activity of chloroplast Hsp70 chaperone or Clp protease systems both prevented growth of <i>Arabidopsis thaliana</i> seedlings after treatment with the oxidative agent methyl viologen. Besides showing a role for these particular protein quality control components on the protection against oxidative stress, we provide evidence supporting the existence of a yet undiscovered pathway for Clp-mediated degradation of the damaged proteins.</p

Blockage of PGE results in protein aggregation in chloroplasts.

<p>(A) TGX Stain-Free gel showing protein fractions from isolated Arabidopsis chloroplasts treated with LIN for the indicated times. An untreated control is also shown. Following ultracentrifugation of chloroplast lysates, supernatant and pellet fractions were collected (corresponding to non-aggregated and aggregated proteins, respectively) and separated by SDS-PAGE. Bands of aggregated proteins whose intensity increased after LIN treatment are marked with black arrowheads; the white arrowhead marks a major band whose intensity did not increase. (B) Quantification of total protein levels from TGX Stain-Free gel runs corresponding to chloroplast lysates before ultracentrifugation (labeled as “total”) and after separation of non-aggregated and aggregated protein fractions. Protein levels are represented relative to those in untreated controls and correspond to the mean and SEM values of n = 3 independent experiments. Asterisks marks statistically significant difference (<i>t</i> test: p<0.05) relative to the untreated sample. (C) Confocal microscopy detection of GFP (green) and chlorophyll (red) fluorescence in chloroplasts of siblings harboring the same T-DNA insertion with the <i>35S</i>:<i>DXS-GFP</i> construct in WT or <i>svr8-2</i> mutant backgrounds. The images were obtained with the same confocal parameters and are to the same scale. They correspond to chloroplasts from the cotyledons of 10-day-old seedlings grown in the presence of absence of 15 μM LIN. (D) Quantification of DXS protein levels detected by immunoblot analysis of insoluble protein fractions isolated from leaves infiltrated with the indicated inhibitors. Results correspond to the mean and SEM values of n = 3 independent experiments are represented relative to those in NFZ-treated samples.</p

Inhibitors and mechanisms modulating metabolic flux to isoprenoids in chloroplasts.

<p>(A) Schematic representation of: (1) MEP pathway and derived products, with the position of enzymes (DXS, DXR) and inhibitor (FSM, NFZ) targets; (2) Hsp70-dependent pathways for misfolded and aggregated forms of DXS to be degraded (via ClpC1 and the Clp protease) or, alternatively, reactivated (via ClpB3); and (3) proposed mechanism by which interference with PGE (e.g. with LIN) impacts the activity of the Clp protease complex, based on the production of the plastome-encoded ClpP1 subunit. Red arrow represents the stress-induced refolding pathway. Dashed arrows represent multiple steps. GAP, glyceraldehyde 3-phosphate; DXP, deoxyxylulose 5-phosphate. See text for other acronyms. (B) Quantification of the inhibitor resistance phenotype estimated from chlorophyll levels in the absence (100%) or presence of inhibitors. (C) Representative images of Arabidopsis WT (Columbia) seedlings germinated and grown for 10 days under LD in the presence of the indicated concentrations of inhibitors.</p

Pre-treatment with a chemical chaperone desensitizes the molecular response to LIN.

<p>Quantitative (qPCR) analysis of transcript levels of the indicated genes in 7-day-old WT plants grown in the presence or absence of glycine betaine (GB) and then transferred for 2h to fresh medium supplemented with 400 μM LIN. Data correspond to the mean and SEM values of n = 3 independent experiments and they are represented relative to the levels before the LIN treatment. For all the genes tested, statistically significant differences (<i>t</i> test: p<0.05) were found between the LIN-triggered induction of control and GB-grown samples.</p

Resistance to FSM and NFZ is improved by disrupting PGE with LIN.

<p>(A) Resistance of WT plants and the indicated mutants was estimated by quantifying SE after germination and growth on media supplemented with 15 μM LIN (L), 30 μM FSM (F), or both (F+L) relative to non-supplemented medium. (B) Resistance of WT plants quantified as CHL levels in media supplemented with 15 μM LIN (L), 30 μM FSM (F), 35 nM NFZ (N) or the indicated combinations relative to non-supplemented medium. Data correspond to the mean and SEM values of n≥3 independent experiments.</p

Interference with PGE promotes the accumulation of soluble DXS protein.

<p>(A) Immunoblot analysis of Hsp70, ClpB3, ClpC and DXS levels in WT plants and PGE-defective mutants <i>rif10-2</i> and <i>svr8-2</i>. PGE was also blocked in WT plants germinated and grown in the presence of 15 μM LIN (WT+LIN sample). Representative images of the plants used for immunoblot analysis are shown on top (bar, 5 mm). Graph shows the quantification of immunoblot data from n≥3 experiments represented as mean and SEM values relative to untreated WT plants. Asterisks mark statistically significant differences (<i>t</i> test: p<0.05) relative to WT samples. (B) Immunoblot analysis with the indicated antibodies of <i>35S</i>:<i>DXS-GFP</i> plants grown on media with (+) or without (-) 15 μM LIN. Arrowhead marks the position of the DXS-GFP protein. (C) DXS and DXR protein distribution in soluble and insoluble fractions isolated from the indicated samples. A Coomassie-Blue (C) staining of the blots is shown for reference.</p

Model for the cpUPR mechanism in Arabidopsis.

<p>LIN treatment represses PGE and this eventually causes a reduced activity of the Clp protease. Normal Clp protease activity removes misfolded proteins and hence prevents protein aggregation. When Clp protease activity is compromised, however, the misfolded proteins that fail to be degraded (including DXS) aggregate. Build-up of protein aggregates somehow sends an unknown retrograde signal to upregulate the expression of <i>HsfA2</i>, a gene that can also be induced (at much higher levels) by heat stress episodes causing protein aggregation in other cell compartments. <i>HsfA2</i> encodes a transcription factor that in turn induces the expression of target genes encoding chloroplast chaperones such as <i>Hsp21</i> and <i>ClpB3</i>. As a result, more of these chaperones are synthesized and imported into plastids, eventually contributing to alleviate protein folding stress in this organelle.</p

Pre-treatment with a chemical chaperone desensitizes the molecular response to LIN.

<p>Quantitative (qPCR) analysis of transcript levels of the indicated genes in 7-day-old WT plants grown in the presence or absence of glycine betaine (GB) and then transferred for 2h to fresh medium supplemented with 400 μM LIN. Data correspond to the mean and SEM values of n = 3 independent experiments and they are represented relative to the levels before the LIN treatment. For all the genes tested, statistically significant differences (<i>t</i> test: p<0.05) were found between the LIN-triggered induction of control and GB-grown samples.</p

Distribution of GFP-tagged isoprenoid enzymes in chloroplasts of agroinfiltrated <i>N</i>. <i>benthamiana</i> leaves.

<p>Images show representative mesophyll chloroplasts from leaves collected at different days (from 1 to 7) after agroinfiltration with the indicated constructs. For each construct, GFP fluorescence (left columns), chlorophyll autofluorescence (middle columns) and merged images (right columns) are shown. Bars, 5 μm.</p