Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis

Què he entès? Interpretació de la retroalimentació en l’assignatura de Biologia i Geologia

[cat] Mitjançant l’avaluació reguladora es recullen dades, s’analitzen i es prenen decisions per avançar en el procés d’ensenyament i aprenentatge. Dins d'aquesta presa de decisions un punt rellevant és la retroalimentació que es rep i es dona. Diversos estudis apunten que, en molts casos, la retroalimentació que un docent dona i la percepció que en tenen els alumnes no coincideix, i això pot suposar una barrera a l’aprenentatge. Per aquest motiu, aquí es planteja el disseny de l’assignatura de Biologia i Geologia de 1r d’ESO donant una importància cabdal a la interpretació de la retroalimentació a través de diferents plataformes i, així, fer-ho de manera inclusiva a la vegada que cada alumne pugui seleccionar quina forma de representació li és més útil. A més, també es posarà èmfasi en què els alumnes siguin capaços de donar una retroalimentació efectiva als seus companys. Tot això dins un enfocament de l’assignatura que s’acosta a la realitat actual treballant els continguts a partir dels objectius de desenvolupament sostenible (ODS) amb la finalitat de motivar encara més als alumnes

New insights into plant isoprenoid metabolism

Isoprenoids are a hugely diverse family of compounds derived from the C5 precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Although all free-living organisms synthesize isoprenoids, they are particularly abundant and diverse in plants, with tens of thousands structures known to date. The highest variety of plant isoprenoids are specialized (secondary) metabolites that participate in the interaction of plants with their environment. These include pigments, volatiles, and defense compounds, some of which have applications in industry and agriculture. For example, isoprenoid drugs are used against cancer (taxol) or malaria (artemisin). But plants also synthesize isoprenoids with essential (primary) functions in respiration (ubiquinone), photosynthesis (carotenoids, chlorophylls, tocopherols, phylloquinones, plastoquinone), membrane architecture (sterols), and growth regulation (brassinosteroids, cytokinins, gibberellins, abscisic acid, strigolactones). Despite their economic importance and biological relevance, our knowledge of the core pathways for the production of the universal isoprenoid precursors IPP and DMAPP in plant cells remained incomplete until the mid-1990s. Impressive progress in the last decade has resulted in the complete elucidation of several isoprenoid pathways, the identification of regulatory mechanisms, the discovery of new functions and properties of specific isoprenoids, and the successful manipulation of isoprenoid biosynthesis in a number of metabolic engineering approaches. In this update article, we will discuss some of the most recent advances in the plant isoprenoid field, focusing on the pathways supplying the C5 precursors in plant cells and the novel insights into regulatory matters.Work in our lab is supported by grants from the Spanish Dirección General de Investigación (BIO2011-23680 and PIM2010IPO-00660), Consejo Superior de Investigaciones Científicas (2010CL0039), Generalitat de Catalunya (2009SGR-26), Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (IBERCAROT), and European Union FP7 (TiMet, contract 245143).Peer reviewe

Salinity is a prevailing factor for amelioration of wheat blast by biocontrol agents

Plants exposed to combined abiotic and biotic stress conditions may show enhanced pathogen resistance. Biocontrol agents (BCAs) can further contribute to ameliorate plant responses to these complex situations. In recent years, wheat production has been challenged by the expansion of salt-affected soils as well as by severe outbreaks of emerging diseases such as wheat blast. Here, the role of two BCAs, Pseudomonas stutzeri AN10 and Trichoderma harzianum Th56, was examined in salt-stressed seedlings infected with two Pyricularia oryzae isolates of contrasting aggressiveness. BCAs did not enhance plant tolerance to high salt stress. However, BCAs improved performance of salt-stressed wheat plants infected with the less aggressive Pyricularia isolate. The lower infection in salt-stressed BCAs-treated plants could be due to a salt-induced priming state required to trigger an early expression of plant defence genes.Fil: Cabot, Catalina. Universitat de Les Illes Balears; EspañaFil: Bosch, Rafael. Universitat de Les Illes Balears; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Martos, Soledad. Universitat de Les Illes Balears; EspañaFil: Poschenrieder, Charlotte. Universitat de Les Illes Balears; EspañaFil: Perello, Analia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentin

Differential subplastidial localization and turnover of enzymes iInvolved in isoprenoid biosynthesis in chloroplasts

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts

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

Effect of concanamycin A on DXR-GFP localization.

<p>Pictures show representative images of GFP fluorescence (left columns), chlorophyll autofluorescence (central columns), or both (right columns) in guard cells of <i>35S</i>:<i>DXR-GFP</i> (H line) plants either exposed (+) or not (-). to 10 μM concanamycin A for 24 h. Bars = 5 μm.</p

Differential distribution of DXR-GFP and GGPPS11-GFP proteins in chloroplasts of transgenic Arabidopsis plants.

<p>(<b>A</b>) Immunoblot analysis of protein extracts from <i>35S</i>:<i>DXR-GFP</i> (L line) and <i>35S</i>:<i>G11-GFP</i> (11) plants with an anti-GFP antibody. Results with two different protein extract amounts are shown to illustrate that these lines have very similar levels of the corresponding GFP-tagged protein. (<b>B</b>) Representative images of stomata (upper rows) and mesophyll cells (lower rows) from leaves of the plants analyzed in (A). Images show GFP fluorescence (left columns), chlorophyll autofluorescence (central columns), or both (right columns). Bars, 5 μm (stomata) and 10 μm (mesophyll).</p

Characterization of transgenic Arabidopsis lines producing DXR-GFP.

<p>(<b>A</b>) Relative levels of <i>DXR</i> transcripts in 30-day-old soil-grown wild type plants (C, white column) and <i>35S</i>:<i>DXR-GFP</i> lines (grey and black columns) (n = 6 per group). The box on the right shows images of merged chlorophyll and GFP fluorescence signals in chloroplasts from the guard cells of lines representative of low (L), medium (M) and high (H) transgene expression levels (black columns). (<b>B</b>) Immunoblot analysis of DXR-GFP levels with an anti-GFP antibody in protein extracts (10 μg) from 10-day-old seedlings of the indicated lines. (<b>C</b>) Representative pictures of seedlings of the indicated lines germinated and grown for 10 days in the presence of fosmidomycin (50 μM). (<b>D</b>) Quantification of the phenotype observed in (C) as the percentage of seedling establishment (SE) and chlorophyll content (CHL) in the presence of fosmidomycin relative to those in the absence of inhibitor.</p