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

Catalina Perello,Pablo Pulido,Miriam Ortiz-Alcaide,Vincent Burlat,Michael A Phillips,Ernesto Llamas,Manuel Rodriguez-Concepcion,Miguel A Blazquez

doi:10.1371/journal.pone.0150539

Abstract

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.

Highlights

Plant chloroplasts are active metabolic machines that fix carbon using the energy of sunlight to produce myriad compounds that support plant growth and development and contribute to their environmental adaptation
The first two enzymes of the methylerythritol 4-phosphate (MEP) pathway, deoxyxylulose 5-phosphate synthase (DXS) and DXP reductoisomerase (DXR), have been proposed to contain bipartite sorting sequences for subplastidial targeting to the thylakoid membrane or lumen [15,16]
As a first step to test the computational analysis predictions, we investigated whether the endogenous Arabidopsis enzymes were associated to thylakoids or associated structures such as plastoglobules by separating them using flotation centrifugation with sucrose density gradients [32] and analyzing the presence of DXS and DXR in the fractions by immunoblot analysis (Fig 1A)

Summary

Introduction

Plant chloroplasts are active metabolic machines that fix carbon using the energy of sunlight to produce myriad compounds that support plant growth and development and contribute to their environmental adaptation. The enzymes that participate in these metabolic pathways are typically encoded by genes in the nuclear genome and they must be imported into plastids for biological activity. Complexes at the outer and inner chloroplast envelope membranes import most plastidial proteins into the stroma [1]. After proteolytic removal of the N-terminal plastid targeting sequence, the mature proteins with additional targeting signals are further directed to specific subplastidial compartments, including thylakoid membranes and lumen [2,3]. When normal activity ceases or environmental challenges such as excess light, temperature peaks, PLOS ONE | DOI:10.1371/journal.pone.0150539. When normal activity ceases or environmental challenges such as excess light, temperature peaks, PLOS ONE | DOI:10.1371/journal.pone.0150539 February 26, 2016

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