Research article
Percoll-purified and photosynthetically active chloroplasts from Arabidopsis thaliana leaves

https://doi.org/10.1016/j.plaphy.2008.06.009Get rights and content

Abstract

The availability of the complete genome sequence of Arabidopsis thaliana and of large collections of insertion mutants paved the way for systematic studies of gene functions in this organism, thus requiring adapting biochemical and physiological tools to this model plant. For physiological analysis of photosynthesis, methods combining high level of chloroplast purity and preservation of the photosynthetic activity were missing. Here, we describe a rapid method (less than 1 h) to obtain Percoll-purified and photosynthetically active chloroplasts from Arabidopsis leaves retaining almost 90% of the Vmax of photosynthesis measured in the starting leaves from plants grown under a light intensity of 150 μmol photon m−2 s−1 and 80% of their initial photosynthetic rate after 3 h of storage.

Introduction

Arabidopsis thaliana is a small flowering plant that is now widely used as a model organism in plant biology. It offers important advantages for basic research in genetics and molecular biology. Since the complete sequencing of its genome and the improvement of efficient transformation methods, a large number of Arabidopsis mutants have been generated. In order to bring insight into the function of mutated genes, existing mutants can be characterized at the physiological level. For instance, the analysis of photosynthetic properties can be useful for the characterization of Arabidopsis mutants affected in the expression of nuclear encoded chloroplast proteins. However, some of these analyses can only be achieved using pure and intact chloroplasts that have conserved their metabolic activities and functional envelope membranes.

Photosynthetically active chloroplasts can be easily purified from leaves of well-established biochemical model plants like spinach or pea [4], [8]. Somerville et al. [19] have described the extraction of active chloroplasts from Arabidopsis protoplasts, but the whole procedure is rather long for routine experiments since collection of stripped leaves and digestion of cell walls already requires 2 h. Furthermore, this method, based on differential centrifugations, allows preparation of an enriched fraction of crude chloroplasts but does not rely on the use of a Percoll gradient. In fact, Mourioux and Douce [15] demonstrated that, more than providing pure preparation of organelles devoid of contaminating intact cells or mitochondria, Percoll-purified chloroplasts maintain their photosynthetic activity over a much longer time when compared to crude preparations. More recently, Kunst [13] published a protocol for the Percoll purification of physiologically active Arabidopsis chloroplasts from leaves. However, no qualitative or quantitative data were presented regarding photosynthetic activity of the purified chloroplast and, in our hands, this method is not compatible with photosynthesis measurements. Finally, extensive attempts have been made by several workers to isolate intact Arabidopsis chloroplasts, compatible with in vitro protein import assays [1], [10], [17]. However, no information is available about the photosynthetic properties of chloroplasts purified using these protocols. Furthermore, the osmoticum concentration used in these methods (0.33 M sorbitol) is not appropriate for the recovery of photosynthetically active chloroplasts (see Section 2.1).

Here, we describe a rapid method, to isolate pure and photosynthetically active chloroplasts from Arabidopsis, by homogenization of leaves followed by isopycnic centrifugation. This was achieved testing the impact of (i) the age of the plants, (ii) the osmoticum concentrations and (iii) the composition of purification and reaction buffers on the integrity of the Percoll-purified chloroplasts and the stability of their photosynthetic properties.

Section snippets

General comments

We tested various conditions for Percoll purification of Arabidopsis chloroplast and systematically investigated these conditions for their impact on the preservation of the photosynthetic activity of the purified organelles. The following parameters were found to be critical to obtain photosynthetically active chloroplasts:

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    When compared to chloroplasts purified from spinach or pea leaves, Arabidopsis chloroplasts appear to be particularly fragile and therefore, all steps of the procedure must

Plant material

Arabidopsis plants, Wassilevskija background (Ws), were grown in growth chambers at 23 °C (12-h light cycle) with a light intensity of 150 μmol photon m−2 s−1. Plants were grown in soil for 3–4 weeks in normal atmospheric conditions, and watered with running water. The yield of photosynthetically active chloroplasts was higher when starting from young leaves. In order to reduce the starch content of plastids, plants were stored in a cold room (in the dark) for 12 h before use.

Purification of intact chloroplasts by isopycnic centrifugation

Arabidopsis chloroplasts

Acknowledgements

We thank Ariane Atteia for helpful discussion and for critical reading of the manuscript. Brigitte Gontero is acknowledged for the gift of the GAPDH antibody.

References (21)

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