BRIEF REPORT |
Uptake of a Fluorescent Dye as a Swift and Simple Indicator of Organelle Intactness : Import-competent Chloroplasts from Soil-grown Arabidopsis
Plant Biochemistry Laboratory (JK,HVS,AM) and Plant Anatomy and Physiology Laboratory (AS), The Royal Veterinary and Agricultural University, Copenhagen, Denmark
Correspondence to: Alexandra Mant, Plant Biochemistry Laboratory, The Royal Veterinary & Agricultural University, 40, Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark. E-mail: ama{at}kvl.dk
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Summary |
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Key Words: carboxyfluorescein diacetate intact organelles Arabidopsis chloroplast glucosinolates protein import
UNDERSTANDING organelle biogenesis relies on the isolation of intact organelles for characterization of import and targeting of nuclear-encoded precursor proteins. Much attention has focused on the biogenesis of chloroplasts. Although these organelles are plant-specific, their protein targeting machinery shares features in common with ER, mitochondria, and the prokaryotic plasma membrane, and they provide an excellent model system in which to study protein-membrane interactions. Indeed, the TAT (twin-arginine translocase) export system was identified first in thylakoids and then in the E. coli plasma membrane. Few species yield import-competent chloroplasts, so most work has exploited pea as a model system. The complete sequencing of the Arabidopsis genome and the ease with which this plant can be manipulated genetically enable us to identify and create useful mutants with altered or missing components of the protein translocation machinery. Progress with Arabidopsis has been hampered by low yields of intact chloroplasts, relatively inefficient protein uptake, and a high degree of nonspecific binding of precursor proteins to the outside of the organelles. These issues have been addressed recently in two studies, the first of which described isolation of chloroplasts from protoplasts (Fitzpatrick and Keegstra 2001) and the second homogenization of entire seedlings (Aronsson and Jarvis 2002
). However, both methods use seedlings grown on sterile medium, which is laborious. We report improved import efficiency of intact chloroplasts isolated from soil-grown Arabidopsis. This rapid method is useful because active chloroplasts can be isolated from batches of plant material used for parallel types of study, e.g., measurements of photosynthetic activity.
Arabidopsis thaliana ecotype Columbia was grown on compost under low light (100 µmol photon m2 sec1) with an 8-hr photoperiod at 20 ± 2C for 45 weeks. Chloroplasts were isolated using a linear Percoll gradient essentially according to the barley method described in Brock et al. (1993) with the following modifications: 10 g healthy leaves were homogenized in 200 ± 50 ml grinding buffer containing 50 mM L (+) sodium ascorbate, in four 2-sec pulses in a kitchen blender equipped with disposable razor blades (Kannangara et al. 1977
), and all centrifugation except the Percoll gradient step was reduced to 1000 x g. Larger quantities of leaf material (2040 g tissue) required 100 mM sodium ascorbate in the initial grinding buffer. Typical yields were 20 ± 5 µg chlorophyll per g FW, which are greater than published yields from similar tissue [5 µg/g FW (Fitzpatrick and Keegstra 2001
); 46 µg/g FW (Rensink et al. 1998
)]. Traditional grinding buffers have sometimes included 110 mM sodium ascorbate or isoascorbate as a general-purpose anti-oxidant (e.g., Walker et al. 1987
). We propose an additional role for sodium ascorbate in protecting Arabidopsis chloroplasts from damage by the breakdown products of glucosinolates. Glucosinolates are abundant in Arabidopsis; tissue damage leads to their hydrolysis by myrosinases (Wittstock and Halkier 2002
). It is well documented that glucosinolate breakdown products are biologically active and can cause enzyme and organelle damage (e.g., Lykkesfeldt and Møller 1993
). L-Ascorbic acid is a specific inhibitor of myrosinases when used at 50100 mM but activates myrosinases at 0.110 mM (Ohtsuru and Hata 1973
). Therefore, it is important to use ascorbate at concentrations
50 mM.
Chloroplast intactness, necessary for protein import assays, is often estimated by phase-contrast microscopy (Walker et al. 1987). Intact chloroplasts are surrounded by a more pronounced halo (Figures 1A and 1C)
compared with broken chloroplasts (Figure 1B). One drawback with this method is that chloroplast envelopes can break, releasing stromal contents, and then reseal, retaining the appearance of intactness (Walker et al. 1987
). Arabidopsis chloroplasts were equilibrated for 5 min with an equal volume of carboxyfluorescein diacetate (CFDA; Molecular Probes, Leiden, The Netherlands), final concentration 0.0025% w/v, and were examined with a Zeiss Photomikroskop II equipped with FITC filters (excitation filter BP 455490, beam splitter FT 510, and emission filter, either "fluorescein-specific" BP 520560 or "nonspecific" LP 520). CFDA fluoresces strongly when de-esterified to carboxyfluorescein (CF). "Ester loading" of nonpolar esterified derivatives of fluorescein is a standard test for cell viability, depending on an intact membrane that prevents leakage of the polar dye into the medium and on the presence of esterases (Oparka and Read 1994
). Chloroplasts that had taken up CFDA fluoresced intensely green (Figure 1E) and corresponded exactly to the ones delineated by a bright halo under phase-contrast (Figure 1D). Up to three Arabidopsis carboxylesterases are predicted to be targeted to the chloroplast stroma (Swissprot Q9M899, Q8L8W1, Q9LT10; Emanuelsson et al. 2000
), implying that the green-fluorescing organelles retain stromal contents as well as an intact envelope. Interestingly, the non-carboxylated dye fluorescein diacetate did not cause fluorescence of chloroplasts (data not shown). Chloroplasts that did not take up CFDA (Figures 1D1F, marked with arrows) were red when all emission wavelengths (F) or only chlorophyll autofluorescence (not shown) were detected using appropriate filters. Red chloroplasts corresponded exactly to the ones without a marked halo under phase-contrast. With the nonspecific filter settings, intact chloroplasts appeared yellowish owing to the presence of both green fluorescein and red autofluorescence. Chloroplasts isolated in the absence of ascorbate (Figures 1G1I) were not visibly damaged and showed a similar degree of intactness as those isolated in the presence of ascorbate (Figures 1D1F).
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We conclude that inclusion of 50 mM ascorbate in the grinding buffer enables the isolation of import-competent chloroplasts. The use of CFDA to identify intact chloroplasts may also be extended to other isolated organelles containing carboxylesterases.
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Acknowledgments |
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We thank Mitsuru Akita and Kenneth Keegstra for giving the clone encoding pea pSSU, Birger L. Møller for invaluable advice and Lynda Fitzpatrick for helpful discussions.
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Footnotes |
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