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BIOENERGETICS AND PHOTOSYNTHESIS

Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by an ATG Gene-Dependent Autophagic Process^1,[W],[OA]

Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981–8555, Japan (H.I., M.I., Y.Y., A.M., T.M.); Department of Cell Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444–8585, Japan (K.Y., Y.O.); and Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 (D.R., M.R.H.)

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H⁺-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.

² Present address: RIKEN Plant Science Center, Suehiro-cho 1–7–22, Tsurumi-ku, Yokohama 230–0045, Japan.

³ Present address: Bitplane AG, Badenerstrasse 682, CH–8048 Zurich, Switzerland.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hiroyuki Ishida (hiroyuki{at}biochem.tohoku.ac.jp).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.122770

^* Corresponding author; e-mail hiroyuki{at}biochem.tohoku.ac.jp.

Received May 10, 2008; accepted July 1, 2008; published July 9, 2008.

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