Abstract
We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.
Highlights
Plants have various ways of responding to limitations in their nutrient supply to ensure their survival
We showed previously that a protein aggregate formed of the fusion protein of cytochrome b5 (Cyt b5) and red fluorescent protein (RFP) (Cyt b5-RFP) was a good substrate for autophagy in tobacco BY-2 cells (Toyooka et al, 2006)
Many of the aggregates were spherical at 0.5–2 μm in diameter, similar to what we observed for Cyt b5-RFP aggregates (Toyooka et al, 2006)
Summary
Plants have various ways of responding to limitations in their nutrient supply to ensure their survival One such response is the induction of autophagy, which is the digestion by a cell of its own intracellular contents. In contrast to the in planta responses that require days after exposure to Pi-starvation, the induction of autophagy of BY-2 cells under nutrient-depleted conditions including Pi-starvation requires less than 12 h (Toyooka et al, 2006). The loss of Pi in the medium induces autophagy (Toyooka et al, 2006), and causes cell cycle arrest of BY-2 cells at the G1 phase (Sano et al, 2004). In contrast to Pi-starvation, the deprivation of other major nutrients does not stop the cell cycle at a specific phase, cell growth is arrested as in the case of Pi depletion (Sano et al, 2004). In the present study we tested whether the induction of autophagy under conditions of deprivation of different nutrients would interact with each other early in the process, or whether there are specific sensing steps for each nutrient in the cell
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