Autophagy in Tobacco Suspension-Cultured Cells in Response to Sucrose Starvation

Abstract

The response of tobacco (Nicofiana fabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated.. When the cells that were grown i n Murashige-Skoog medium containing 3% (w/v) sucrose were transferred t o the same medium without sucrose, 30 t o 45% of the intracellular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were degraded nonselectively. With the same treatment, protease adivity in the cell, which was measured at p H 5.0 using fluorescein thiocarbamoyl-casein as a substrate, increased 3-t o 7-fold after 1 d. When the cysteine protease inhibitor (2S,3S)-frans-epoxysuccinyl-~-leucylamido-3-methyl-butane (10 p~) was present in the starvation medium, both the protein degradation and the increase i n the protease activity were effectively inhibited. Light microscopy analysis showed that many small spherical bodies accumulated in the perinuclear region of the cytosol 8 h after the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 t o 6 p m in diameter that contained severa1 particles. Quinacrine stained these vesicles and the central vacuole; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1 -naphthylphosphate and P-glycerophosphate as substrates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions. Almost all cellular proteins tum over (Huffaker and Peterson, 1974;Goldberg and St. John, 1976;Davies, 1982; Hershko and Ciechanover, 1982). In mammalian cells the mechanisms underlying cellular protein degradation are divided into lysosomal and nonlysosomal pathways. The lysosomal pathway is thought to be responsible for the degradation of longlived cytosolic proteins and for enhanced protein degradation under stressed conditions. The contribution of the lysosomal pathway to the total cellular protein degradation has been shown to vary from as low as 10 to 30% to as high as 85 to 100% by different researchers using different cells under different conditions (Dice, 1987). In plant cells knowledge about the mechanism of protein degradation is extremely limited compared with that in animal cells. Recently, a ubiquitinproteasome system, which is thought to be a main contributor ' This work was supported by grants-in-aid for scientific research (no. 06740619, no. 07640867) from the Ministry of Education, Science and Culture of Japan to Y.M.* Corresponding author; e-mail moriyasuQmomo1.u-shizuokaken.ac.jp; fax 81-54-264-5099.of the nonlysosomal pathway in mammalian and yeast cells, has been shown to be present in plant cells (Vierstra, 1993). However, the existence of a lysosomal pathway in plant cells has not been demonstrated.Autophagy may contribute to growth and differentiation of plants in many aspects. During plant development many phenomena accompanying autolysis occur at the cellular, tissue, and organ levels,...

Evidence for autophagy-dependent pathways of rRNA turnover inArabidopsis

“…52,76 Autophagy is upregulated during the developmental processes of senescence and cell death as well as during biotic and abiotic stresses such as oxidative stress, drought, nutrient starvation, and pathogen infection. 6,36,[77][78][79][80][81] Upregulation of autophagy in rns2-2 could be caused by a lack of ribosome turnover that results in the accumulation of toxic products and oxidative stress, or it could be the result of a starvation response, since rRNA represents a large proportion of cellular resources. The turnover of ribosomes through autophagy or other mechanisms may be important for homeostasis and the resources recycled through this process, such as bases, nucleosides, nitrogen, or energy, may be needed to maintain housekeeping functions in normal cells.…”

Barley Aleurone Cells Contain Two Types of Vacuoles: Characterization of Lytic Organelles by Use of Fluorescent Probes

“…Evidence from electron microscopy suggests that protein storage vacuoles originated de novo from the rough endoplasmic reticulum and not from fragmentation of the vegetative vacuole . In suspension-cultured tobacco BY-2 cells subjected to sucrose starvation in the presence of cysteine protease inhibitors, acidic organelles containing acid phosphatase activity accumulated in the cytosol (Moriyasu and Ohsumi, 1996). These organelles were seen in cells with well-formed central vacuoles, and the authors speculated that they might be lytic organelles that function in autophagy.…”

“…In sucrose-starved tobacco BY-2 cells treated with cysteine protease inhibitors, a specialized acidic vacuole 1 to 6 m in diameter arose de novo in the cytosol. Although this compartment was not shown to contain proteolytic activities, it appeared coincident with an increase in cellular proteases and with increased protein degradation, and it contained acid phosphatase activity (Moriyasu and Ohsumi, 1996). Acid phosphatase is an enzyme closely associated with lysosomes in animal cells.…”

“…Our data allow us to conclude that barley aleurone protoplasts have at least two types of vacuoles, of which one appears to have features and functions in common with lysosomes. The existence of lysosomes in plants has long been debated (see, e.g., Moriyasu and Ohsumi, 1996). Matile (1975) recognized that catabolic enzymes were essential for sustained biological activity and that these enzymes must be compartmentalized to prevent their indiscriminate hydrolysis of biopolymers.…”

Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene

“…In rice (Oryza sativa)-, sycamore (Acer pseudoplatanus)-, and tobacco (Nicotiana tabacum)-cultured cells, autophagy is induced by Suc starvation (Chen et al, 1994;Aubert et al, 1996;Moriyasu and Ohsumi, 1996). Net protein degradation is induced by carbon starvation in whole maize (Zea mays) plants, most likely by an autophagic process (Brouquisse et al, 1998).…”