Figure 4: Interference of OsGAS2 expression induced autophagy in rice suspension cells.

(a) Real-time quantitative RT-PCR analysis of OsGAS2 expression in wild type and RNAi rice suspension cells. (b) Cytosolic Ca²⁺ level in wild type and RNAi cell lines under DTT treatment. Protoplasts from wild type and RNAi lines were treated with 6.4 mM DTT for 18h, then stained with 5 μM FLuo 3-AM, the cytosolic Ca²⁺ probe. protoplasts were imaged with a PerkinElmer UltraVIEW VoX confocal microscope. Bar = 5 μm. (c) Quantitative analysis of FLuo 3-AM fluorescence intensity in (b). The fluorescence intensity (%) of the protoplasts was measured by delimiting the individual protoplast and the mean fluorescence of the protoplasts was measured. Data represent the mean ± standard error of 8–10 cells. (d) Detection of autophagy in wild type and RNAi protoplasts. Protoplasts were treated with 6.4 mM DTT with or without 5 mM 3-MA for 18h, and then stained with 50 nm Lytracker red for 20 min at 37 °C. Protoplasts were imaged with a PerkinElmer UltraVIEW VoX confocal microscope. Bar = 5 μm. (e) Detection of autophagosomes in wild type and RNAi protoplasts. Protoplasts were treated with or without 6.4 mM DTT treatment, and then observed under electron microscope. *starch grains in the plastid. n, nucleus. v, vacuole. black arrowheads indicated numerous vesicles which were accumulated in the cytoplasm. Enlarged inset showed a typical double-membrane bound autophagosomes, and white arrowheads point to the inner and outer membranes of the autophagosome. Bar = 2 μm, and represented 0.5 μm in enlarged inset.