Article Figures & Data
Figures
- Figure 1.
Induction of senescence in IDLs of wild-type and atg4a4b-1 plants. A, Photographs of wild-type and atg4a4b-1 plants before (day 0) and after (day 3) the 3-d treatment and their control leaves and IDLs after the 5-d treatment (day 5). The third and fourth rosette leaves, in order of appearance (indicated on the day 0 plants), were individually covered just after bolting and the plants grown for a further 5 d. B, Expression of SAG12, SEN1, RBCS2B, and CAB2B in control leaves and IDLs of wild-type and atg4a4b-1 plants during the 5-d treatment. Total RNA from third and fourth leaves of each plant was isolated and subjected to semiquantitative RT-PCR using gene-specific primers. 18s rRNA was used as an internal standard.
- Figure 2.
Changes in the chlorophyll, nitrogen, soluble protein, and Rubisco protein concentrations in control leaves and IDLs of wild type (white circles) and atg4a4b-1 (black circles) over the treatment period. Data are means ± sd (n = 3).
- Figure 3.
Expression of ATGs in control leaves and IDLs of wild-type and atg4a4b-1 plants over the treatment period. Total RNA from third and fourth leaves of each plant was isolated and subjected to semiquantitative RT-PCR using gene-specific primers. 18s rRNA was used as an internal standard. DAT, Days after treatment.
- Figure 4.
Visible chloroplast catabolism proceeds in IDLs of wild-type plants, but is suppressed in those of atg4a4b-1. A, Differential interference contrast images of chloroplasts in mesophyll cells separated from leaves of wild-type (a) and atg4a4b-1 (d) plants before the treatment, from control leaves of wild-type (b) and atg4a4b-1 (e) plants after the 5-d treatment, and from IDLs of wild-type (c) and atg4a4b-1 (f) plants after the 5-d treatment. Leaves were cut into small pieces, fixed with 3.5% glutaraldehyde, and mesophyll cells were individually dispersed on the glass plate and observed by microscopy. Bars = 10 μm. B and C, Changes in the number (B) and the area (C) of chloroplasts in control leaves and IDLs of wild-type (white columns) and atg4a4b-1 (black columns) plants during the treatment. Chloroplasts in mesophyll cells were identified by those exhibiting chlorophyll autofluorescence during LSCM. The number of chloroplasts per cell and their length and breadth were counted on differential interference contrast images (shown as A) of mesophyll cells separated from leaves. Chloroplast area was calculated using the assumption that chloroplasts were ovals. Data are means ± sd (n = 50 in the no. per cell; n = 45 in the area). Statistical analysis was performed by Tukey-Kramer's HSD test. Different letters in each graph denote differences at P ≤ 0.01.
- Figure 5.
Visualization of stroma-targeted DsRed and chlorophyll autofluorescence in living mesophyll cells of wild-type plants by LSCM. A and B, Fresh control leaves (A) and IDLs (B) excised from plants after the 5-d treatment and observed immediately. C, IDLs from plants after the 5-d treatment incubated with 1 μm concanamycin A in 10 mm MES-NaOH (pH 5.5) at 23°C for 20 h in darkness. Stroma-targeted DsRed appears green and chlorophyll fluorescence appears red. In merged images, overlap of DsRed and chlorophyll fluorescence appears yellow. Vesicles with DsRed are represented by white arrowheads. Chloroplasts having only chlorophyll fluorescence are represented by arrows. D and E, Magnifications of vesicles and chloroplasts having only chlorophyll fluorescence indicated by the dashed-line areas in B and C, respectively. Bars = 25 μm.
- Figure 6.
Detection of chloroplasts exhibiting chlorophyll fluorescence in vacuoles isolated from wild-type IDLs. Vacuoles were released from the thermally lysed protoplasts, which were prepared from 5-d-treated IDLs of plants expressing stroma-targeted DsRed, and were fractionated by a Ficoll density gradient. Stroma-targeted DsRed appears green, chlorophyll fluorescence appears red, and an image by differential interference contrast (DIC) is shown in gray. Some isolated vacuoles accumulated both stroma-targeted DsRed and chloroplasts shown as the overlap of DIC images and chlorophyll fluorescence. Merged images are taken from Supplemental Video S1. Bar = 10 μm.
- Figure 7.
Distinction between RCBs and chloroplasts incorporated into the vacuole by fluorescence spectra. A, Visualization of RCBs and chloroplasts in the vacuole of excised IDLs by LSCM with a spectral detector. Emission between 565 and 615 nm reflecting DsRed appears pseudo-colored green, and emission between 650 and 710 nm reflecting chlorophyll fluorescence appears pseudo-colored red. The merged image is shown in which the overlap of DsRed and chlorophyll appears in yellow. Bar = 10 μm. B, Difference in fluorescence spectra of RCBs and chloroplasts in the vacuole. Spectra between 550 and 710 nm of a chloroplast in the cytoplasm (indicated by circle a in A), a chloroplast in the vacuole (circle b in A), and a RCB (circle c in A) are obtained at 5-nm resolution and are shown as yellow squares, green triangles, and red circles, respectively. Chlorophyll fluorescence of a wild-type chloroplast (gray circles) and DsRed fluorescence of a chlorophyll-free plastid in roots of the transgenic plants (gray triangles) are shown as references. C, Difference in the ratio of peak of DsRed fluorescence (592 nm) per peak of chlorophyll fluorescence (682 nm) of RCBs and chloroplasts in the vacuole. Data are means ± sd (n = 10). Statistical analysis was performed by t test. Different letters denote differences at P ≤ 0.01.
Tables
- Table I.
Quantitative analysis of vacuolar transfers of RCBs, chloroplasts, and stroma-targeted DsRed
RCB and chloroplast columns indicate cell numbers having RCBs or chloroplasts in the vacuole per 100 cell sections observed by LSCM. Intensity of DsRed in the vacuole of wild-type background plants represents the relative intensity of DsRed accumulated in the vacuole as the IDL 5-d + concanamycin A (conA) 20-h criterion. Fluorescence intensity is measured at cell center spots of images captured using the same laser power and detector gain. Values are means ± sd (n = 50). Different letters represent significant difference statistically analyzed by Tukey-Kramer's HSD test (P ≤ 0.01). The correlation coefficient between treatment time and RCB observability is 0.9867.
Treatment Span for Treatment RCB Whole Cp Intensity of DsRed in Vacuole Wild Type atg4a4b-1 Wild Type atg4a4b-1 Control 5 d 0 0 0 0 4.1 ± 2.6d IDLs 1 d 21 0 0 0 20.0 ± 8.1c 3 d 49 0 4 0 32.6 ± 13c 5 d 66 0 5 0 68.3 ± 20b 5 d + conA 20 h 97 0 43 0 100.0 ± 37a
Supplemental Data
Supplemental Figures and Movie
Files in this Data Supplement:
- Supplemental Data - Supplemental Legends
- Supplemental Data - Supplemental Figure 1
- Supplemental Data - Supplemental Figure 2
- Supplemental Data - Supplemental Movie
