Supplemental Data. Wang et al. (2014). Plant Cell 10.1105 ... · Supplemental Figure 3....
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Supplemental Figure 1. The dynamics of CCV localization (A) The transient expression of CV-GFP in cotyledon mesophyll cells of Col-0 and transgenic plants expressing stroma-targeted DsRed (CT-DsRed); Bar=20cm (B) CV-GFP is localized in both chloroplast and cytosols in epidermal cells (white arrow) of cotyledons. Bar=20cm (C) Time lapse observation of CCV released from chloroplast. The photography lasts for 10 s and the interval is 2 s. Blue arrows indicate the movement of CCV departing from the chloroplast. Bar=10μm. (D) DEX-induced expression of CV in mesophyll cells of true leaves from transgenic plant DEX-CV-GFP/CT-DsRed. The seedlings were cultured in liquid MS/2 media with 10μM DEX for 24h. Yellow arrows indicate that CCVs is localized in the cytosol (outside of chloroplast). Bar=20μm. Supplemental Data. Wang et al. (2014). Plant Cell 10.1105/tpc.114.133116 1
Transcript of Supplemental Data. Wang et al. (2014). Plant Cell 10.1105 ... · Supplemental Figure 3....
Supplemental Figure 1. The dynamics of CCV localization
(A) The transient expression of CV-GFP in cotyledon mesophyll cells of Col-0 and transgenic plants expressing stroma-targeted DsRed (CT-DsRed); Bar=20cm (B) CV-GFP is localized in both chloroplast and cytosols in epidermal cells (white arrow) of cotyledons. Bar=20cm (C) Time lapse observation of CCV released from chloroplast. The photography lasts for 10 s and the interval is 2 s. Blue arrows indicate the movement of CCV departing from the chloroplast. Bar=10μm. (D) DEX-induced expression of CV in mesophyll cells of true leaves from transgenic plant DEX-CV-GFP/CT-DsRed. The seedlings were cultured in liquid MS/2 media with 10μM DEX for 24h. Yellow arrows indicate that CCVs is localized in the cytosol (outside of chloroplast). Bar=20μm.
Supplemental Data. Wang et al. (2014). Plant Cell 10.1105/tpc.114.133116
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Supplemental Figure 2. Alignment of CV homologous genes in the plant kingdom
The sequences were assembled by Cobalt Constraint-based Multple Protein Alignment Tool (http://www.ncbi.nlm.nih.gov/tools/cobalt/). AT: Arabidopsis thaliana (AT2G25625) ; BD:Brachypodium distachyon (BD2G15690); FV: Fragaria vesca (FV2G40830); GM: Glycine max (GM04G07440); MD: Malus domestica (MD00G406410); ME: Manihot esculenta (ME00847G01190); MT: Medicago truncatula (MT3G107890); VV: Vitis vinifera (GenBank: CAN65725.1); OS:Oryza sativa (OS05G49940); PT: Populus trichocarpa (PT06G24730); RC: Ricinus communis (RC29912G02840) ; SL: Solanum lycopersicum (Solyc08G067630); SB: Sorghum bicolor (SB09G029300); TC: Theobroma cacao (TC09G013320)
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Supplemental Figure 3. Immunolabeling TEM of CV-GFP in DEX-CV-GFP and Col-0
Detection of GFP by immuno-labeling TEM in cotyledon mesophyll cells of DEX-CV-GFP transgenic plants (A) and wild type Col-0 (B, C) cultured in liquid MS/2 media containing 10μM DEX for 24h. Blue frame indicated the position cropped for Figure 2E. Bar=1μm. (D) Distribution of gold particles representing CV protein in chloroplast (including disassembled chloroplast and CV-containing vesicles) and vacuole (including other organelles than chloroplasts). We quantified total 1276 gold particles from three different observations.
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Supplemental Figure 4. Detection of CV-HA and PsbO1 by double immunolabeling TEM analysis
(A, B) Double immuno-labeling of CV-HA with anti-HA antibody (small dots, 5nm gold particles), as indicated by red arrow in (B), and PsbO with anti-PsbO antibody (large dots, 20nm gold particles), as indicated by blue arrow in (B). Red frame in (A) indicated the position of (B). The 5-day-old plants of transgenic line DEX-AtCV-HA (DEX-3) were cultured in MS/2 media with 10μM DEX for 1 week. The leaf sections were double immunolabeled with anti-HA antibody (from mouse) and anti-PsbO antibody (from rabbit) overnight. Then, leaf sections were treated subsequently with 5nm gold-conjugated goat anti-mouse IgG and 20nm gold-conjugated goat anti-rabbit IgG for 1h. (A) Bar=2μm. (B) Bar=0.5μm.
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Supplemental Figure 5. CCVs contain Tic20-II
(A) Transient expression of Tic20-II-RFP in cotyledon cells of Col-0 plants. Bar=10 μm. (B) Transient co-expression of CV-GFP and Tic20-II-RFP in cotyledon cells. White arrows indicate the CCVs departing from chloroplast or aggregating in the cytosol. Orange arrows indicate CV-GFP localized in chloroplasts. Bar=10 μm
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Supplemental Figure 6. Autophagy is not involved in formation and trafficking of CCVs
(A) Transient expression of CV-RFP in cotyledon cells of transgenic plants expressing GFP-ATG8a. The green bodies, as indicated by green arrows, are autophagosomes. The red vesicles, as indicated by red arrows, are CCVs. Bar=20μm. (B) Transient expression of CV-GFP in cotyledon cells of autophagy-defective mutant atg5-1. Yellow arrows indicate the CCVs released from chloroplasts. Bar=10 μm
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Supplemental Figure 7. CCVs do not overlap with SAVs
(A) Transient expression of CV-GFP in cells of cotyledon stained by fluorescence dye Lysotracker-Red. Yellow arrows indicated the single or aggregations of CCVs. Bar=20μm. (B) Co-transient expression of CV-GFP and SAG12-RFP in cotyledon cells. Bar=10 μm
Supplemental Data. Wang et al. (2014). Plant Cell 10.1105/tpc.114.133116
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Supplementary Figure 8. CV overexpression enhanced salt stress-induced chloroplast degradation (A, B) The morphology (A) and total chlorophyll content (B) of the seedling from Col-0 and DEX-CV-GFP were cultured in the MS/2 media in absence or presence of DEX and 50mM NaCl for 3 weeks. Asterisk indicates significant difference at P<0.001 compared to CV-GFP plants at control conditions.
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Supplemental Figure 9: Silencing CV increased chloroplast stability
(A) 36-day-old plants of Col-0 and CV artificial-miRNA-silencing lines (amiR-1, amiR-2, and amiR-3). White bar=5cm. (B, C) Morphology (B) and total chlorophyll content (C) of young seedlings of Col-0 and AtCV silencing lines (amiR-1, amiR-2, and amiR-3) were cultured in MS/2 media with or without 5μM methyl viologen. Asterisk indicates significant difference at P<0.001 as compared to Col-0 plants treated with MV. Black bar=5cm.
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Supplemental Figure 10: Silencing CV increased drought tolerance
(A) Plants of Wild type Col-0 and CV-silenced transgenic lines (amiR-1,-2 and -3) were subjected to water stress for 14 days and rewatering for 4 days. Bar=10cm. (B) Survival rate was determined 4 days after rewatering, as described in (A). 15 plants for each line were evaluated and Mean±SD were obtained from three independent experiments. Asterisk indicate significant difference at P<0.001 compared with Col-0. (C) CV expression in leaves from Col-0 and amiR-1 plants during drought treatment were determined by quantitative RT-PCR.
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Supplemental Figure 11: CCVs contain thylakoid proteins CYP20-2 and FtsH1 but not plastoglobule protein
(A) Transient expression of CV-YFP in cotyledon cells of transgenic plants expressing CYP20-2-CFP. Bar=10μm. (B) Transient expression of CV-GFP in cotyledon cells of transgenic plants expressing FtsH1-CFP. Whit arrow indicated the CCV outside of chloroplasts. Bar=10μm. (C) Transient expression of CV-RFP in cotyledon cells of transgenic plants expressing PGL34-YFP. Yellow and red arrow indicated PGL34-YFP and CV-RFP, respectively. Bar=10μm
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Figure 12. A proposed model of CV-mediated chloroplast degradation
Senescence and abiotic stress can activate CV expression. CV protein (green) targets chloroplast and are mostly associated with membranes of chloroplast envelope and thylakoids. CV destabilizes chloroplast and induces vesicle formation. The CV-containing vesicles (CCVs) are formed from the disrupted chloroplast membrane. CCVs also contain stromal protein (Red) and thylakoid proteins (Blue) and will be released from chloroplasts. CCVs could be aggregated in pre-vacuolar compartment and eventually transported to vacuole for degradation.
Supplemental Data. Wang et al. (2014). Plant Cell 10.1105/tpc.114.133116
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Function Protein Name Accession Number Number of
peptides identified
Subcellular Localization
CV (Bait) AT2G25625 17 Photosystem II PsbO1, Photosystem II subunit O-1 AT5G66570 16 Thylakoid lumen
PsbO2, Photosystem II subunit O-2 AT3G50820 3 PsbC (CP43), Photosystem II reaction center protein C ATCG00280 9
Thylakoid membrane
PsbA(D1), Photosystem II reaction center protein D1 ATCG00020 4 NAD(P)H dehydrogenase complex
CYP20-2, Cyclophilin 20-2 AT5G13120 14 Thylakoid lumen ndhI, NAD(P)H dehydrogenase complex subunit I ATCG01090 8 Thylakoid membrane ndhJ, NAD(P)H dehydrogenase complex subunit J ATCG00420 6 ndhB.1, NAD(P)H dehydrogenase complex subunit B.1 ATCG00890 3
Protein degradation DegP1, DegP protease 1 AT3G27925 8 Thylakoid membrane FTSH 1, FTSH protease 1 AT1G50250 4
Chloroplast mobilization CHUP1, Chloroplast Unusual Positioning 1 AT3G25690 4
Outer envelope membrane
Protein folding HSP70-2, chloroplast Heat Shock Protein 70-2 AT5G49910 10 stroma Carbon metabolism FBA2, Fructose-bisphosphate aldolase 2 AT4G38970 6
CA1, Carbonic anhydrase 1 AT3G01500 3 Cytoskeleton TUA2, Tubulin alpha-2 chain AT1G50010 4 cytosol
ACT2, Actin-2 AT3G18780 4
Supplemental Data. Wang et al. (2014). Plant Cell 10.1105/tpc.114.133116
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Supplemental Table 1: Proteins interacting with CV as identified by coimmunoprecipitation and Mass SpectrometryThree independent immunoprecipitations were performed. The proteins in Table I were identified in three replicates of Co-IP samples from DEX-
CV-HA-3 plants but not in samples from Col-0. The Number of peptides in the table is the average of three replicates
Protein degradation RD21A, Cysteine proteinase RD21 AT1G47128 4 Vacuole VHA-A, Vacuolar ATPase subunit A AT1G78900 5 Vacuolar membrane
VAB1, Vacuolar ATPase subunit B1 AT1G76030 3
Supplemental Data. Wang et al. (2014). Plant Cell 10.1105/tpc.114.133116
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Supplemental Table 2: Primers designed for this study
F: Forward primer; R: Reverse primer
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