Supplemental Data. Wang et al. (2013). Plant Cell /tpc

Transcription

1 Supplemental Data. Wang et al. (2013). Plant Cell /tpc Supplemental Figure 1. 3-MA Treatment Reduces the Growth of Seedlings. Two-week-old Nicotiana benthamiana seedlings germinated on MS plates containing or not containing 5 mm 3-MA.

2 Supplemental Figure 2. Reduced Expression Levels of ATG Genes in the Silenced Plants. We used the tobacco rattle virus (TRV)-based vectors to silence ATG genes in Nicotiana benthamiana. RT-PCR analyses of ATG transcripts in the leaves of the silenced plants were performed using the gene-specific primers listed in Supplemental Table 2. EF1A was used as an internal control.

3 Supplemental Figure 3. Autophagosomes Are Rarely Observed in ATG6-Silenced Plants Imaged by Laser-Scanning Confocal Microscopy.

4 (A) Visualization of autophagosomes during the night by MDC staining. Leaves were harvested from ATG6-silenced plants exposed to darkness for 0 h, 4 h, or 8 h. Structures that incorporated MDC are green, whereas chloroplasts are red. Scale bars = 10 μm. (B) Visualization of autophagosomes during the night using the autophagy marker, CFP-ATG8f. Leaves were harvested from ATG6-silenced plants exposed to darkness for 0 h, 4 h, or 8 h. The CFP-ATG8f fusion protein appears cyan and chloroplasts appear red. Scale bars = 10 μm.

5 Supplemental Figure 4. Characterization of Starch Granules and SSGLs. (A) Average number of the visible starch granules per chloroplast. Leaves exposed to darkness for 0 h, 2 h, 4 h or 8 h were harvested for the TEM (Transmission Electron Microscope) analysis. More than a hundred chloroplasts in about ten mesophyll cells were used for the quantification of visible starch granule numbers and calculation of average number of visible starch granules per chloroplast. Values represent means±se. (B) Distribution of starch granule diameter at the end of the day. The diameter of

6 starch granules accumulated in chloroplasts was measured by using the TEM images. Sixty-five data points (triangles) were collected and presented. The black solid line represents the mean of the data. (C) Distribution of SSGL diameter observed outside of the chloroplast. The diameter of SSGLs was measured using TEM images of leaves exposed to darkness for 2 h and 4 h. Fifty-five data points (diamonds) were collected and presented. The black solid line represents the mean of the data.

7 Supplemental Figure 5. The Starch-Excess Phenotype in ATG6-Silenced Plants Is Due to the Reduced Starch Degradation During the Night. (A) Iodine staining of leaves harvested at the end of the day showed that ATG6-silenced leaves accumulate more starch than the non-silenced ones. At least two replicate leaf samples were used for the staining. (B) and (C) Destarch assay revealed that starch overaccumulation in ATG6-silenced leaves is caused by reduced degradation rather than enhanced synthesis. After

8 exposure to darkness for 60 h, ATG6-silenced and non-silenced plants were returned to a regular 16-h light/8-h dark cycle (time point 0 h). The leaf starch content was measured during the next two days at different time points. (B) Leaf starch contents of ATG6-silenced (red circles) and non-silenced plants (black squares). Values represent means±se from at least two replicate leaf samples. White and black bars on the bottom indicate days and nights, respectively. (C) Percentage of starch degraded in ATG6-silenced (red column) and non-silenced (black column) plants during each night as a percentage of the starch synthesized during the day. The values are determined according to the starch content measured at each time point in (B).

9

10 Supplemental Figure 6. Autophagosome Formation Is Reduced in Other ATG-Silenced Plants Examined by Laser-Scanning Confocal Microscopy. We monitored the autophagic activity in leaves of ATG-silenced and non-silenced plants using the autophagy marker, CFP-ATG8f. Enhanced autophagosome formation was usually detected in the leaves of non-silenced plants exposed to darkness for 4 h (VIGS-Vector), while few autophagosomes were observed in the ATG-silenced plants. The CFP-ATG8f fusion protein appears cyan and chloroplasts appear red. Scale bars = 10 μm.

11 Supplemental Figure 7. Ultrastructural Analysis of Silver Proteinate-Stained SSGLs (A) Control treatments for demonstration of starch components in SSGLs with PA-TCH-SP method. No silver grains were deposited on the starch granules and SSGLs (arrows) in the control sections (stained with TCH-SP or PA-SP or SP) and therefore they appeared electron-translucent, similar with the appearance of starch components in the unstained sections. However, intensive staining of the starch granules and SSGLs was observed in the sections treated with PA-TCH-SP. The results clearly demonstrated the SSGLs were starch components. The blue, cyan, red arrows indicate the unstained SSGLs in the vacuole, cytoplasm and autophagosome-like structures, respectively. The yellow arrow indicates the silver-stained SSGL in the vacuole. Osmiophilic granules (magenta arrowheads) were often observed in the chloroplasts. They appeared electron-dense in the non-oxidized sections (Unstained, TCH-SP, SP) and electron-lucent in the oxidized sections (PA-SP,

12 PA-TCH-SP) as the result of removal of the tissue-bound osmium from the sections by PA oxidation. The red asterisk refers to an isolation membrane (the autophagosome precursor). (B) Three types of silver proteinate-stained SSGLs in the vacuole. The red, yellow, and blue arrows refer to SSGLs engulfed by a single-membrane vesicle, SSGLs located directly in the vacuole, and SSLGs that had almost completely degraded, respectively. Areas enclosed by the magenta dashed line indicate the representative diffuse silver depositions which are supposed to be stained glucans released from the SSGLs. S, starch; V, vacuole; Cp, chloroplast; M, mitochondrion; CW, cell wall. Scale bars = 500 nm.

13

14 Supplemental Figure 8. Developmental Phenotypes and Leaf Starch Content in Wild-Type Arabidopsis and atg Mutantst. (A) Two-week-old atg2, atg5, and atg9 seedlings germinated on rich medium showed no significant difference in growth compared with wild-type Columbia seedlings. Scale bars = 1mm. (B) Quantification of starch content in the atg mutant seedlings germinated on rich medium at the end of the night. Approximately 25 seedlings were harvested and regarded as one sample for the starch quantification test. Values are means ± SE from four replicate samples. DW, dry weight. (C) The six-week-old soil-grown atg2, atg5 mutants showed reduced growth and early senescence while atg9 showed no significant difference compared with wild-type Columbia. Scale bars = 1cm. (D) Quantification of leaf starch content in the soil-grown atg mutants at the end of the night. Leaf samples were harvested from the six-week-old Arabidopsis plants grown under a 16-h light/8-h dark cycle Values are means±se from two more replicate samples. FW, fresh weight.

15 Supplemental Figure 9. Ultrastructure of Fragmented Starch Granules in Leaves Exposed to Darkness. Two fragmented starch granules (indicated by blue squares) in the silver proteinate-stained sections are shown in (A) and (B), respectively. The arrowheads refer to the Small starch granule-like structures (SSGLs) segmented from the main body of the starch granule. S, starch; V, vacuole. Scale bars = 500 nm.

16 Supplemental Table 1. List of Primers Used in This Study Purpose Gene name Primer name Primer sequence(5 to 3 ) ATG2 ATG2-F GCGTAAGGACAGCCTCAATC RT-PCR ATG2-R CTGAAACTAATGGCATGGTC for confirming ATG3 ATG3-F TACTGTCGCAGAAGATTCAC gene silencing ATG3-R CTTTCTTTAGGTTTCCCATG ATG5 ATG5-F AAGCTCATACGCATTCAGGG ATG5-R GCTTCGGACCTTTGCTACCT ATG6 ATG6-F CCTTCTTCTTCATACAATGGCTCA ATG6-R AGACGGTTGCAGAAAGTGGC ATG7 ATG7-F TTGCTCCTATTGCATCAGCC ATG7-R ATTATCCCAGTCCAGCGTGT PI3K PI3K-F TTAGCAACTGGGCACGATGA PI3K-R CGGTGGAAAGGGCTTAGGAT VTI12 VTI12-F CAAGTGACAGAATCAGGGAG VTI12-R AAAGTAAACCCGTAGAGCAT EF1A EF1A-F CCCCTTCGTCTTCCACTTCA EF1A-R GCTTGGTCGGCATCATCTTA Real-time RT-PCR ATG1c ATG1c-F TGGAAAGTCCCTCATCTGCACCTG ATG1c-R GCCTGCCTACCTCAACCTTCTCAT ATG2 ATG2-F GCAATTGGGCTTGGAGTGCATTTG ATG2-R CCTGTCGGGCATCTCTAGGTTGAT ATG4 ATG4-F GGCGAAGCTGACTGGATACCTGTT ATG4-R ATCATCCTGCACGCCGACAATGTA ATG6 ATG6-F ACCTGCGTAAAGGAGTTTGCTGAC ATG6-R AGAGCTTTGGTCCAACTTTCCTGC ATG7 ATG7-F CCAGCAGTGGAAGCAGAAGGTCTT ATG7-R GCCACCGACTTTCCCGTGTATCA ATG9 ATG9-F TCCGGTGGATTTGCTGCTGTTCT ATG9-R ACATCGCTCCCTGTGGGTCAAG PI3K PI3K-F GCTGTGCTGGTTACTCCGTCATC PI3K-R ACTGACTTTCCGCTCCACCCATA ACTIN ACTIN-F CCCAGAGAGGAAATACAGTG SEX1 ATG6 ACTIN-R SEX1-F-for silencing test SEX1-R-for silencing test ATG6-F-for silencing test ATG6-R-for silencing test CAATAGACGGACCAGATTCG GTTATGCCGGTGCTGGCCTCTAT TTCCATCCCTCACTACACCCTCGA CCTCTGTCAGAACTGCCACAATCC TCCCTGGTCCCTGATTTCTTTGCT

17 Overlapping PCR amplification of CFP-ATG8f Overlapping PCR amplification of GBSSI-YFP CFP CFP-F CgACgACAAgACCgTgACCATGGTGAG CAAGGGCGAGGAGCTGT CFP-R CTTGTACAGCTCGTCCATGCCGAGA ATG8f ATG8f-F ATGGACGAGCTGTACAAGATGGCAA AGAGTTCATTCAAGCAAG ATG8f-R gaggagaagagccgtttacaccaagttg AGGTCGCCAAAT GBSSI GBSSI-F CgACgACAAgACCgTgACCATGGCAAG CATCACAGCTTCACACT GBSSI-R AGGAGTGGCCACATTTTCCTTGGCA YFP YFP-F GAAAATGTGGCCACTCCTATGGTGAG CAAGGGCGAGGAGCTGT YFP-R gaggagaagagccgttcacttgtacagct CGTCCATG