Supplemental Data. Álvarez et al. (2012). Plant Cell /tpc oas-a1.1. Ponceau!

Transcription

1 A! oas-a1.1 B! oas-a1.1 Col-0 des1-1! Free ATG8 proteins! ATG8-PE adducts! α-atg8! Ponceau! Supplemental Figure 1 online. Detec'on of SAVs (A) and (B) immunoblot analysis of ATG8 accumula'on in oas- a1.1 mutant plants. For the detec'on of SAVs, the plants were grown in soil for four weeks, and senescence- associated vacuoles were visualized by staining of protoplasts isolated from leaves with Lysotracker Red. The yellow fluorescence due to specific staining is superimposed on the chlorophyll- specific red fluorescence. The experiment was repeated at least four 'mes with different batches of plants, and the results were consistent across all replicates. For the immunoblot analysis of ATG8 accumula'on in leaves of Col- 0 wild- type and oas- a1.1 and des1.1 mutant plants, the total lysates prepared from leaves were centrifuged at low speed, and the supernatants were resolved by 15% SDS- PAGE and subjected to immunoblot analysis with an'- ATG8 an'bodies. The Ponceau staining is shown as the protein loading control.

2 A 1.4 Relative gene expression! B 1.40 Relative gene expression! SULTR1.2 SULTR2.1 SULTR 2.2 SULTR3.1 APR1 APR2 APR3 Supplemental Figure 2 online. Relative expression levels of sulfur-responsive genes in Col-0 wild-type plants treated with exogenous sulfide. Real-time RT-PCR analysis of expression of the SULTR1.2 (At1g78000), SULTR2.1 (At5g10180), SULTR2.2 (At1g77990), SULTR3.1 (At3g51895), APR1 (At4g04610), APR2 (At1g62180), and APR3 (At4g21990) was performed in leaves from 20-day-old wild-type plants grown in soil and irrigated with water or 200 µm Na 2 S in water for 10 days (A) and in leaves from 20-day-old wild-type Col-0 plants subjected to carbon starvation in the absence or presence of 200 µm Na 2 S (B). Carbon starvation was induced by placing the plants in darkness for 3 days and allowing them to recover for an additional 5 days before leaves were collected. The transcript levels were normalized to the internal constitutive UBQ10 gene. Data shown are the means ± SD for three independent analyses and represent the transcript levels of each gene in the sulfide-treated Col-0 plants relative to the transcript levels in the untreated plants.

3 Fold change! !! !! !! !! !! 0! 0.00! NIA1! NIA2! NTR1.1! NTR2.1! Supplemental Figure 3 online. Relative expression of nitrate assimilation pathway genes in des1-1 mutant plants.! Real-time RT-PCR analysis of expression of NIA1 (At1g77760), NIA2 (At1g37130), NTR1.1 (At1g12110), and NTR2.1 (At1g08090) was performed in leaves from 30- day-old des1-1 mutant and Col-0 wild-type plants grown in soil. The transcript levels were normalized to the internal constitutive UBQ10 gene. Data shown are the means ± SD for three independent analyses and represent the transcript levels of each gene in the des1-1 mutant plants relative to the wild type plants. ANOVA was performed and asterisks represent statistical differences in gene expression between mutant and wild type,,p< 0.05.!!

4 A! Col-0 des1-1! No-0 des1-2! No addition! Exogenous addition of 200 µm Na 2 S! B! Col-0!! des1-1! No addition! Exogenous addition of 200 µm NH 4 Cl! Supplemental Figure 4 online. Effect of exogenous sulfide and ammonium on the phenotype of Arabidopsis plants under dark-induced carbon starvation. Twenty-day-old wild-type Col-0 and No-0 ecotypes and des1-1 and des1-2 mutant plants grown in soil were subjected to carbon starvation in the absence or presence of 200 µm Na 2 S (A) and in the absence or presence of 200 µm NH 4 Cl (B). Carbon starvation was induced by placing the plants in darkness for 3 days and allowing them to recover for an additional 5 days. The plants were photographed, and a representative image is shown. The experiment was repeated at least three times, and the results are similar across all replicates.

5 Tetrapyrrole synthesis DNA Co-factor and vitamine metabolism Phothosyntesis Metal handling Cell wall Minor CHO metabolism Nucleotide metabolism Redox Major CHO metabolism Lipid metabolism Cell Amino acid metabolism Hormone metabolism Development Stress Signalling Secondary metabolism Miscellaneous RNA Transport Protein Not assigned Number of genes Supplemental Figure 5 online. Gene categorization according to biological processes for the genes with altered transcript levels (induced, black bars; repressed, white bar) in the des1-1 mutant compared to Col-0 plants grown for 30 days in soil under long-day conditions.

6 XERICO! ATG8B! ATG12A! SKP1! AFR! FBW2! SKP2A! UBC11! MMZ2! UBC32! Supplemental Figure 6 online. Diagram of ubiquitin- and autophagy-dependent degradation pathways. Small blue and red boxes represent up-regulated and downregulated genes, respectively, in des1-1 mutants relative to wild-type Col-0 plants grown for 30 days. Some representative genes are highlighted. The diagram was generated using the MapMan application.

7 Log 2 (fold change) Log 2 (fold change) Supplemental Data. Álvarez et al. (2012). Plant Cell /tpc des1-1 vs Col-0 PORB GERMIN3 LTP6 Cys SAG29 LHY1 Galactinol proteinase synthase Cys SAG29 LHY1 Galactinol proteinase synthase des1-2 vs No-0 PORB GERMIN3 LTP Gly-rich Gly-rich -2-6 Cys SAG29 LHY1 Galactinol proteinase synthase Cys SAG29 LHY1 Galactinol proteinase synthase des1-1 + S 2- vs Col-0 + S 2- PORB GERMIN3 LTP6 des1-2 + S 2- vs No-0 + S 2- PORB GERMIN3 LTP6 Gly-rich Gly-rich Supplemental Figure 7 online. Relative expression levels of selected genes in the des1-1 and des1-2 mutant plants. Real-time RT-PCR analysis was performed for Cys proteinase (At2g27420), SAG29 (At5g13170), LHY1 (At1g01060), Galactinol synthase (At1g56600), PORB (At4g27440), GERMIN3 (At5g20630), LTP6 (At3g08770) and Gly-rich protein (At4g29020) in leaves from the des1-1 and des1-2 mutants and their respective wild-type ecotypes. The plants were grown under identical long-day conditions in soil for 30 days (left panels) or for 20 days followed by treatment with 200 µm Na 2 S for 10 additional days (right panels). The transcript levels were normalized to the constitutive UBQ10 gene. Data shown are the means ± SD for three independent analyses and represent the transcript levels in the des1 mutant plants relative to the transcript levels in the respective wild-type ecotypes., P<0.05.

8 3500! SA content (ng/g FW)! 3000! 2500! 2000! 1500! 1000! 500! a b c d 0! Col-0 des1-1 No-0 des1-2! Supplemental Figure 8 online. SA accumula,on in des1 mutant plants. Total SA was quan,fied by HPLC coupled to mass spectrometry in leaf extracts of the des1 mutant and the respec,ve wild- type plant grown in soil for four weeks under long- day condi,ons. Data shown are from three independent experiments and 2- factor ANOVA was conducted. Same lejers indicate no sta,s,cal differences. P< 0.05.

9 Supplemental Table 1 online. List of differentially induced genes in des1-1 mutant plants grown for 20 days in soil under long-day conditions. Locus Fold Change logratio (M) FDR Gene Symbol Gene Title AT2G Plant defensin protein (PDF1.2b) AT3G Cation exchanger 3 (CAX3) AT5G Plant defensin protein (PDF1.2a) AT5G Vegetative storage protein 2 (VSP2) AT2G Glycine-rich protein (GRP3S) AT3G Hevein-like protein (HEL) AT1G Expressed protein AT2G Histone H1-3 (HIS1-3) AT3G Encodes a member of the ERF/AP2 transcription factor family (RAP2.3) AT2G Trypsin inhibitor, putative AT1G Encodes a member of the NAC transcription factor gene family, NAP AT1G HXXXD-type acyl-transferase family protein AT4G Senescence-associated gene 21 (SAG21) AT5G Glutathione S-transferase 16 AT4G Monooxygenase, putative (MO2) AT1G Myb family transcription factor/ production of anthocyanin pigment 2 protein (PAP2)

10 Supplemental Table 2 online. List of differentially regulated genes in des1-1 mutant plants grown for 30 days in soil under long-day conditions categorized within the ubiquitin- and autophagy- dependent degradation functional groups Protein Function Locus Gene Symbol / Gene Title Fold Change Autophagy protein.degradation.autophagy At4g04620 ATG8B 2.37 protein.degradation.autophagy At1g54210 ATG12A 2.08 E2 protein.degradation.ubiquitin.e2 At3g08690 UBC11; ubiquitin-conjugating enzyme protein.degradation.ubiquitin.e2 At1g70660 UEV1B; ubiquitin E2 variant 1B 2.33 protein.degradation.ubiquitin.e2 At3g17000 UBC32; ubiquiting-conjugating enzyme 32 3,33 SCF protein.degradation.ubiquitin.e3.scf.skp At5g57900 SKP1 INTERACTING PARTNER protein.degradation.ubiquitin.e3.scf.fbox At3g12350 F-box family protein 2.09 protein.degradation.ubiquitin.e3.scf.fbox At2g24540 AFR (ATTENUATED FAR-RED RESPONSE) 7.33 protein.degradation.ubiquitin.e3.scf.fbox At3g07870 F-box family protein 4.02 protein.degradation.ubiquitin.e3.scf.fbox At1g78100 F-box family protein 2.14 protein.degradation.ubiquitin.e3.scf.fbox At1g80440 kelch repeat-containing F-box family protein protein.degradation.ubiquitin.e3.scf.fbox At1g70590 F-box family protein 2.28 protein.degradation.ubiquitin.e3.scf.fbox At1g67480 kelch repeat-containing F-box family protein 2.09 protein.degradation.ubiquitin.e3.scf.fbox At1g23390 kelch repeat-containing F-box family protein protein.degradation.ubiquitin.e3.scf.fbox At3g59940 kelch repeat-containing F-box family protein protein.degradation.ubiquitin.e3.scf.fbox At4g08980 F-box family protein (FBW2) 2.1 protein.degradation.ubiquitin.e3.scf.fbox At1g21410 SKP2A 4.59 Ring/ U-box Hect protein.degradation.ubiquitin.e3.ring At4g28270 zinc finger (C3HC4-type RING finger) family protein 5.16 protein.degradation.ubiquitin.e3.ring At5g47050 protein binding / zinc ion binding 3.86 protein.degradation.ubiquitin.e3.ring At2g17450 RHA3A; protein binding / zinc ion binding protein.degradation.ubiquitin.e3.ring At5g01520 zinc finger (C3HC4-type RING finger) family protein 2.66 protein.degradation.ubiquitin.e3.ring At1g18660 zinc finger (C3HC4-type RING finger) family protein 2.12 protein.degradation.ubiquitin.e3.ring At1g49200 zinc finger (C3HC4-type RING finger) family protein 4.69

11 protein.degradation.ubiquitin.e3.ring At1g73760 zinc finger (C3HC4-type RING finger) family protein protein.degradation.ubiquitin.e3.ring At5g41400 zinc finger (C3HC4-type RING finger) family protein 5.15 protein.degradation.ubiquitin.e3.ring At1g79110 protein binding / zinc ion binding 3.0 protein.degradation.ubiquitin.e3.ring At3g47160 protein binding / zinc ion binding protein.degradation.ubiquitin.e3.ring At5g10380 RING1; protein binding / ubiquitin-protein ligase 2.25 protein.degradation.ubiquitin.e3.ring At4g19670 zinc finger (C3HC4-type RING finger) family protein 2.17 protein.degradation.ubiquitin.e3.ring At3g46620 zinc finger (C3HC4-type RING finger) family protein protein.degradation.ubiquitin.e3.ring At1g10650 protein binding / zinc ion binding 3.11 protein.degradation.ubiquitin.e3.ring At1g15100 RHA2A; protein binding / ubiquitin-protein ligase 5.92 protein.degradation.ubiquitin.e3.ring At2g40830 RHC1A; protein binding / zinc ion binding 2.05 protein.degradation.ubiquitin.e3.ring At5g22920 zinc finger (C3HC4-type RING finger) family protein protein.degradation.ubiquitin.e3.ring At2g17730 zinc finger (C3HC4-type RING finger) family protein 2.32 protein.degradation.ubiquitin.e3.ring At5g47610 zinc finger (C3HC4-type RING finger) family protein protein.degradation.ubiquitin.e3.ring At1g55530 zinc finger (C3HC4-type RING finger) family protein 2.1 protein.degradation.ubiquitin.e3.ring At5g17600 zinc finger (C3HC4-type RING finger) family protein 3.89 protein.degradation.ubiquitin.e3.ring At2g04240 XERICO; protein binding / zinc ion binding 2.37 protein.degradation.ubiquitin.e3.ring At1g19310 zinc finger (C3HC4-type RING finger) family protein protein.degradation.ubiquitin.ubiquitin At4g05050 UBQ11 (UBIQUITIN 11) 2.01

12 Supplemental Table 3 online. List of differentially regulated genes in des1-1 mutant plants grown for 20 days in soil under long-day conditions and treated with 200 µm Na 2 S for 10 additional days. Locus Fold Change logratio (M) FDR Gene Symbol Gene Title AT4G AT2G AT5G AT5G AT5G AT5G AT2G AT3G AT2G AT3G AT5G AT4G AT1G AT2G AT2G TAZ zinc finger family protein / BTB/POZ domaincontaining protein Plant defensin-fusion protein, putative (PDF1.2b) Plant defensin protein, putative (PDF1.2a) AP2 domain-containing transcription factor family protein Expressed protein TCH4 / xyloglucan:xyloglucosyl transferase / xyloglucan endotransglycosylase Cell wall protein precursor, putative LTP6 / lipid transfer protein 6 (LTP6) Protodermal factor 1 (PDF1) 60S acidic ribosomal protein P2 (RPP2C) Glycosyl hydrolase family protein 17 Coronatine-responsive tyrosine aminotransferase / tyrosine transaminase Expressed protein Pathogenesis-related protein 1 (PR-1) Plant defensin-fusion protein, putative (PDF2.2)

13 Supplemental Table 4 online. Oligonucleotides used in the present study. DES1 (At5g28030) UBQ10 (At4g05320) SULTR1.2 (At1g78000) SULTR2.1 (At5g10180) SULTR2.2 (At1g77990) SULTR3.1 (At3g51895) APR1 (At4g04610) APR2 (At1g62180) APR3 (At4g21990) NIA1 (At1g77760) NIA2 (At1g37130) NTR1.1 (At1g12110) NTR2.1 (At1g08090) Cys Proteinase (At2g27420) DES1F DES1R UBQ10F UBQ10R SULTR1.2F SULTR1.2R SULTR2.1F SULTR2.1R SULTR2.2F SULTR2.2R SULTR3.1F SULTR3.1R APR1F APR1R APR2F APR2R APR3F APR3R NIA1F NIA1R NIA2F NIA2R NTR1.1F NTR1.1R NTR2.1F NTR2.1R CysProtF CysProtR CACCATGGAAGACCGCGTCTTGAT TCATTCAACTGGCAAATTCTCAGCTT GGCCTTGTATAATCCCTGATGAATAAG AAAGAGATAACAGGAACGGAAACATAGT GGATCCAGAGATGGCTACATGA TCGATGTCCGTAACAGGTGAC ATTGTTGCTCTAACCGAGGCGATT TGTACCCTTTTATTCCGGCGAACG CGACATGTCTTGCGTGATGGGCG GCTCGCTTCAATTTGTGAAGTACCCT CCAGATCCAAATTCATCGGTGA CGGTTCGGTTTTAAACGTGTG CATTGGAGCCAAAAGTTTCGC TCCTCAATCTCAACCACATCAAC GAGCTCCACGGGCTATTAAGTA TTCTCTTTCATTCTTCATCACCC GGAAGAGATCCTCCGTGAAAGC CTGTAACCTCAGAAGCAACAATGGA GGTGCTGGTGTTTCTGGTCACT GGGTCTGGTCGGGTGTTC CGAGACACACAACAGCAACGC GCAACGGGACAGGGGTGA AAAGCTGCCACACACTGAAC ATTGTGCGACTGATAATGTCGT GGCTAACGTGGATGGGAGTG CGGCGTAGCAGCAGAGC TGGCCATTCT TGCTTTCTAT TCCATTTGCACGAAAACAT

14 SAG29 (At5g13170) LHY1 (At1g01060) Galactinol synthase (At1g56600) PORB (At4g27440) SAG29F SAG29R LHY1F LHY1R GalactinolsynF GalactinolsynR PORBF PORBR GTGGAGGAGCCG TCAA GCCCTGACGAGCCGAGAG TCGATTGGAAGGAGAAGCTT AAAGAAATGTTCGGAAGGAA CGAG CTCGTTGCTG CATAC GTGGAAATTTAAAAAATCCTCC AGGTGGTGAG TGATCCAAGC AGTGAGAAGCTCGTGGGC GERMIN3 (At5g20630) LTP6 (At3g08770) Gly-rich protein (At4g29020) SAG21 (At4g02380) SAG12 (AT5G45890) NAP (At1g69490) PR1 (At2g14610) GERMIN3F GERMIN3R LTP6F LTP6R Gly-richF Gly-richR SAG21F SAG21R SAG12F SAG12R NAPF NAPR PR1F PR1R CGTTCCTTAG CGATGCAGAA TCGAAATAACAAAACGCACA CCTTCCAC TGACTGCGAC GTATAGGGTGGAAGCATGGA GCGGTGCAGGTGGTCTA CGACCGATCTTTGGTTTATAG ATTACAGACCCGAAACCG GCACCAGATCATTACCCA CGTCTGGTGTGTTCACTGGA TCATATATCCACTTTCTCCCCA CGTCGATGAAACTTCCAAGG CCGAACCAACTAGACTCCGAAT TCAGTGAGACTCGGATGTG CCTGCATATGATGCTCCTT