Supplemental Data. Pan et al. Plant Cell. (2014) /tpc

Transkript

1 Plants Non-plant eukaryotes 93 yanobacteria Signal 1 Signal 2. THLIN MIYRS--LRKLVEINHRKTRPFFTTSGGTV-SLTPPQFSPLFPHFSHRLSPLSK-WFVPLNGPLFLSSPPWKLLQSTPLHWRGNGSVLKKVELN S. EREVISIE MIQMVP--IYSSLLRR TI-PKR PFYHVLSGLTV-R FKV N PQLN H. SPIENS MLLRV--RGSWGLRG WPGTRPSKRR-WLLPPVPLGLE-R WRLRP LG X. LEVIS MSVLLFLSRSPLGLRR LGVTKTW-SRRRHV-FPPLSVLSQLETRPER-----QL------WTRRT LLTG * : *.. * : :.. THLIN -LRLDRIRSRTRFPRQLG-LQ-SVVPNILTVDRNDSKE EDGGKLVKSFVNVPNMISMRLVSGPVLWWMISNEMYSSFLGLVSGSDWLD S. EREVISIE -YNLFRDLTRREY TNP SKTPHIKSKLLNIPNILTLSRIGTPFIGLFIITNNLTPLGLFFSSITDFMD H. SPIENS -LRLPGIGQRNH-SG---G-KPRP-GGEPGGQWGPSTPSLYENPWTIPNMLSMTRIGLPVLGYLIIEEDFNILGVFLGLTDLLD X. LEVIS SPPLPRTFTRIRLSSSRDGHHRPKPK-GHEGDSQ SSLTHLYENPWTIPNMLSMTRIGLSPVLGYLVVGEDFNLLGLFFGMTDLLD * *. :. :...:**:::::*: *.: :: :. *: :*.:. :* :*. THLIN GYVRRMK-INSVVGSYLDPLDKVLIGVVMVQ---KDLLHPGLVGIVLLRDVLVGGVYLRLNLDW---KWKTWSDFFNLDGSSPQKVEPLFIS S. EREVISIE GYIRKYG-LKTIGTILDPLDKLLMITTTLLSVPSGPQIIPVSIIILGRDVLLISLFIRYSTLKLKYPGRVWNSYWDIVRYPSEVRPSQLS H. SPIENS GFIRNWNQRSLGSLDPLDKILISILYVSLTY---DLIPVPLTYMIISRDVMLIVFYVRYRTLPT----PRTLKYFNP-YTRLKPTFIS X. LEVIS GYIRNWNQKSLGSLDPLDKILISVLYVLTY---HLIPVPLTIMIILRDVLISVFYVRYKTLPP----PKTLSRYFNP-YTQLEPTFIS *::**..: *: *******:*: :.:.:: :. ::: *** *...::*.* : :::...:.* :*. THLIN KVNTVFQLTLVGILQPEFGN PDTQTWITYLSWLVSTTMSTYGVQYWKKRPISMIK-RS S. EREVISIE KWNTFFQMVYLGSGVLLLLYEKEEGEKTEEDFEDRKQDFQKFSYLGYVTTTTIMSGVSY---LKRNFKLL---K H. SPIENS KVNTVQLILVSLPVFNY DS-IYLQILWFTFTTSYSYY--HYGRKTVQVIK--D X.LEVIS KMNTVQLILVSLPVFNY VDS-IYLQTLWFITFTTSYSYY--HYGQETVKVLRDDK * **.*: :...: : * : *..* ** * :* :...::. Signal 1 Signal 2. THLIN MIYRSLRKLVEI--NHRKTRPFFTT-SGGTVS-LTPPQFSPL-----FPHF--SH-RLSPLSKWF----VPLNGPLFLSSPPWKLLQSTPLHWRG P. PERSI MVVFRSLKLIE---NPKRSRTFLTTSY--ISPLQYPTLSYPLSHPLFRLPSQISRTFLSPLSNWI----IPFHGPLFLSPPWKLSQSTPLYLRG P. TRIHORP MTIYRSIKTLISKIPNSNKSRSFFTTNST-----II-QSPF--PFHYYYYYPPS--SP-PNRFLSKWIF-LNPFQGPLFLSSPPWKLLQSSPLYLRG R. OMMUNIS MVIYKSLRTLITKN-NNRNRSFVTVTNSII-PSPYTTSPLHY SRFVSKWVSQFQQGQGPLFLSFPPWKLLQSTNPLYLRG *.:::*:: *:.:.*.*.*. :: * *: :*:*. :****** ***** **: **: **. THLIN -NGSVLKKVE-LNLRLDRIRSRTRFPRQLGLQSVVPNILTVDRNDSKEEDGGKLVKSFVNVPNMISMRLVSGPVLWWMISNEMYSSFLGLVSGSD P. PERSI -NGVVLRKIESLSLNLIRRRP--SFPLPFEVGSLSPPTVLDRGVGLKE----SDDFVNLPNLISISRMVSGPLLGWMINEWYSSMVGLISGTD P. TRIHORP -NIVLRKVE-FNLHLLKSRVGSGYVGQGVLSDRVDLVKEEEVDDGGDKDG--ILESFVNLPNFISISRLVSGPLIGWMITNDMYSSFVLIGSD R. OMMUNIS KNVVVLKKVE-LN--LLRSRVGPGLINQRVVDSVQEELKEHLD-----EG--LWKSFINLPNFVSFTRLVSGPVIGWMITNEMYSSFVGLISGTD * **:*:** :. * : * :.....*:*:**::*::*:****:: ***:*: ****::.**::**:*. THLIN WLDGYVRRMKINSVVGSYLDPLDKVLIGVVMVQKDLLHPGLVGIVLLRDVLVGGVYLRLNLDWKWKTWSDFFNLDGSSPQKVEPLFISKVNT P. PERSI WLDGYMRRMKINSVVGSYLDPLDKVLIGVLMVHEGLLHPGLVGLIVFRDVGLVGGVYQRSNLEWKWKSWSDFFNIGGTRPEKVEPLFISKLNT P. TRIHORP WLDGYVRKMRIDSVVGSYLDPLDKVLIGSVLMVHMDLLHPGLVGLVVLRDVLVGVYHRSSLGWKWTSWYDFFNLDGTRPEKVEPLFISKVNT R. OMMUNIS WLDGFIRKMRINSVVGSYLDPLDKVLIGSVLMVHMDLLHPGLVGLVVLRDILVGGIYHRNSLGWKWNSWYDFFNLDGTRPEKVEPLFISKVNT ****::**:*:*:*****************.**:***:.********::::**:.** **:* **.* ***.:* ****:.*: *:*********:**. THLIN VFQLTLVGILQPEFGNPDTQTWITYLSWLVSTTMSTYGVQYWKKRPISMIKRS P. PERSI VFQLILVLLQPEFGTQDTQIYITYL R P. TRIHORP VFQLVLVLIQPEFGTQETLPYITYLSWLVGTTVSTYGKYM-NRPLLRKS R. OMMUNIS VFQLVLVLLQPEFGTEETQSYITYLSWLVTTTVSTGYGVQYMKNRYSLLSKS **** ***.*::*****. :* :****

2 Supplemental Figure 1. Sequence comparison of LS proteins from different species. () phylogenetic tree of LS sequences from different species. The tree was created using the online tools in Phylogeny.fr ( Dereeper et al., 2008; 2010). Protein sequences were aligned using MUSLE (3.7) program (Edgar 2004) with the maximum number of iterations of 16, and then adjusted manually. The alignment curation was done with Gblocks 0.91b program (astresana 2000) (see Supplmental Data Set 1 online). The phylogeny analysis was done using PhyML v3.0 program (Guindon and Gascuel 2003). The statistical test for branch support was done using pproximate Likelihood-Ratio Test (alrt) with the setting of SH-Like (nisimova and Gascuel 2006). In alrt, the model of amino acids substitution was WG, the number of taxa was 14, the log-likelihood was , the discrete gamma model was yes, the number of categories was 4, the gamma shape parameter was 1.796, and the proportion of invariant was Tree Rendering was done using TreeDyn program (hevenet et al., 2006). Mid-point rooting was used. Scale bar, 1.0 amino acid substitutions per site. ( and ) Sequence alignment between rabidopsis LS and LS homologs from non-plant species () and plant species (). Sequence alignment was performed using the lustalw2 program. Signal 1 and signal 2 on rabidopsis LS, which direct the protein to mitochondria and mitochondria/chloroplasts, respectively, are highlighted. The conserved enzymatic activity domain (DP-alcohol phosphatidyltransferase) is underlined by blue dashed lines. ccession numbers for the LS proteins: rabidopsis thaliana (T4G04870), Ricinus communis (XP_ ), Populus trichocarpa (XP_ ), Vitis vinifera (XP_ ), Prunus persica (EMJ ), ucumis sativus (XP_ ), Homo sapiens (NP_ ), Mus musculus (NP_ ), Danio rerio (NP_ ), Xenopus laevis (NP_ ), Drosophila melanogaster (NP_ ), Phaeodactylum tricornutum (EZ ), Saccharomyces cerevisiae (NP_ ). Nostoc sp. P 7120 (NP_ ).

3 YFP-LS LS-YFP-H LS-YFP-H E S P Supplementary Figure 2. LS-YFP-H is processed upon mitochondrial targeting. () Immunoblot analysis of transiently expressed YFP-LS and LS-YFP-H proteins in tobacco leaves, using α-gfp antibodies. rrow heads point to the putative precursor and mature form of LS-YFP-H. Loading control, large subunit of Rubisco stained by Ponceau S. () Immunoblot analysis of LS-YFP-H in different subcellular crude fractions. Tobacco leaves transiently expressing LS-YFP-H were homogenized on ice in grinding buffer (450 mm Sucrose, 1.5 mm EGT, 0.2% S, 0.6% PVP-40, 10 mm DTT, 0.2 mm PMSF, and 15 mm MOPS/KOH, ph7.4). The homogenized solution is considered crude extract (E). hloroplasts and other organelles and particles were sedimented by centrifugation for 10 min at 3,500 g (5 min each time) and 5 min at 6,000 g. Finally, the solution was centrifuged for 10 min at 17,000 g to get a supernatant fraction (S) and a pellet fraction (P) enriched in mitochondria. rrow heads point to the putative precursor and mature form of LS-YFP-H. Protein markers in kda are indicated.

4 Merged hlorophyll Merged OX4-FP LS truncation -YFP DP-alcohol phosphatidyltransferase domain LS ER Merged LS protein 1-20 YFP 1-41 YFP YFP YFP YFP LS Golgi Merged YFP YFP Signal 1: 1-20 aa Signal 2: aa LS region Mitochondrial localization (Leaf epidermal cells) hloroplast localization (Leaf mesophyll cells) Supplementary Figure 3. nalysis of organelle targeting signals on LS. () Schematics of the LS protein and deletion constructs. lack box, transmembrane helices predicted by TMHMM server ( () onfocal images of the localization of fusion proteins between various LS fragments and YFP transiently expressed in tobacco leaves together with the mitochondrial marker OX4-FP. o-localization between OX4-FP and LS-YFP fusion proteins were examined in epidermal cells. o-localization between chloroplast autofluorescent signals and LS-YFP fusion proteins were examined in mesophyll cells. Scale bars, 5 μm. () Localization of LS YFP to the ER and Golgi. The YFP fusion protein was transiently expressed in tobacco leaves with the ER marker twk2-fp or Golgi marker GmMan1-FP (Nelson et al., 2007). Scale bar, 5 μm.

5 YFP-PTS1 D WT cls-1 ol-0 ol-0 cls-1 cls-1 primer 2 T-DN 5 3 WT cls-1 cls-1 (rescued) H 2 O primers 1 & 3 primers 1 & bp primer 1 primer 3 Supplemental Figure 4. More characterization of cls-1. (-) Five-week-old plants () and siliques (). () Genotyping of cls-1 mutant and rescued lines. Schematics of the rabidopsis LS gene, position of the T-DN insertions in cls-1, and primers used in the genotyping are shown on the left. Genotyping of two independent lines for each genotype is shown on the right. (D) Peroxisomal morphology in five-week-old cls-1 and wild-type plants expressing the peroxisome marker YFP-PTS1 (SKL). onfocal images were taken from leaf mesophyll cells. Scale bar, 10 μm.

6 Germination Silique Seed Dry seed Seed development stages Supplemental Figure 5. Expression profile of the rabidopsis LS gene. () LS expression data from Genevestigator ( () LS expression data from rabidopsis efp browser (bar.utoronto.ca). Developmental stages include: 1, Dry seed; 2, Imbibed seed, 24 h; 3, 1st Node; 4, Flower Stage 12, Stamens; 5, auline Leaf; 6, otyledon; 7, Root; 8, Entire Rosette fter Transition to Flowering; 9, Flower Stage 9; 10, Flower Stage 10/11; 11, Flower Stage 12; 12, Flower Stage 15; 13, Flower Stage 12, arpels; 14, Flower Stage 12, Petals; 15, Flower Stage 12, Sepals; 16, Flower Stage 15, arpels; 17, Flower Stage 15, Petals; 18, Flower Stage 15, Sepals; 19, Flower Stage 15, Stamen; 20, Flowers Stage 15, Pedicels; 21, Leaf 1 + 2; 22, Leaf 7, Petiole; 23, Leaf 7, Distal Half; 24, Leaf 7, Proximal Half; 25, Hypocotyl; 26, Root; 27, Rosette Leaf 2; 28, Rosette Leaf 4; 29, Rosette Leaf 6; 30, Rosette Leaf 8; 31, Rosette Leaf 10; 32. Rosette Leaf 12; 33, Senescing Leaf; 34, Shoot pex, Inflorescence; 35, Shoot pex, Transition; 36, Shoot pex, Vegetative; 37, Stem, 2nd Internode; 38, Mature Pollen; 39, Seeds Stage 3 w/ Siliques; 40, Seeds Stage 4 w/ Siliques; 41, Seeds Stage 5 w/ Siliques; 42, Seeds Stage 6 w/o Siliques; 43, Seeds Stage 7 w/o Siliques; 44, Seeds Stage 8 w/o Siliques; 45, Seeds Stage 9 w/o Siliques; 46, Seeds Stage 10 w/o Siliques; 47, Vegetative Rosette.

7 atdrp3 MTIEEVSGETPPSTPPSSSTPSPSSSTTNPLGSSVIPIVNKLQDIFQLGSQS--TI 58 atdrp3 MSVDDLP PSSSVT---PLGSSVIPIVNKLQDIFQLGSQS--TI 42 hsdlp MELIPVINKLQDVFNTVGD---IIQ 24 mmdrp MELIPVINKLQDVFNTVGD---IIQ 24 dmdrp MELIPVINKLQDVFNTVGSD---SIQ 24 scdnm MSLEDLIPTVNKLQDVMYDSGID---TLD 27 scvps MDEHLISTINKLQDLPLGGGSQSPID 28 atr TRMEVSKSVTVEEMEEDDIEERWSLYEYNELHLQELETPFE 52 atdrp3 LPQVVVVGSQSSGKSSVLELVGRDFLPRGNDITRRPLVLQLLQTKS RN 109 atdrp3 LPQVVVGSQSSGKSSVLELVGRDFLPRGNDITRRPLRLQLVQTKP SSD 93 hsdlp1 LPQIVVVGTQSSGKSSVLESLVGRDLLPRGTGIVTRRPLILQLVHVSQ-----EDKRKTT 79 mmdrp1 LPQIVVVGTQSSGKSSVLESLVGRDLLPRGTGVVTRRPLILQLVHVSP-----EDKRKTT 79 dmdrp1 LPQIVVLGSQSSGKSSVIESVVGRSFLPRGTGIVTRRPLVLQLIYSPL-----DDRENRS 79 scdnm1 LPILVVGSQSSGKSSILETLVGRDFLPRGTGIVTRRPLVLQLNNISPNSPLIEEDDNSV 87 scvps1 LPQITVVGSQSSGKSSVLENIVGRDFLPRGTGIVTRRPLVLQLINRRPKKSEHKVNQT 88 atr5 PVLVVGQQTDGKSLVELMGFQFNHVGGGTKTRRPITLHMKYDPQ atdrp3 GGSD DEWGEFR-HLPETRFYDFSEIRREIEET 141 atdrp3 GGSD EEWGEFLHHDPVRRIYDFSEIRREIEET 126 hsdlp1 GEEN GVEEEWGKFL-HTKNKLYTDFDEIRQEIENET 115 mmdrp1 GEENDPTWKNSRH LSKGVEEEWGKFL-HTKNKLYTDFDEIRQEIENET 128 dmdrp1 ENG TSNEEWGRFL-HTK-KFTDFDEIRKEIENET 114 scdnm1 NPHDEVTKISGFEGTKPLEYR--GKERNHDEWGEFL-HIPGKRFYDFDDIKREIENET 144 scvps1 NELIDLNINDDDKKKDESGKHQNEGQSEDNKEEWGEFL-HLPGKKFYNFDEIRKEIVKET 147 atr QFPLHLGSDD-DPSVSLPKSLSQIQYIEEN 133 atdrp3 NRLVGE-NKGVDTQIRLKISSPNVLNITLVDLPGITKVP--VGDQPSDIERIRTMILS 198 atdrp3 NRVSGE-NKGVSDIPIGLKIFSPNVLDISLVDLPGITKVP--VGDQPSDIERIRTMILT 183 hsdlp1 ERISGN-NKGVSPEPIHLKIFSPNVVNLTLVDLPGMTKVP--VGDQPKDIELQIRELILR 172 mmdrp1 ERISGN-NKGVSPEPIHLKVFSPNVVNLTLVDLPGMTKVP--VGDQPKDIELQIRELILR 185 dmdrp1 ERGS-NKGIPEPINLKIFSTHVVNLTLVDLPGITKVP--VGDQPEDIEQIKELVLK 171 scdnm1 RIGK-DKGISKIPINLKVFSPHVLNLTLVDLPGITKVP--IGEQPPDIEKQIKNLILD 201 scvps1 DKVTG-NSGISSVPINLRIYSPHVLTLTLVDLPGLTKVP--VGDQPPDIERQIKDMLLK 204 atr5 MRLEQEPSPFSKEIIVKVQYKYPNLTIIDTPGLIPPGLKNRLQVQRVELVR 193 atdrp3 YIKQDTLILVTPNTDLNSDLQISIVDPDGHRTIGVITKLDIMDKGTDRKLLLG 258 atdrp3 YIKEPSLILVSPNTDLNSDLQIGNDPDGHRTIGVITKLDIMDRGTDRNHLLG 243 hsdlp1 FISNPNSIILVTNTDMTSELKISREVDPDGRRTLVITKLDLMDGTDMDVLMG 232 mmdrp1 FISNPNSIILVTNTDMTSELKISREVDPDGRRTLVITKLDLMDGTDMDVLMG 245 dmdrp1 YIENPNSIILVTNTDMTSELKLKDVDPDGRRTLVVTKLDLMDGTDIDILG 231 scdnm1 YITPNLILVSPNVDLVNSESLKLREVDPQGKRTIGVITKLDLMDSGTNLDILSG 261 scvps1 YISKPNIILSVNNTDLNSDGLKLREVDPEGTRTIGVLTKVDLMDQGTDVIDILG 264 atr5 KMQHKEFIILLEDSSDWSITTRRIVMQVDPELSRTIVVSTKLDTKIPQFSSSDVEV 253 atdrp3 NVVPLRLGYVGVVNRQEDILLNRTVKELLEEKFFRSHPVYHGLD--RLGVPQLKK 316 atdrp3 KTIPLRLGYVGVVNRSQEDILMNRSIKDLVEEKFFRSRPVYSGLTD--RLGVPQLKK 301 hsdlp1 RVIPVKLGIIGVVNRSQLDINNKKSVTDSIRDEYFLQKK--YPSLN--RNGTKYLRT 288 mmdrp1 RVIPVKLGIIGVVNRSQLDINNKKSVTDSIRDEYFLQKK--YPSLN--RNGTKYLRT 301 dmdrp1 RVIPVKLGIIGVMNRSQKDIMDQKHIDDQMKDEFLQRK--YPTLT--RNGTPYLKT 287 scdnm1 KMYPLKLGFVGVVNRSQQDIQLNKTVEESLDKEEDYFRKHPVYRTIST--KGTRYLKL 319 scvps1 RVIPLRYGYIPVINRGQKDIEHKKTIRELENERKFFENHPSYSSKH--YGTPYLKK 322 atr5 FLSPPSLDSSLLGDSPFFTSVPSGRVGYGQDSVYKSNDEFKQVSLREMEDISLEKK 313 Supplementary Figure 6. lignment of the N terminus (containing the GTPase domain) of mitochondrial/peroxisome division DRP proteins from various species. The conserved L-interacting rg and the three other conserved residues, Lys, Ser and Thr, in the GTPase domain are highlighted. Sequence alignment was performed using the lustalw2 program. ccession numbers for proteins sequences used for the alignment: DRP3 (t4g33650), DRP3 (t2g14120), DRP5/R5 (t3g19720), hsdlp1 (O ), mmdrp1 (Q8K1M6.2), dmdrp1 (Q9VQE0.1), scdnm1(p ), scvps1(p ).

8 Peroxisomes DRP3 R273E -FP mock DRP3 R258E -FP D HsDRP1 tdrp3 FP-DRP3 FP-DRP3 FP-DRP3 R273E FP-DRP3 R258E tdrp3 Merged DRP3-FP FP-DRP3 28 YFP-PTS1 RubiscoL FP-DRP3 R273E FP-DRP3 R258E DRP3 273E -FP DRP3 R258E FP Mock Supplementary Figure 7. Functional association of L and DRP3 in mitochondrial fission. () Structural modeling of the GTPase domain from rabidopsis DRP3 (60 to 321 aa) and DRP3 (44 to 306 aa) and human DRP1 (2 to 314 aa). The protein model was generated by SWISS-MODEL ( based on the structure of Dynamin 3 GTPase domain, and visualized with the PyMOL software. The cardiolipin-interacting rg (R247) is shown in red and indicated by an arrow. () Epifluorescent images showing mitochondrial morphology in tobacco leaf epidermal cells transiently overexpressing wild-type FP-DRP3 or FP-DRP3, together with the mitochondrial marker OX4-YFP. Scale bar, 10 μm. () Epifluorescent images showing peroxisome morphology in tobacco leaf epidermal cells transiently overexpressing DRP3 R273E -FP, DRP3 R258E -FP, FP-DRP3 R273E, or FP-DRP3 R258E, together with the peroxisomal marker YFP-PTS1. Scale bar, 10 μm. (D) Immunoblot analysis of DRP3 fusion proteins in crude protein extracts from tobacco leaves coexpressing each FP-DRP3 (or DRP3-FP) fusion protein and the peroxisomal marker YFP-PTS1. The proteins were detected by the GFP antibody. Protein size markers in kda are indicated.

9 Mitochondria Peroxisomes Mitochondria (OX4-YFP) WT drp3-2 drp3-2 drp3-2 Mitochondria (OX4-YFP) 35S pro :FP-DRP3 R273E 35S pro :FP-DRP3 K72 35S pro :FP-DRP3 S73N 35S pro :FP-DRP3 T93 drp3-2 drp3-2 drp3-2 drp3-2 Peroxisomes (YFP-PTS1) WT drp3-2 drp3-2 drp3-2 Peroxisomes (YFP-PTS1) 35S pro :FP-DRP3 R273E 35S pro :FP-DRP3 K72 35S pro :FP-DRP3 S73N 35S pro :FP-DRP3 T93 drp3-2 drp3-2 drp3-2 drp3-2 FP-DRP3 K72 FP-DRP3 S73N FP-DRP3 T93 D FP-DRP3 K72 FP-DRP3 S73N FP-DRP3 T93 Supplemental Figure 8. nalysis of the role of a few conserved residues in mitochondrial and peroxisomal fission. (-) Mitochondrial () and peroxisomal () morphologies in leaves of rabidopsis plants in various genetic backgrounds (left) or in drp3-2 transiently expressing mutant FP-DRP3 proteins (right). (-D) Tobacco leaf epidermis overexpressing FP-DRP3 K72, FP-DRP3 S73N or FP-DRP3 T93 and the mitochondrial () or peroxisomal (D) marker.

10 WT cls-1 WT cls-1 (line 1) cls-1 (line 2) H2O H-Y DRP3 DRP3 DRP3 (R258E) DRP3 (R258E) YFP-X DRP3 DRP3 (R273E) DRP3 (R258E) DRP3 (R273E) IP: α-gfp lot: α-gfp Input lot: α-h IP: α-gfp lot: α-h DRP3 DRP3 ctin DRP3 DRP3 25 OXII Supplemental Figure 9. nalyses of DRP3 R->E protein selfinteraction in tobacco and DRP3 transcript and protein levels in cls-1. () o-ip analysis of YFP- and H-tagged DRP3 proteins coexpressed in tobacco leaves. IP, immunoprecipitation. lot, immunoblot. () RT-PR analysis of DRP3 and DRP3 transcripts in wildtype and cls-1 plants. ctin mrn level was included as a loading control. () SDS-PGE followed by immunoblot analysis of endogenous DRP3 and OXII in cls-1, detected by their respective antibodies.

11 amirn-1 amirn-2 WT amirn-1 amirn-2 WT Leaves showing no necrotic area (%) DI Guard cells Leaf mesophyll Leaf epidermis OX4-YFP DI OX4-YFP DI OX4-YFP 65 D min WT amirn-1 amirn (hr) 37 : 0 hr 24 hr 22 : 0 hr 0 hr 4 hr 12 hr 24 hr 36 hr 48 hr 60 hr Supplemental Figure 10. Mitochondrial phenotypes of LS amirn lines and characterization of L s role in plant response to PD-inducing stresses. () onfocal images showing mitochondrial morphology in the two amirn lines used in this study. Scale bars, 10 μm. () Electrophoresis of genomic DN from wild-type plants incubated at 65 for various lengths of time. () Three-week-old rabidopsis seedlings subjected to 37 heat treatment for 24 hours and then left at 22. (D) Quantitative analysis of the susceptibility of wild type and cls mutants to 24-hr heat stress at 37. Percentage of leaves showing no signs of chlorosis or withering was calculated.

12 Supplemental Table 1. Primer used in this study. Primer Sequence Purpose LS-Fw-tt1 LS-Re-tt2-N GGGGGTTTGTGGG TTTGGGTTTGTTTG GGGGTTTGTGGTGG GTTTGTTTTTTGTT GG For cloning full length LS; for RT-PR analysis of full length LS & LS front 453 bp For cloning full length LS with stop codon; for RT-PR analysis of full length LS LS-Re-front GTTGTTTTTGG For RT-PR analysis of LS front 453 bp LS-Re-tt2- DRP3-Fw-att1 DRP3-Fw-att2-N DRP3-Re-att2- DRP3-Fw-att1 DRP3-Re-att2-N DRP3-Re-att2- LS-partial -Re-1 LS-partial-Re-2 LS-partial-Re-3 LS-partial-Fw-4 LS-partial-Fw-5 LS-partial-Fw-6 DRP3-R273E-Fw DRP3-R273E-Re DRP3-R258E-Fw GGGGTTTGTGGTGG GTTGTTTTTTGTTGG GGGGGTTTGTGGG TTTGTTTGGGTTTG GGGGTTTGTGGTGG GTTTGTGTTTTTTGGTG GGGGTTTGTGGTGG GTGTGTTTTTTGGTG GGGGGTTTGTGGG TTTGTGTGGTT GGGGTTTGTGGTGG GTTTTTGGGTGTT GGGGTTTGTGGTGG GTTTGGGTGTT GGGGTTTGTGGTGG GTTGGTTTGTTTTGGTGTTG GGGGTTTGTGGTGG GTGGGGTGGGTG G GGGGTTTGTGGTGG GTTTTGTTTTT GGGGGTTTGTGGG TTTGGTTTTGG TG GGGGGTTTGTGGG TTTGGTTGTTTTTT GGGGGTTTGTGGG TTTGGGTTTTGTTTGTGG GTTGGTGGGGGTTTTTG TG TGGTTTTTGTT GT GGTTGTTGGGTGGGT TTTTGTG For cloning full length LS without stop codon For cloning full length DRP3 For cloning full length DRP3 with stop codon For cloning full length DRP3 without stop codon For cloning full length DRP3 For cloning full length DRP3 with stop codon For cloning full length DRP3 without stop codon For cloning partial LS For cloning partial LS For cloning partial LS For cloning partial LS For cloning partial LS For cloning partial LS Overlapping primer for cloning DRP3 with R273E mutation Overlapping primer for cloning DRP3 with R273E mutation Overlapping primer for cloning DRP3 with R258E mutation

13 DRP3-R258E-Re LS I mir-s LS II mir-a LS III mir*s LS IV mir*a TTTTGTTTTTT GTT GTTGTGTT TTTTTGTTT GGGGTTTTTGGGTGTGTT GGTTG GGGTTTTTGGTGTGTTT GGTGTGTTG GTGGTGTTT TTTTTT Overlapping primer for cloning DRP3 with R258E mutation For cloning amirn LS For cloning amirn LS For cloning amirn LS For cloning amirn LS UQ10-1 TTTTTTGTGTGTG For RT-PR analysis of UQ10 UQ10-2 GGTGTGTTTGG For RT-PR analysis of UQ10 RT-3-F RT-3-R RT-3-F RT-3-R GTGTGGGTGTGGTGG G TTTGTGTTGTGTTG TGGTGGGGTGG G GTTGTTTGGGTGT For RT-PR analysis of DRP3 For RT-PR analysis of DRP3 For RT-PR analysis of DRP3 For RT-PR analysis of DRP3 P67_ct7_FP TTTGTTGTGT For RT-PR analysis of T7 (actin 7) P68_ct7_RP TGTGTGGTG For RT-PR analysis of T7 (actin 7) PR-1-L1 TGTTTTTGGTTT For RT-PR analysis of PR-1 PR-1-R1 TGTTGGGG For RT-PR analysis of PR-1 PR-2-L1 GTTTTTT For RT-PR analysis of PR-2 PR-2-R1 TGTTTTGGGG For RT-PR analysis of PR-2 eif1α-l1 GGTTTGTG For RT-PR analysis of eif1 eif1α-r1 TGGGTTGGTT For RT-PR analysis of eif1 LS-genotyping-1 GTTTGTTTTGG For genotyping of cls-1and rescued line LS-genotyping-2 TTTTGGTTTGG For genotyping of cls-1and rescued line LS-genotyping-3 TTGGGGGTGTTTTG For genotyping of cls-1and rescued line DRP3-K72-fw DRP3-K72-re DRP3-S73N-fw DRP3-S73N-re DRP3-T93-fw DRP3-T93-re GTGGGGTTGGTTTGG TGTG GTGGGTGTTTGGTT GGGTGGTTTGGT GTGGG GGTTTTTGTGTTTGGT T TTGGGGTGTTTTGTTT GT GGGGGTTTTG GGGG Overlapping primer for cloning DRP3 with K72 mutation Overlapping primer for cloning DRP3 with K72 mutation Overlapping primer for cloning DRP3 with S73N mutation Overlapping primer for cloning DRP3 with S73N mutation Overlapping primer for cloning DRP3 with T93 mutation Overlapping primer for cloning DRP3 with T93 mutation

14 Supplemental Table 2. Vector used in this study Vector and reference Description onstruct Selection of transgenic plants pdonor 207 (Invitrogen) Donor vector all donor plasmids pearleygate 100 (Earley et al., 2006) For amirn LS construction amirn LS ST pearleygate 101 (Earley et al., 2006) For fusing YFP to N-terminal end of the gene. YFP-LS. ST pearleygate 104 (Earley et al., 2006) For fusing YFP-H to -terminal end of the gene. LS-YFP-H ST pearleygate 201 (Earley et al., 2006) For fusing H to N-terminal end of the gene. H-DRP3 ST pdest-35s-6xhis-y YFP-DRP3, YFP-DRP3, Kanamycin FP-X (Reumann et For fusing YFP to N-terminal YFP-DRP3 R273E, al., 2009) end of the gene. YFP-DRP3 R258E LS YFP, LS YFP, Kanamycin LS YFP, LS YFP, pdest-35s-x-yfp- LS YFP, 6xHis (Reumann et For fusing YFP to -terminal LS YFP, al., 2009) end of the gene. LS YFP FP-DRP3, FP-DRP3, Hygromycin FP-DRP3 R273E, FP-DRP3 R258E pgw544 FP-DRP3 K72, (Nakagawa et al., For fusing FP to -terminal FP-DRP3 S73N, 2007) end of the gene. FP-DRP3 T93 pgw545 (Nakagawa et al., 2007) For fusing FP to N-terminal end of the gene. DRP3 R273E -FP, DRP3 R258E -FP Hygromycin

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