Supplemental Figures

Figure S1: Primers used for genotyping icp55 insertion lines, full length cDNA amplification and qRT-PCR.

Primers for Genotyping:

SALK-BP1.3 ATTTTGCCGATTTCGGAACSALK_001742-LP AAATCCTTGTTATTGTCCCGCSALK_001742-RP AATTCCACACTTCAGAATGCGSALK_123635/7-LP ACTCCAGCAACTTCTCCTTCCSALK_123635/7-RP CTTCTTCTACAGCAGCCATCG

SAIL_672_D05-LP TCTTGGGCGTTTAGTGTTACGSAIL_672_D05-RP CTTTTCTACATACCCCCTGCCSAIL Lb1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC

Primers for FL cDNA amplification and for PCR confirmation after transformation:

FLcDNA-Forward: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCAGTTTCTTGCGCGGAATCTFLcDNA-Reverse: GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAAACAATCTTCTTGGGTTAC

Compl-Forward: GAGTTGTTGTGTGATGGACTGATCompl-Reverse: GTACGTCATCCTCGATCCTTATG

Primers for qRT-PCR:

F12 TCAGCCCAGGTTGAATATGAGTGCR1 CACCGACTACAGGATTGAACGCR2 ACCACCGACTACAGGATTGAACGF3 TGATTACGATTGAGCCAGGAGR3 TCAGTACGTCATCCTCGATCCF4 GTGATTACGATTGAGCCAGGAR4 GATCCTTATCCCAATCCCTTG

Housekeeping genes for qRT-PCR:PPase 2A (At1g13320): Forward: TAACGTGGCCAAAATGATGC Reverse: GTTCTCCACAACCGCTTGGTYls8 (At5g08290): Forward: GGGATGAGACCTGTATGCAGAT Reverse: GCTCGTACATGGTGTTGAAGTCClathrin (At5g46630): Forward: TCGATTGCTTGGTTTGGAAGAT Reverse: GCACTTAGCGTGGACTCTGTTT

SALK_123635/7-LP ACTCCAGCAACTTCTCCTTCCSALK_123635/7-RP CTTCTTCTACAGCAGCCATCGSALK-BP1.3 ATTTTGCCGATTTCGGAAC

SAIL_672_D05-LP TCTTGGGCGTTTAGTGTTACGSAIL_672_D05-RP CTTTTCTACATACCCCCTGCCSAIL Lb1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC

SALK_123635

SALK_123637

Blank

Primer pair: SALK_123635/7-LP; SALK_123635/7-RP

Primer pair: SALK_123635/7-RP; SALK-BP1.3

T-DNA is NOT amplified.

SAIL_672_D05

Primer pair: SAIL_672_D05-LP; SAIL_672_D05-RP

Primer pair: SAIL Lb1 ; SAIL_672_D05-RP

T-DNA is amplified.

SALK-BP1.3 ATTTTGCCGATTTCGGAACSALK_001742-LP AAATCCTTGTTATTGTCCCGCSALK_001742-RP AATTCCACACTTCAGAATGCG

SALK_001742

Primer pair: SALK_001742-LP; SALK_001742-RP

Primer pair: SALK-BP1.3; SALK_001742-RP

T-DNA is NOT amplified.

Figure S2: PCR genotype of four T-DNA insertion lines for ICP55. Only Sail_672_D05 has T-DNA insertion amplification.

A

B

Figure S3: Response of WT and icp55 to high temperature and high light. (A) Plants grown at 4oC for 3 days and then moved to long-day conditions for 3 day. After treated at 40oC for 0, 12, 24 and 48 h, plants grown under long-day conditions for 5 days for recovery. (B) Plants grown under continuous light (left) and long day light (right) for 3 weeks and then treated with 40oC for 12 h. After 5 days recovery in continuous light (left) and long-day conditions (right), pictures were taken. (C) Plants grown under continuous light (left) and long day light (right) for 3 weeks were then treated with 40oC for 24 h. After 5 days recovery in continuous light (left) and long-day light (right), pictures were taken. (D) Plants grown under short day conditions for 40 days (top) and then exposed to high light (700 µmol E m-2 s-1) at high temperature (37oC) under short days for 7 days (middle). Plants were transferred to short-day for 7 days of recovery (bottom).

C

WT | icp55 WT | icp55 D

WT icp55

Figure S4: Differential in gel electrophoresis (DIGE) analysis of the proteome of mitochondria isolated from leaves of 3 weeks –old hydroponically grown plants. WT (red) and icp55 (green) were overlaid. The highlighted region indicate d the pI movement of a protein spot identified as At5g13450 (ATP synthase delta subunit) by MALDI-TOF/TOF. Analysis of three replicate gels found that only the spots indicated for At5g13450 were differentially abundant between the genotypes.

At5g13450

- + - + - + - + - + - + 0h 16h 20h 24h 0h 16h 20h 24h

WT icp55

Figure S5: In vitro protein degradation of freshly isolated mitochondrial proteins from WT and icp55 when incubated with (+) or without (-) proteinase inhibitors. Samples from 0h, 16h, 20h and 24h of incubation.

97

66

45

30

20

14

1 MAMAMALRRL SSSIDKPIRP LIRSTSCYMS SLPSEAVDEK ERSRVTWPKQ 51 LNAPLEEVDP EIADIIEHEK ARQWKGLELI PSENFTSVSV MQAVGSVMTN101 KYSEGYPGAR YYGGNEYIDM AETLCQKRAL EAFRLDPEKW GVNVQPLSGS151 PANFHVYTAL LKPHERIMAL DLPHGGHLSH GYQTDTKKIS AVSIFFETMP201 YRLDESTGYI DYDQMEKSAT LFRPKLIVAG ASAYARLYDY ARIRKVCNKQ251 KAVMLADMAH ISGLVAANVI PSPFDYADVV TTTTHKSLRG PRGAMIFFRK301 GVKEINKQGK EVLYDFEDKI NQAVFPGLQG GPHNHTITGL AVALKQATTS351 EYKAYQEQVL SNSAKFAQTL MERGYELVSG GTDNHLVLVN LKPKGIDGSR401 VEKVLEAVHI ASNKNTVPGD VSAMVPGGIR MGTPALTSRG FVEEDFAKVA451 EYFDKAVTIA LKVKSEAQGT KLKDFVSAME SSSTIQSEIA KLRHEVEEFA501 KQFPTIGFEK ETMKYKN

Figure S6: Peptides (red) detected in spot 15 in Figure 4B and their positions in the SHMT (At4g37930) protein sequence. The two serines (SS) are highlighted in green and indicate the start of the mature protein after the presequence is cleaved.

Observed Mr(expt) Mr(calc) Delta Peptide Ion Score

999.4465 998.4392 998.4457 -0.0065 YSEGYPGAR 713018.6356 3017.6283 3016.5672 1.0612 WGVNVQPLSGSPANFHVYTALLKPHER 321660.8414 1659.8342 1659.8331 0.0011 ISAVSIFFETMPYR 461676.8484 1675.8412 1675.8280 0.0132 ISAVSIFFETMPYR Oxidation (M) 541822.7838 1821.7765 1821.7615 0.0150 LDESTGYIDYDQMEK Oxidation (M) 921091.6126 1090.6053 1090.6135 -0.0082 LIVAGASAYAR 73 800.3844 799.3771 799.3864 -0.0093 LYDYAR 37

Match to At4g37930: SERINE TRANSHYDROXYMETHYLTRANSFERASE 1 (SHMT1)

Total ion score: 357; Coverage: 15%; Peptides matched: 7

KF921304 MQFLARNLVRRVSRTQVVSRNAYSTQTVRDIGQPTPASHPHLMAEGEVTPGIRIEEYIGR At1g09300 MQFLARNLVRRVSRTQVVSRNAYSTQTVRDIGQPTPASHPHLMAEGEVTPGIRIEEYIGR ************************************************************ KF921304 RKKLVELLPENSLAIISSAPVKMMTDVVPYTFRQDADYLYLTGCQQPGGVAVLSDERGLC At1g09300 RKKLVELLPENSLAIISSAPVKMMTDVVPYTFRQDADYLYLTGCQQPGGVAVLSDERGLC ************************************************************ KF921304 MFMPESTPKDIAWEGEVAGVDAASEVFKADQAYPISKLPEILSDMIRHSSKVFHNVQSAS At1g09300 MFMPESTPKDIAWEGEVAGVDAASEVFKADQAYPISKLPEILSDMIRHSSKVFHNVQSAS ************************************************************ KF921304 QRYTNLDDFQNSASLGKVKTLSSLTHELRLIKSPAELKLMRESASIACQGLLKTMLHSKG At1g09300 QRYTNLDDFQNSASLGKVKTLSSLTHELRLIKSPAELKLMRESASIACQGLLKTMLHSKG ************************************************************ KF921304 FPDEGILSAQVEYECRVRGAQRMAFNPVVGGGSNASVIHYSRNDQRIKDGDLVLMDMGCE At1g09300 FPDEGILSAQVEYECRVRGAQRMAFNPVVGGGSNASVIHYSRNDQRIKDGDLVLMDMGCE ************************************************************ KF921304 LHGYVSDLTRTWPPCGKFSSVQEELYDLILQTNKECIKQCKPGTTIRQLNTYSTELLCDG At1g09300 LHGYVSDLTRTWPPCGKFSSVQEELYDLILQTNKECIKQCKPGTTIRQLNTYSTELLCDG ************************************************************ KF921304 LMKMGILKSRRLYHQLNPTSIGHYLGMDVHDSSAVGYDRPLQPGFVITIEPGVYIPSSFD At1g09300 LMKMGILKSRRLYHQLNPTSIGHYLGMDVHDSSAVGYDRPLQPGFVITIEPGVYIPSSFD ************************************************************ KF921304 CPERFQGIGIRIEDDVLITETGYEVLTGSMPKEIKHIETLLNNHCHDNSARS--FASFSL At1g09300 CPERFQGIGIRIEDDVLITETGYEVLTGSMPKEIKHIETLLNNHCHDNSARTSPVSLCKV ***************************************************: .: .: KF921304 QG----------- At1g09300 KGLHTNRNPRRLF :*

Figure S7: Predicted amino acid sequence alignment of amplified full length cDNA (KF921304) and the TAIR At1g09300 CDS sequence. The position of the T-DNA insertion is indicated is highlighted in green. The two peptides quantified by mass spectrometry are highlighted in yellow: peptide1 is LVELLPENSLAIISSAPVK (64-82); peptide 2 is YTNLDDFQNSASLGK (183-197).

1.00 1.07 1.05 0.95 0.73 0.92

WT

Icp5

5Co

mpl

WT

Icp5

5Co

mpl

O h 20 h

1.00 1.13 1.08 1.04 0.63 0.95

1.00 1.05 1.03 0.93 0.55 0.75

Figure S8: Quantification of protein degradation after 20 h in mitochondria from 3 week old shoot from WT, icp55 and complemented line. Coomassie stained 1D-SDS-PAGE gel of WT, icp55 and complemented line proteins after 0 and 20 h room temperature incubation of mitochondria. The quantification of selected bands using ImageJ are given for comparison.

At1g09300 50 PGIRIEEYIGRRKKLVELLPE---------NSLAIISSAPVK--MMTDVVPYTFRQDADY 98 P I +E + G RKKL+E + + ++ K TD FRQ++ +At4g29490 7 PPIPMELHAGNRKKLLESIRRQLSSSNRSLDGFVLLQGGEEKNRYCTDHTE-LFRQESYF 65 At1g09300 99 LYLTGCQQPGGVAVLSDERGLCMFMPESTPKDIA-WEGEVAGVDAASEVFKADQAYPISK 157 YL G ++P + G + P D A W GE+ + E + D + + +At4g29490 66 AYLFGVREPDFYGAIDIGSGKSILFIPRLPDDYAVWLGEIKPLSHFKETYMVDMVFYVDE 125 At1g09300 158 LPEILSDMIRHSSK----VFHNVQSASQRYTNLDDFQNSASL-GKVKTLSSLTHELRLIK 212 + ++ ++ + S K + H + + S ++ F+ + TL + E R+IKAt4g29490 126 IIQVFNEQFKGSGKPLLYLLHGLNTDSSNFSKPASFEGIDKFETDLTTLHPILAECRVIK 185 At1g09300 213 SPAELKLMRESASIACQGLLKTMLH-SKGFPDEGILSAQVEYECRVRGAQRMAFNPVVGG 271 S EL+L++ + I+ + ++ M + G + + S + + G + ++ + At4g29490 186 SSLELQLIQFANDISSEAHIEVMRKVTPGMKEYQMESMFLHHSYMYGGCRHCSYTCICAT 245 At1g09300 272 GSNASVIHYSR----NDQRIKDGDLVLMDMGCELHGYVSDLTRTWPPCGKFSSVQEELYD 327 G N++V+HY ND+ +DGDL L+DMG E H Y SD+T ++P GKF+S Q +Y+At4g29490 246 GDNSAVLHYGHAAAPNDRTFEDGDLALLDMGAEYHFYGSDITCSFPVNGKFTSDQSLIYN 305 At1g09300 328 LILQTNKECIKQCKPGTTIRQLNTYSTELLCDGLMKMGILKS-------RRLYHQLNPTS 380 +L + I KPG ++ + +++ + L K IL +RL P At4g29490 306 AVLDAHNSVISAMKPGVNWVDMHKLAEKIILESLKKGSILTGDVDDMMVQRLGAVFMPHG 365 At1g09300 381 IGHYLGMDVHDSSAV--GYDRPLQPGF-------------VITIEPGVYIPSSFDCP--- 422 +GH++G+D HD+ G +RP +PG VIT+EPG Y + P At4g29490 366 LGHFMGIDTHDTGGYPKGVERPKKPGLKSLRTARDLLEGMVITVEPGCYFIKALLFPAMA 425 At1g09300 423 -------------ERFQGI-GIRIEDDVLITETGYEVLTGSMPKEIKHIETLL 461 ERF+ G+RIE D+++T G + +T ++P+E IE ++At4g29490 426 NATTSKFFNRETIERFRNFGGVRIESDLVVTANGCKNMT-NVPRETWEIEAVM 477

Figure S9: Sequence similarity of ICP55 and At4g29490. Alignment of At1g09300 (ICP55) and At4g29490 using NCBI blast. Bit score, E-value and % identity and similarity are shown.

Score Expect Identities Positives Gaps

162 bits(410) 4e-48 122/473(26%) 217/473(45%) 63/473(13%)