Col-0 Col-0 Col-0 Col-0 Col-0 Col-0 Col-0 Col-0 Col-0 A B

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Parents and crossing direction are shown for each cross together with F1 progeny ... emerging from msh1 TDNA line, T4 generation. ... (B) Sample plant showing aerial rosettes at a later stage of development. .... reciprocal crossing with wild type Col-0 (white). (B) Self-pollinated progeny of parents and reciprocal F1 progeny ...
B Curly

Col-0

Curly

Col-0 Curly

Col-0

----------------------------------------Col-0 x curly ------------------------------------ F1

------------------------------------- -----Col-0 x wrinkled wrinkled Col-0 1-19- F1

------------------------------------------curly x Col-0 ---------------------------------- ------------------------------------------wrinkled x Col-0 wrinkled Col-0 1-19- F1 F1

A Col-0

Col-0

Col-0

Col-0 Col-0

Col-0

Supplemental Figure 1. Maternal Inheritance of wrinkled and curly phenotypes (A) Reciprocal cross with wrinkled type. (B) Reciprocal cross with curly type. Parents and crossing direction are shown for each cross together with F1 progeny

A

msh1 T4 TDNA mutant

B

Normal wrinkled/curly Supplemental Figure 2.Wrinkled and curly types emerging from msh1 TDNA line, T4 generation. (A) Wrinkled T4 plant (circled) in msh1 TDNA mutant line. (B) Selfed progeny from wrinkled selection produced normal, wrinkled and curly types

A

B

C

Supplemental Figure 3. Plastid-altered phenotype at 12-hr photoperiod. (A) Phenotypic range in the mitochondrial hemi-complemented line AOX::MSH1::GFP1 at early stage of development. (B) Sample plant showing aerial rosettes at a later stage of development. Photograph taken at approximately 3 months. This “perennial-like” phenotype arises in approximately 10% of plants. (C) Confocal micrograph showing AOX::MSH1::GFP transgene expression product localizing in mitochondria (arrows). Brightness was adjusted to enhance visibility of the low level expression product.

Supplemental Figure 4. Non-photochemical quenching measurements. The msh1 and ppd3 mutants exhibit an increase in the development of NPQ in the light and slower loss of NPQ in the dark. These findings are consistent with a more highly reduced PQ pool. Lines tested are Col-0 wild type, msh1 null mutant, AOX1a::MSH1 as a mitochondrial targeted MSH1 hemi-complementation line, OE MSH1 overexpression line, SSU::MSH1 as a plastid targeted MSH1 hemi-complementation line, ppd3-gabi T-DNA mutant in exon, ppd3-sail T-DNA mutant in promoter.

A

B

T1 Col-0 msh1 T2 T3

Supplemental Figure 5 . Co-suppression of MSH1. (A) Full-length MSH1 genomic DNA was cloned in association with the CaMV 35S promoter, fused to GFP at its carboxyl end and transformed to Col-0. From a total of 109 T1 plants, 37 showed evidence of variegation (co-suppression). (B) PCR-based mitochondrial DNA recombination assay of cosuppressed plants.

A

C

Col-0 RNAi Null

Total PQ

αTC

Leaf Area

Total Leaf Area mm2

B

γTC ------Different F2 Populations------ Col-0

Supplemental Figure 6. Comparative assays of redox behavior. (A) Analysis of physiological changes in plastid-depleted (AOX::MSH1) and mitochondriallydepleted lines (SSU::MSH1-normal (N), -wrinkled (W), -curly(C)). Error bars represent the results of 4-6 replications. (B) Stem assay results with a MSH1 RNAi transgene-null line. Error bars represent the results of 6 replications. PQH2 and plastochromanol-8 were nondetectable. (C) F2 populations derived from crossing curly leaf and wrinkled leaf hemicomplementation lines (SSU::MSH1) to Col-0. Population 5 (blue) showed delayed germination. Each F2 population consisted of 10 plants, and the experiment was carried out twice.

A

B

C

D

Percentage of plasCds

E

20 15 10 5 0 Stems

leaves

SSU-MSH1-GFP Supplemental Figure 7 . Fluorescence Activated Cell Sorting (FACS) analysis from Col-0 and plastid-complemented SSU-MSH1 plants. (A) and (B) Total plastids from stems of Col-0 and SSU-MSH1 were ground in chloroplast extraction buffer and analyzed by FACS. (C) and (D) same assay from total leaves. (E) Average percentage of GFP-expressing plastids from 8 different sorting experiments. Chlorophyll autofluorescence and GFP signal were captured under PerCP (585/35) and FITC (530/30) detector. Percentage of MSH1-containing plastids in SSU-MSH1 samples was calculated from shift in FITC signal relative to Col-0 samples.

E

FYE/FYA (T-2)

F

FYE/LCA (T-31)

Days to flower

D

Col-0 Empty FYE/FYA FYE/LCA

Supplemental Figure 8. MSH1 localization behavior depends upon its DNA binding motif, and conserved glutamate is essential for plastid targeting. (A) and (B) Genetic complementation assay with FYE/FYA point and FYE/LCA triple mutations, early stage [A] and later stage [B]. Col-0 and empty vector controls are the same as shown in Figure 8. (C) PCR-based mitochondrial DNA recombination assay. (D) Days to flowering in FYE/FYA and FYE/LCA lines compared to wild type Col-0 and empty vector. (E) and (F) Confocal microscopy of stable transgenic lines carrying FYE/FYA and FYE/LCA transgene.

A

B Red Green Merge

Red Green Merge

At-MSH1(80aa)-GFP

At-MSH1(200aa)-GFP

At-MSH1(120aa)-GFP

At-MSH1(500aa)-GFP

At-MSH1(500aa)-GFP

C

P::TP::D2-full::GFP

A

B

C

D

Supplemental Figure 10. The PPD3 mutant and its phenotype. (A) A PPD3 gene diagram showing T-DNA insertion sites. (B) PCR-based assay allowed genotyping for the PPD3 TDNA mutations. (C) RT-PCR assays for PPD3 gene expression in ppd3 TDNA insertion mutants. (D) ppd3-gabi produces aerial rosettes (circles) and extended growth over 5 months. This phenotype, evident in about 5-10% of the mutants. Two ppd3-gabi plants are shown here. ppd3-sail-2: homozygous ppd3 mutant, PPD3-sail, #10: heterozygous PPD3, ppd3-gabi: homozygous ppd3 mutant plants

E

F

G

Col 0

NLS::MSH1

Supplemental Figure 11. Complementation testing in msh1 by an MSH1 transgene lacking transit peptide or fused to NLS. (A) MSH1 gene construction without transit peptide (77 aa), controlled by its native promoter, and fused to mGFP. (B) to (E). T2 generation of T-12, T-18 and T-8 independent transgenic lines [B], Empty vector [C], PCR based mitochondrial DNA recombination assay [D], Confocal microscopy [E]. (F) to (G). Complementation with NLS fused to MSH1, confocal image showing nuclear localization but not targeted to plastid or mitochondria [F], msh1 plants containing the NLS::MSH1 transgene showing dwarf, wrinkled and variegation phenotypes [G].

A

C

B Rosette diameter (cm)

--------F1 reciprocals-

Days to bolt

--------F1 reciprocals-

D

Supplemental Figure 12 . Maternal inheritance of plastid-altered msh1 phenotypes. (A) A range in intensity (1-3) of AOX1:MSH1 hemi-complemented phenotypes was selected for reciprocal crossing with wild type Col-0 (white). (B) Self-pollinated progeny of parents and reciprocal F1 progeny (correspondingly color-coded) were evaluated for rosette diameter. (C) Days to bolt. (D) F1 progeny from reciprocal crosses showing phenotypic difference for variegation and plant size. All F1 progeny were genotyped to confirm heterozygosity.

Supplemental  Table  1.  Inheritance  of  wrinkled  and  curly  leaf  plants  from  self-­‐pollination  of   plastid  (SSU::MSH1,  line  104-­‐15)  hemi-­‐complemented  plants   Plant

Generation

Total  plants

   %  wrinkled  or  curly

1(w)-­‐13(w)-­‐

S2

50

70.00%  wrinkled  and  variable  in  size; 5%  curly  

1(w)-­‐18(w)-­‐

S2

53

39.72%  wrinkled  and  variable  in  size

2(w)-­‐1(n)-­‐

S2

48

37.50%  wrinkled  and  variable  in  size

2(w)-­‐2(w)-­‐

S2

57

89.47%  wrinkle  and  variable  in  size

2(w)-­‐4(w)-­‐

S2

54

83.33%  wrinkled  and  variable  in  size

2(w)-­‐5(w)-­‐

S2

56

64.29%  wrinkled  and  variable  in  size

1(w)-­‐13(w)-­‐3(c)-­‐

S3

36

100%  curly  and  uniform  in  size

1(w)-­‐13(w)-­‐4(c)-­‐

S3

36

100%  curly  and  uniform  in  size

2(w)-­‐2(w)-­‐2(w)-­‐

S3

72  

78%  wrinkled  and  variable  in  size

2(w)-­‐2(w)-­‐10(w)-­‐

S3

70  

82%  wrinkled  and  variable  in  size

1(w)-­‐13(w)-­‐3(c)-­‐1(c)-­‐

S4

54

100%  curly  and  uniform  in  size

1(w)-­‐13(w)-­‐3(c)-­‐2(c)-­‐

S4

54

100%  curly  and  uniform  in  size

2(w)-­‐2(w)-­‐2(w)-­‐1(w)-­‐

S4

108

80%  wrinkled  and  variable  in  size

  Abbreviation:  w;  wrinkled,  c;  curly,  n;  normal,  S;  selfing.  All  plastid  hemi-­‐complemented  (mitochondrial-­‐   depleted  MSH1)  plants  are  derived  from  a  single  line  (104-­‐15)  

Supplemental  Table  2.    Inheritance  of  wrinkled  and  curly  population  derived  from  crossing   SSU::MSH1  (normal  looking,  lines  104-­‐15)  x  Col-­‐0   Family  

Generation  

Total  plants  

F-­‐2(w)-­‐  

F2  

22  

41.67%  wrinkled,  variable  in  size  

F-­‐12(w)-­‐  

F2  

38  

68.42%  wrinkled,  variable  in  size  

F-­‐15(w)-­‐  

F2  

24  

25.00%  wrinkled,  variable  in  size  

F-­‐18(w)-­‐  

F2  

21  

63.64%  wrinkled,  variable  in  size  

F-­‐30(w)-­‐  

F2  

30  

66.67%  wrinkled,  variable  in  size  

F-­‐12(w)-­‐5(w)-­‐    

F3  

60  

90%  wrinkled,  variable  in  size  

F-­‐12(w)-­‐6(w)-­‐  

F3  

72  

85%  wrinkled,  variable  in  size  

F-­‐10(C)-­‐  

F2  

36  

100%  curly,  uniform  in  size  

F-­‐10(C)-­‐40(C)-­‐    

F3  

17  

100%  curly,  uniform  in  size  

F-­‐10(C)-­‐40(C)-­‐4(C)-­‐  

F4  

25  

100%  curly,  uniform  in  size  

F-­‐10(C)-­‐40-­‐(C)-­‐5(C)-­‐  

F4  

22  

100%  curly,  uniform  in  size  

F-­‐10(C)-­‐40-­‐(C)-­‐5(C)-­‐22(C)-­‐  

F5  

25  

100%  curly,  uniform  in  size  

F-­‐10(C)-­‐40-­‐(C)-­‐5(C)-­‐16(C)-­‐  

F5  

24  

100%  curly,  uniform  in  size  

F-­‐10(C)-­‐40-­‐(C)-­‐5(C)-­‐22(C)-­‐2(C)-­‐  

F6  

36  

100%  curly,  uniform  in  size  

Abbreviation:  w  wrinkled;  C  curly  

%    wrinkled  or  curly  

Supplemental  Table  3.  Genetic  complementation  assay  with  point  mutations  in  DNA  binding   motif  of  MSH1   Construct  

Targeting  

 

 

Empty   vector       (FYE/FCE)  

Cytosolic  

    FYE/LYE           FYE/FYA     FYE/LCA          

    Mitochondria  and   plastids       Mitochondria  and   plastids           Mitochondria;   undetected  in  plastids     Mitochondria;     undetected  in  plastids          

Transgenic   independent  lines    

T2  (only  transgene  positive  segregants)   Variegation  

Mitochondrial  DNA   recombination  

    T-­‐10   T-­‐29    

 

 

26  /26   29  /29    

26/26   29/29    

T-­‐27   T-­‐34    

0  /19   0  /21    

0/19   0/21    

T-­‐1   T-­‐20   T-­‐76   T-­‐78    

25/26   21/26   18/26   20  /23    

26/26   26/26   26/26   23/23    

T-­‐7    

49  /49    

0/49    

T-­‐7   T-­‐31   T-­‐50   T-­‐56  

26  /30   20  /20   22/27   17  /27  

30  /30   20  /20   27/27   27  /27    

Supplemental Table 4. In silico prediction of transmembrane/hydrophobic topology in MSH1

  Transmembrane/ hydrophobic stretches

MSH1 conserved domains

DAS

TMpred

TopPred

386-404aa

383-403aa

Phobius

MEMSAT-SVM

692-707aa

1

Domain 3

2

Domain 4

687-698aa

687-704aa

685-705aa

685-702aa

3

Domain 5

782-791aa

781-801aa

780-800aa

777-796aa

GENE

AT1G76450 (PPD3)

AT5G66570 (PSBO1) AT3G50820 (PSBO2) AT4G03280 (PetC) ATCG00020 (D1) AT2G24820 (TIC55) AT5G13430 (Rieske ironsulphur protein) AT2G45200 (ATGOS12) AT1G34130 (STT3B) AT1G04690 (KAB1) AT4G23800 (HMG1/2)

AT4G37980  (ATCAD7)

Localization/Function

chloroplast, chloroplast thylakoid lumen, chloroplast thylakoid membrane apoplast, chloroplast photosystem II, stroma, thylakoid lumen, thylakoid membrane, oxygen evolving complex, plastid thylakoid membrane, plastoglobule. poly(U) RNA binding chloroplast photosystem II, chloroplast stroma, chloroplast thylakoid, thylakoid lumen, thylakoid membrane, oxygen evolving complex, plastoglobule. poly(U) RNA binding chloroplast envelope, thylakoid, thylakoid membrane, plasma membrane. constituent of cytochrome b6f complex chloroplast thylakoid, thylakoid membrane, lightharvesting complex, membrane, photosystem II, plastoglobule. Chlorophyll binding chloroplast, chloroplast envelope Ubiquinol-cytochrome C reductase iron-sulfur subunit, membrane, mitochondrial respiratory chain complex III, mitochondrion Golgi apparatus, cytosol, endosome, integral to membrane, intracellular, trans-Golgi network, vacuole.   SNARE binding Endoplasmic reticulum, membrane, plasma membrane.   oligosaccharyl transferase activity Cytoplasm, membrane, plasma membrane, plasmodesma. Oxidation reduction, potassium ion transport, transmembrane transport Nucleus, functions in DNA binding Cytoplasm, cinnamyl-alcohol dehydrogenase activity, nucleotide binding, oxidoreductase activity, transferase activity, transferring acyl groups other than amino-acyl groups, zinc ion binding Note: PetC, PSBA, Tic55 were identified by Co-IP

 

Interaction with MSH1 full gene and Sub-domains Msh1 full gene D 1 D2 D3 D4 D5 D6 Yes

No

Yes

Yes

No

Yes

Yes

Yes

No

No

Yes

No

No

No

Yes

No

No

Yes

No

No

No

No

No

No

Yes

No

No

No

No No

No No

No No

Yes Yes

No No

No No

No No

Yes

No

Yes

Yes

Yes

No

Yes

Yes

No

No

No

No

No

No

Yes

No

Yes

Yes

No

No

Yes

Yes

No

Yes

Yes

No

No

Yes

Yes

No

No

Yes

No

No

No

Yes

Yes

No

No

No

No

Yes

Supplemental Table 6. Primers used for cloning and genotyping   Gene AT3G24320 (MSH1)

Forward

Reverse

AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1)

MSH1-P3 MSH1-P2 5’AGTCACTAGTCTCTGTTTATGAGTCTT 5’GTCACTAGTTAAGATGCTGACTACGT TTC 3’ CTG 3’ MSH1-Phe/Leu-For MSH1-Phe/Leu-Rev 5’TTTGTTTCTCCAGGTAGGAGAACTTTA 5’TCTATTCCAATAGCCTCATAAAGTTCT TGAGGCTATTGGAATAGA-3’ CCTACCTGGAGAAACAAA-3’ MSH1-Tyr/Cys-For MSH1-Tyr/Cys-Rev 5’TTTCTCCAGGTAGGAGAATTTTGTGAG 5’AGCATCTATTCCAATAGCCTCACAAAA GCTATTGGAATAGATGCT-3’ TTCTCCTACCTGGAGAAA-3’ MSH1-Glu/Ala-For MSH1-Glu/Ala-Rev 5’CTCCAGGTAGGAGAATTTTATGCGGC 5’ACAAGCATCTATTCCAATAGCCGCATA TATTGGAATAGATGCTTGT-3’ AAATTCTCCTACCTGGAG-3’ MSH1-Phe-Tyr-Glu/leu-Cys-Ala-For MSH1-Phe-Tyr-Glu/leu-Cys-Ala-For 5’TGTTTCTCCAGGTAGGAGAACTTTGTG 5’TGTTTCTCCAGGTAGGAGAACTTTGT CGGCTATTGGAATAGATGCT-3’ GCGGCTATTGGAATAGATGCT-3’ At-Fwd-SpeI At-C5R-SpeI 5’ATTGACTAGTGCATAGTCGTCGTCTTC 5’ACTGACTAGTGAGAACATCACTTGAC TGG3’ GTCTTCAC3’ At-Fwd-SpeI At-C6R-SpeI 5’ATTGACTAGTGCATAGTCGTCGTCTTC 5’TTGAACTAGTCTGCAACATCTCCCAGT TGG3’ TGA3’ At-Fwd-SpeI A-C7R-SpeI 5’ATTGACTAGTGCATAGTCGTCGTCTTC 5’ACTGACTAGTTTTACGGGAGCGTGCT TGG3’ GGT3’ At-Fwd-SpeI At-C8R-SpeI 5’ATTG 5’ TCAC ACTAGT ACTAGTGCATAGTCGTCGTCTTCTGG3’ TGTATCAGACGCCCAATGAC3’ MSH1 Endo 951aa-Forward MSH1 full Reverse 5’AGTCCCATGGGCGCAAAAGACGCATC 5’AGTCCTCGAGTAAGATGCTGACTACG AGCTGAAG-3’ TCTGA-3’ PEND-ATG-For PEND-88aa-Rev 5’AGTCACTAGTATGCACTCTCTTAAGAC 5’AGTCACTAGT TAC-3’ ACCAGGACCAAGGACTCTATTTT-3’ pTAC2-ATG-For pTAC2-Full-Rev 5’AGTCACTAGTATGAACCTAGCAATTCC 5’AGTCACTAGTAGCTGTGCTCCCTGCT AAATCC3’ AGTT3’ MFP1-Full-For MFP1-Full-Rev: 5’AGTCACTAGTATGGGTTTCCTGATAGG 5’AGTCACTAGTAGAACTGGTACTGCTC GG-3’ TTTCTCC-3’ PPD3promoterEco-F AT1G76450 PPD3-R 5’AGTCGAATTCGGATTTGCCGGTGAATT 5’AGTCAGATCTGATGAATCTGAAAGAC TGATGAG-3’ TTGACT-3’ New CHM-Forward New CHM-Wt-Reverse 5-TTAACTTTGATCTCTCTCCTGC-3’ 5GCGTGGAAACCTTGACTTAAATTG-3’ New CHM-Forward New CHM-Mut-Reverse 5-TTAACTTTGATCTCTCTCCTGC-3’ 5’GCGTGGAAACCTTGACTTAAATTA-3' RL-P1 5'GTATAATTGTCGACCCGAGATGTTAGAA-3'

AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1)

RL-P2 5'G G CTAG ATAGCACCATTGTGTCA-3' RL-P3 5'CTATATTTCGTACGTTTCGGATATAGCA-3' RD-P1 5’GCTACGCAACACTTCCTTGCAACT-3’

AT3G24320 (MSH1)

RD-P2 5’TAAACAGGAGTGCTTCCGTTCCCT-3’

AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G24320 (MSH1) AT3G52170 (PEND) AT1G14410 (pTAC2) AT3G16000 (MFP1) AT1G76450 (PPD3)

Purpose Full length cloning into pBlueScript KS(+) and pCambia 1302c vector FYE/LYE single mutation FYE/FCE single mutation FYE/FYA single mutation FYE/LCA triple mutation Truncated clone (80aa) into 1302C GFP binary vector Truncated clone (120aa) into 1302C GFP binary vector Truncated clone (200aa) into 1302C GFP binary vector Truncated clone (500aa) into 1302C GFP binary vector MSH1 endonuclease cloning for antibody Cloning into 1302C GFP/RFP binary vector Cloning into 1302C GFP/RFP binary vector Cloning into 1302C GFP/RFP binary vector Cloning into 1302C GFP/RFP binary vector chm1-1 genotyping chm1-1 genotyping Mitochondrial DNA recombination assay (P1 + P2 + P3)

Mitochondrial DNA recombination assay (P1 + P2 + P3)

AT3G24320 (MSH1) AT1G76450 (PPD3) AT1G76450 (PPD3) AT3G24320 (MSH1) SAIL T-DNA backbone SALK T-DNA backbone GABI T-DNA backbone AT3G18780 (Actin 2) Nuclear localization signal sequence

RD-P3 5’TAATAGCTTGCTTCTCGGGTGGCT-3’ PPD3 SAIIL-641C02-LP PPD3-SAIL641C02-RP 5’’TGGATGACCAACTACTGCTGG3’ 5’GGAAAGCTTCTGATGCATCTG 3’ PPD3-Gabi-TM19: PPD3 Gabi-Rev 5'TGCAATTGAAACAAACAAACAAA-3' 5’-GATGAATCTGAAAGACTT 3’ MSH1 Sail-LP MSH1 Sail-LP ACGGAAAAAGTTCTTTCCAGG ACGGAAAAAGTTCTTTCCAGG Sail LB1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC

PPD3 Sail T-DNA genotyping PPD3 GABI-Kat T-DNA genotyping MSH1 T-DNA genotyping SAIL T-DNA

Salk LBb1.3 ATTTTGCCGATTTCGGAAC

SALK T-DNA

Salk LBb1.3 ATTTTGCCGATTTCGGAAC

GABI-Kat T-DNA

ACTIN2 RT-PCR Forward 5’GCAACTGGGATGATATGGAAAAGA-3' NLS-b54-F GTCGACATGTCGGTGCTGGGAAAGCGA AAAAGACATCCTAAGGTCGAC

ACTIN2 RT-PCR Reverse 5’CAAACGAGGGCTGGAACAAGACT 3’ NLS-b54-R GTCGACCTTAGGATGTCTTTTTCGCTTT CCCAGCACCGACATGTCGAC

RT-PCR NLS sequence used to target MSH1 to nucleus