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 ...
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
