Supplemental Figures 9-22-2013 - The Plant Cell · Supplemental Figure 1. ... F7J7 T6K22 F18E5 F17L22 T8O5 F1N20 T10I14 F7K2 chromosome 4 SM229_155,3 FIZ4 ... 35 01 WT rcf2-1 RCF2:RCF2-FLAG

WT rcf2-1

A

Supplemental Figure 1. Isolation of the rcf2-1 Mutant and Map-based Cloning of RCF2. (A) Increased CBF2:LUC expression in rcf2-1 plants under cold stress (4C for 20 h). (B) Induction of endogenous CBF genes in the wild type and rcf2-1. (C) Freezing tolerance of wild type and rcf2-1. WT (CA), rcf2-1 (CA), cold-acclimated (4°C for 1 week) wild-type and rcf2-1 mutant plants, respectively. (D) Expression of PRR5 in the wild type and rcf2-1 under cold stress. (E) Expression of RING-H2 Finger A1A and Agamous-like 8 in the wild type and rcf2-1 under cold stress. (F) Positional cloning of RCF2. (a) Positional cloning of RCF2. (b) Structure of the RCF2 gene. Filled boxes are exons, and lines between filled boxes are introns. Positions (relative to the start codon) of the rcf2-1 and fry2-1 mutations in RCF2 are indicated. dsRBM, double-strand RNA-binding motif; NLS, nuclear localization signal. (c) RCF2 protein structure with functional domains indicated. Drawing is not to scale. WT, wild type. Error bars indicate the standard deviation (n = 6 in [B], [D], and [E], 12 in [C]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 1, except (F), are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

PRR5

4C (h)

CBF1 CBF2 CBF34C (h)

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T10I14

F7K2

chromosome 4 SM229_155,3 FIZ4

F9F13-40K(4/1632) F7J7-47K

(4/1632)

F18E5-79K(2/1632)

F17L22-77K(1/1632)

T8O5-3K(2/1632)

F1N20-48K(5/1632) F7K2-28K

(7/1632)RCF2BACS

(# of recombinants/total plants)

(a)

(b)ATG

TAA

rcf2-1(G2156A)

RCF2fry2-1

(G3218A)

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5412

Agamous-like 8

4C (h)

RING-H2 Finger A1A

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(c) RCF2

958 967

Phosphatase catalytic domain

dsRBM dsRBM NLS

Supplemental Data. Guan et al. (2013). Plant Cell 10.1105/tpc.113.118927

1

WT

rcf2

-1

Abnormal transcript #1 in rcf2-1 mutant plants contains one nucleotide deletion of G at nt 1232 in the full-length wild type RCF2 coding sequence corresponding to the last nucleotide G of the 5th exon (highlighted in red color in the wild type). The frame-shifted amino acids in rcf2-1 are underlined. * indicates a stop codon.

Wild type nt 1216 AGA GGT GGA TTT TTC AGG GAT TTT GAT GAT AGT CTG CTA CCA AGG 1260 Wild type aa 406 R G G F F R D F D D S L L P R 420 rcf2-1 nt 1216 AGA GGT GGA TTT TTC AGG ATT TTG ATG ATA GTC TGC TAC CAA GGA 1260 rcf2-1 aa 406 R G G F F R I L M I V C Y Q G 420 Wild type nt 1261 ATT GCT GAA ATT TCT TAT GAG AAT GAT GCT GAG GAT ATT CCT TCT 1305 Wild type aa 421 I A E I S Y E N D A E D I P S 435 rcf2-1 nt 1261 TTG CTG AAA TTT CTT ATG AGA ATG ATG CTG AGG ATA TTC CTT CTC 1305 rcf2-1 aa 421 L L K F L M R M M L R I F L L 435 Wild type nt 1306 CCG CCT GAT GTC AGC CAT TAT TTG GTG TCG GAG GAT GAT ACA TCG 1350 Wild type aa 436 P P D V S H Y L V S E D D T S 450 rcf2-1 nt 1306 CGC CTG ATG TCA GCC ATT ATT TGG TGT CGG AGG ATG ATA CAT CGG 1350 rcf2-1 aa 436 R L M S A I I W C R R M I H R 450 Wild type nt 1351 GGT TTA AAT GGG AAC AAA GAT CCA CTT TCC TTT GAC GGG ATG GCT 1395 Wild type aa 451 G L N G N K D P L S F D G M A 465 rcf2-1 nt 1351 GTT TAA 1381 rcf2-1 aa 451 V * 452

Abnormal transcript #2

Supplemental Figure 2A B

C

Phosphatase catalytic domainWild type

rcf2-1

1

1 451

TUB8

Abnormal transcript #1rcf2-1

(G2156A)

RCF2 RT F

RCF2 RT R

RCF21551

3217

Abnormal transcript #2 in rcf2-1 mutant plants carries the entire 5th intron (in lowercase). * indicates a stop codon.

D

Supplemental Figure 2. The Nature of Two Abnormal RCF2 Transcripts in rcf2-1 Mutant Plants. (A) Positions of a pair of primers used to amplify RCF2 transcripts in wild-type and rcf2-1 plants. (B) Two abnormal RCF2 transcripts (indicated by the arrows) in rcf2-1 plants by RT-PCR analysis. TUB8 was used as a loading control. (C) The nature of abnormal transcript #1 in rcf2-1. Illustration below the sequence comparisons indicates wild-type RCF2 and truncated mutant protein in rcf2-1. (D) The nature of abnormal transcript #2 in rcf2-1. Illustration below the sequence comparisons indicates wild-type RCF2 and truncated mutant protein in rcf2-1. Translation from nucleotides (nt) to amino acids (aa) in (C) and (D) was performed with the BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html; Hall, 1999). dsRBM, double-strand RNA-binding motif; NLS, nuclear localization signal. Values in Supplemental Figure 2, except (A), are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

Wild type

rcf2-1 411

1

1

Phosphatase catalytic domain

Wild type nt 1216 AGA GGT GGA TTT TTC AG------------------------------------- 1232 Wild type aa 406 R G G F F R------------------------------------- 411 rcf2-1 nt 1216 AGA GGT GGA TTT TTC AGg taa gat gtc aga ttt gat aat agt agt 1260 rcf2-1 aa 406 R G G F F R * D V R F D N S S 420 Wild type nt ----------------------------------------------------------- wild type aa ----------------------------------------------------------- rcf2-1 nt 1261 ttg ttt tct tga cat ctc tac tga tca agg gtg ctt cct tag tac 1305 rcf2-1 aa 421 L F S * H L Y * S R V L P * Y 435 Wild type nt --------------------------------------------- G GAT TTT GAT 1242 Wild type aa ------------------------------------------------ D F D 404 rcf2-1 nt 1306 ata ctt acg gtg aaa tgg tat ttg aac ttg gtt aca gGG ATT TTG 1350 rcf2-1 aa 436 I L T V K W Y L N L V T G I L 450

dsRBM dsRBM

967NLS

967dsRBM dsRBM

NLS


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Supplemental Figure 3. Gene Complementation of the rcf2-1 Mutant. (A) RCF2 expression in rcf2-1 plants expressing RCF2:RCF2-FLAG (rcf2-1 complementation plants). (B) CBF2:LUCexpression in wild-type, rcf2-1, and rcf2-1 complementation plants under cold stress (4C for 20 h). (C) Expression of CBF genes in wild-type, rcf2-1, and rcf2-1 complementation plants. Error bars indicate the standard deviation (n = 6). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 3 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

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rcf2

-1

RCF2 :FLAG-FLAG+ rcf2-1 #2

RCF2:RCF2-FLAG+rcf2-1 #4

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Supplemental Figure 4. Sensitivity of rcf2-1 to NaCl and ABA, and Developmental Defects of rcf2-1 and fry2-1 Plants. (A) and (B) Seed germination rates of wild-type and rcf2-1 plants in response to various levels of NaCl (A) or ABA (B). Seeds in which the radical had emerged through the seed coat were considered germinated. There were 80–150 seeds per genotype per biological replicate. (C) Root elongation of wild-type and rcf2-1 seedlings in response to ABA. Four-d-old wild-type and rcf2-1seedlings grown on MS medium were transferred to MS medium supplemented with various levels of ABA and allowed to grow for an additional 7 d. (D) Reduced fertility of rcf2-1 and fry2-1 plants. Arrows indicate the sterile siliques. (E) Quantification of fertility of plants shown in (D). (F) Flowering time of rcf2-1 and fry2-1 plants as determined by total leaf number at flowering. Plants shown in (D)–(F) were grown under a long-day condition (16-h light/8-h darkness). Error bars indicate the standard deviation (n = 10 in [A] and [B], 25 in [C], and 60 in [E] and [F]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 4 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.



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RCF2:RCF2-FLAG + rcf2-1 #4

HSFA7b HSP101

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Supplemental Figure 5. RCF2 Expression Profiles and Heat Stress-Responsive Gene Expression in rcf2-1 Complementation Lines. (A) Expression patterns of RCF2 in various tissues of wild-type plants. (B) Expression of RCF2 in wild-type plants under heat stress. (C) and (D) Expression of HSFA7b and HSP101 in rcf2-1 complementation lines. Error bars indicate the standard deviation (n = 6). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 5 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

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37C (h)

DREB2A HSP18 HSP70B HSP18(flowers)

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C24

fry2-1

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Supplemental Figure 6. Effect of fry2-1 Mutation on Thermotolerance and Expression of Heat Stress-Responsive Genes. (A) Thermotolerance of C24 and fry2-1 plants. Three-week-old soil-grown plants were transferred to incubators at 21C or 37C and allowed to grow for an additional 7 d. (B) Survival rates of C24 and fry2-1 plants as shown in (A) and flowers of separate batches of 1-month-old C24 and fry2-1 plants under heat stress (37C for 4 d). Flowers that failed to produce viable siliques were considered dead. Values are mean survival rates relative to the non-stressed conditions. (C)–(I) Expression profiles of heat stress-responsive genes in 14-d-old or 1-month-old (for HSPs in flowers) C24 and fry2-1 plants subjected to heat stress at 37C for 0 or 1 h. Error bars represent the standard deviation (n = 80 [individual pots where C24 and fry2-1 plants were grown side-by-side] in [B]; n = 6 in [C] –[I]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 6 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.


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WT

nac019

NAC019:NAC019-HA + nac019 #2NAC019: NAC019-HA + nac019 #4

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HSFA6b

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Supplemental Figure 7. NAC019 Expression Profiles and Heat Stress-Responsive Genes in nac019 Complementation Lines. (A) NAC019 expression in different tissues of wild-type plants. (B) NAC019expression in wild-type plants subjected to heat stress. Values presented in (A [b]) and (B [b]) are from publicly available gene expression profiles as described (Kilian et al. 2007). (C) NAC019 expression levels in 14-d-old wild-type and rcf2-1 plants subjected to 37C for 0 or 1 h. (D) NAC019 expression levels in wild-type and nac019 plants. (E) NAC019 expression in nac019 plants expressing NAC019:NAC019-HA (nac019 complementation lines). (F) and (G) Expression of HSFA6b and HSP18 in nac019 complementation lines. Error bars indicate the standard deviation (n = 6 except in A [b] and B [b] where n = 2). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 7, except A (b) and B (b), are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

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WT #2 #4NAC019:NAC019-HA+ nac019 lines

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7

gcatggctatttatccaacCTGGAGAAAAGAGTGTGCATTAAGCTCGTTTGCTGCCATCATGTCAGCCTCAACTCCACCAGCACGCTGTCCGGGGTAGACctttacagagacaagtatgtgagggaaagaatgaatgcaaagcaataaaatatccaaatgaagttttgatgattctaataatgtaaggaggactcgcacagctgaacaaaatatgcccagataatgtagagagactcatgaaaaatggaaactgccttcaggaaaagaacaatattggctgagaagaagaaacaagattgacaaaacatgcagaatcacCTTAAAGACGACAGGCGTTCCTCGAAATGGACCCTGAGATATTCGTCCTTGATATAGTctgcatacaaactgctgtggctaatacatttttaacttgaccaaatatattaccatctaataaattgtctacacaaagccaaaagtgcacCTGATCTTAACGGCACCTTCTCCAATATCTTGAGATTTTAATCGCCCAATATCATCAGACGTATATTCAAAGTCAGATCCTGACTGCAGCCCTGTATAActaggagtatagaagtggtgaaaagctaagtaaacgaagtagaacacagcggcagataaaagagtaaaactagaccaacaaacgtagcataatctagctaaagatgcacaataatcataagaaaggtcaatgtatcccaattcaaatacactaacaaaagtgatttttttgttaaacccttcgaataattgtcaagctcacgtatacattgcaatgagtgcataaacctgttgcaactacagattcacacatacacaaacaatttcgatataaaattagagatttcgttacacgaagacgtagaaacaacacagaagctccagataaagagagaaatcaaaaatccagtgagagtacCTAGTTACATTCATGAATCCGTAGCTTCCGATAAGCCGCCCTACCTCGAACGAGTCAGCACTGGTTATCAAACTCGCTGTCACCGCATGCAACCGCCGAATCGGCCGATTCCGTCCACGCGAATTTACAATCCGACAGCCGGAGAATCCATCGCCTGATTCTCTATGTTTCAAAGAGAAACGCGAAACGCATTCGAAGGAAACGCGATGAATAGCGACCAGATTCATcgcttttgtttgtgtttcgtcgcttcacttcaaattactttccaggatttaacgagtgtcagtgtcagtataagagagagagagataagagccaggaaagttttctctgtgtcttacgtggcttttaatatacataattaaatgatataatcgtgcgtcgtatagataaatatctgagataatcttattaaattttaacggtgtatattatgccacgtatgaaactcaccgagaaaaacagaaaaagctgctatcgatgctacgtgtacttttaaaccgaattaatccgaaccaaatcaaaatacacgaaatgaaaccgtttaatttaggtgacagtcagagtgtctaccaaaactaatcgtacaaaaccgaactgaattgaaccgaaatggccatgcctgcttttcttcagctttaagggaaaagatcaaagattgggtcttcaacacttgagagagtctctgtctctgtaaaagatatttgagcttgagaagaacagaagaaacttccaggatcaatcaatcgatcaactttagtgaactaactcttgatttttcattcgaagttATGGAATCGGTTCCCGAATCCGTACCATCGCCGAACTCGAATACACCGTCAATACCACCGCCGGTGAACTCCGTACCGCCTTTCTTGAGTAAAACCTACGACATGGTTGATGATCCGTTGACCAATGAGGTCGTTTCGTGGAGCA

agccgataatgcttgcactaagacaactataatgacgtaTtttctttctccctaattaaaaacaaaccatgtttataaatgtagccttggccacgtggttgatttctaaatttggacccactgtgtttatacgtatcaaactttatccatatttttgtattcaattttctcgttattgtttttaggtaggttatcgttggtactttgccaccaattttagattttagctgtagggtttttataagatttgtataattcgcctgatggagagtataataatattatttaggttggaagatatttacacctgtttaacctcaaaaacagatatttaagaaaagacagtaagtaatcgaatatgtgtacatttgtttgtgcgctgctcaaaacgcgtacgtacgtgtgttacatatggatgcgtactttgttatatattcacgttctaaattattaataaattagtagcaaaagtttttggattattaaacccaaaaagtaaatgtgatgattatacagtctaatctagtattaagtctgtgggttttccaggttggaatgggtaaagttgtcggcctgataacatgacacgatagcaaaattagaaacactgtgagatatttattcttaattgaaaatttgtttttgatttgtttctaaataatactccaaggactattgacctatcatggtaactaaacgattgtgggtagaagtagagcgagtgctcttactttagcggtatcaacttggaagctaccctgcccattggattggatctaagcttcatatttgtggattaaaaaggccttagtgggactcatgtctcctccggtgggactagtgtcttttacaatgtcaagtttaaaaaattggataacaacaccgataaaaattcacatttgcaaattttattcagtcggaatatatatttgaaacaagttttgaaatccattggacgattaaaattcattgttgagaggataaatatggatttgttcatctgaaccatgtcgttgattagtgattgactaccatgaaaaatatgttatgaaaagtataacaacttttgataaatcacatttattaacaataaatcaagacaaaatatgtcaacaataatagtagtagaagatattaattcaaattcatccgtaacaacaaaaaatcataccacaattaagtgtacagaaaaaccttttggatatatttattgtcgcttttcaatgattttcgtgaaaaggatatatttgtgtaaaataagaaggatcttgacgggtgtaaaaacatgcacaattcttaatttagaccaatcagaagacaacacgaacacttctttattataagctattaaacaaaatcttgcctattttgcttagaataatatgaagagtgactcatcagggagtggaaaatatctcaggatttgcttttagctctaacatgtcaaactatctagatgccaacaacacaaagtgcaaattcttttaatatgaaaacaacaataatatttctaatagaaaattaaaaagggaaataaaatatttttttaaaatatacaaaagaagaaggaatccatcatcaaagttttataaaattgtaatataatacaaacttgtttgcttccttgtctctccctctgtctctctcatctctcctatcttctccatatatacttcatcttcacacccaaaactccacacaaaatatctctccctctatctgcaaattttccaaagttgcatcctttcaatttccactcctctctaatataattcacattttcccactattgctgattcatttttttttgtgaattatttcaaacccacataaaaaaatctttgtttaaatttaaaaccATGGATCCTTCATTTAGGTTCATTAAAGAGGAGTTTCCTGCTGGATTCAGTGATTCTCCATCACCACCATCTTCTTCTTCATACCTTTATTCATCTTCCATGGCTGAAGCAGCCATAAATGATCCAACAACATTGAGCTATCCACAACCATTAGAAGGTCTCCATGAATCAGGGCCACCTCCATTTTTGACAAAGACATATGACTTGGTGGAAGATTCAAGAACCAATCATG

ctgtctctggtactacagaccagatgttgagtacaaacacgtaccatgaggcatcgcggtaatgcatcaagtgtcacttagactgttcgacacatgtttgaggattgcatcaaaaaggtggctacgaagtctacagcttctaggaagagtccaagcggaccaattaggatttgttctatgtatggtcaaaacctatatagaatttgtttgctagcgttgctattatctttttttctttttgttgattaaaaaaaactatgttctccgaatgtaaaaaaaaaacaacaacgaagttctgctccagatactgttttaaccggccaattcagaataaactgatttaaatacatgtacccattcttgaaggtcaaacgacaaaaaatatatacaaattgtttgctagtgaattactattatcttttttcgtgtatttttttgttgattagaaactatgttctacgaatgtaaagaaaaaatacaacaacgaagttgtgctccagatactgttctgaccgaccaattctgaattacctgagtagtccattttaaggcatgtacccattttttctcttttgggttaagggtttaagtttttgctttaaaaagaagaaatgttttgtagccatatattaatataaaagtgtatattttataaatatctctaacttatatattataaagaaaaatacctaagaaatttactagtcagaaattaatggaggccttcgaattgcagggaatttgtcaatcaactcttggagtacttctggttggttttattttagccttaaccaatccggtttagacccaagccaaatgtcaagatgagacaacagccacatcaacattaaaaccaaacttaacccaattacaatttgccatttgcatcactctttgtttttatatttgtaaaacacaaaaggtgggtcctaatactaacaatgtgaaaaccataccgtacagtcctcaattacgaagttccagaagtttcgactattcgaatgtgcatcaaaaaggtccaaaaccgtttccagaagtttctaatttctaacttgagtgtactataataataatttttacaaatattttaatcaaatctaatatcatcactcactttctactcttcctatataaagccacctcaaaattatcttacgtggacattaccttcctcactccatcaactgccataaacgaacttaatccgaccagtttttcaaaatccgaagatgatgaactcaacttcggagactggttaatcgaaatcatcaagaatcttgtctttaatttccaattgggtttttcaaaacccgttacctcaggATGATGAACCCGTTTCTCCCGGAAGGCTGCGATCCACCACCACCACCACAACCAATGGAGGGTTTACACGAAAATGCTCCACCTCCATTTCTGACCAAGACATTTGAGATGGTGGATGATCCAAACACTGACCACATCGTATCTTGGAACAGAGGAGGAACAAGTTTTGTCGTCTGGGATTTGCATTC

Supplemental Figure 8. NAC019 Recognition Sites (CATGT) and Core Binding Sites (CACG) in the Promoter Regions of HSFA1b, HSFA6b, and HSFA7a. NAC019 recognition sites and core binding sites in promoter regions of HSFA1b, HSFA6b, and HSFA7a are highlighted in pink. Upper case bases indicate exons and lowercase bases indicate introns or promoter regions. Lowercase bases in red indicate 5- UTR (untranslated region).

HSFA1b

HSFA6b

HSFA7a



8

cagtaaatggttccgtttaagctgttagatgatttttgagtaaaaactaatgtaaaaaaaacaaaaaaaaaacaatgtagttcataatacatgcatgttttaaagaagtttcttgtttactatcaacttgaatagtatttcacgaagtcaaaattgttcattccgacttttctatgtggagaaaaaaaattctatcattgtgcacaatttaacagaatgtaatttcttgtaaaagaagaggaaacaattcgctgttagtaaatgtgaagtatagaagtctaaaatgagatacctcaactagcttgaattaagaaaaaaaacaaaaactctatcgacatgaaaaaggtcgcaaatatttatcatttatcaatgccaaaggagtatttggttcacaaaatactgaatcatttatatagatatataattagctctaaattctactataacttgcaaaataagtatactgactcaattatatagcgtttaaaaatagacgatttgtatgatgaggtccatatatatggagatgtgcatgcaactatcgacattttcacacgttgatatcgtctttctccaatggagacttgaatttgtgtaaactatgaatactcgtctctctaagaccttttttcttcaaccatgccaactatttaggtaagattttactgtctttgattgatattaaatacttagccgtggcgttatcaatgaatgataataaaaatgcggataaaagccaaaggtgttggaaataaatccaagaatgaagacgtagatgtcgatgggtattttaagaacttgaatttgtcacgactcacacgttaaaatatattatccgaattgtttagtctaaagacacacatatattgaaaaagaaaaggtaaatgaagctcattggtgcctaaatgtgaaatgaagccgaaatgtgttaggtgaacacatttaaatatacaaaaagaaatataatagaaacaaaactaattaacaaagtcgcaatttgtattgtataaaatatctttccgtctcccgtcatatttgaaaaaaaaaaaattacaaatctgttaattttaaaactttctagaaaaacacaagtatataattttctcttttcgtgcgtgtttgttttaaaataacattgttttgattggcgactcaacatattttagcatttacatatttctgcatatattaaatgatttataaactcaactatagattaaaatataatttgacatctaataattttaacaataatataaaatatgagatttataaattacgaatataaatattcaagggagagaaaaagtagaacataattcaaaagataagactttttagacttttttaacaatatttttgatggataaaaattattcaaaagagaagaaagtaagaagaaaagatgtttctgagaatttctagaaacagcatccgtttttataatttaattttcttacaaaggtaggaccaacatttgtgatctataaatcttcctactacgttatatagagacccttcgacataacacttaactcgtttatatatttgttttacttgttttgcacatacacacaaaaataaaaaagactttatatttatttactttttaatcacacggattagctccggcgaagtatggtcgtcgtcttcatcttcttcctccatcatcagatttttccttaaATGGAAGAAACCAAACGAAACTCCGATCTTCTCCGTTCTCGTGTTTTCCTCTCTGGCTTTTATTGCTGGGATTGGGAATTTCTCACCGCTCTCTTGCTTTTTAGTTGCTGAttctttttccttcgactttctatttccaatctttcttcttctctttgtgtattagattatttttagttttatttttctgtggtaaaataaaaaaagttcgccggagATGACGGCTGTGACGGCGGCGCAAAGATCAGTTCCGGCGCCGTTTTTAAGCAAAACGTATCAGCTAGTTGATGATCATAGCACAGACGACGTCGTTTCATGGAACGAAGAAGG

gattctacgcaacgtgtcgaatcagtacttgttcctgagttcctccgtcactaaccagtgcggaccatgtcagtatccaagcacccacacgctcgtcgtctcttgaaaacatctcacacaaatacacaacggctcggactaataacttgccatctaagaaaagctaattgacctaaggctttgtggcacctgcatgtaaaaagcaaacacgaatttgccacgttattacaaagcataccacgtcaccgtctcactgttacactatctcttgttctcctttatttcgctatggacaacaaaaattataaggtggggaagagatgtctcaaaatacttttttatcgtcctctcaatttatgtaactgataataatcgaaatagataaatttaattgtaggttgacaaagcgctaattttaataaaaacatatattagtttggaaaattttgtcgtagtaagtaattaaacctatcaatcttgactaataatttcaagtatcccataaaaatcaaaattgtccttaggttttgaaattgtactactgatagtacgtaattaaacaaacaaaagctatagcttgtggattatagctaatcccatttggcgatgagagtaatatatatattgttcaaataaagttgtaattaagattcgattgaaaaggtaaattatagtttaagaaaaacaaagagaggagacaacaattaaaaaaatgaaaagtggagaactgtactctcgcctatacgaggaggtcattatctccaaggctctcaaggattaaggaccatgctttgattctataaatgttgttttattcaaactatttatattattatatcatacgtaatatgtggttagcatttgacccacgtccccttaattaagtctagattaatcccattaatctatccaattaataattaggaaaattttatataagattgctattggatttctcggtggtggtttaaactataaaatagtaatgcctttgatgcgtgtgattttttattggaacttattggttttaattcttaacctagcaattatatttgtagtagtttatgttaatcattgtttaataatactcctataataactcttgcaagtagcaacacttgtgtggctatttattccagagtccatggatatgtatgtataagaaaataggagtgcttttgcagattgaagagtttgtctataaaatcatgtttacttggcatttagtgttattgtttgcaattttttattttaaattagtcggatattttaagttaaaatctgaatacacagcatgttccttctaacgttttttaacataattgtaaactaaagaaaaattataatataataaattaaattataccctctctattcattgtagaagatttgtttgtttcaccaaatgtgttttttttttataagttgctctcactcttgagagttttggagtattttgaagaggacgaggttcttttgtgaaaaagtgaaagaaacaaatcatgtcaagaattagaaagaaatcatgacaaataattttgtttgtgcagaaattacaaatgaatcatagatatccttctaattcgggatcggaaaccagttgaaaaatcgatggactaagctttctatattgggactagcttacccttagaattagtcaagcagcttaaatcatctatgacttaaaattataattaagaaaaaacaatgcctaaatatGCATATATTTCAAATGTATCACATAACTTGTGACATAAGAAAATATAAACAAAACAAAAAGGGCAAAAAAGACCTGAAAGCTTAGAGGCACACCTGCATAGGTCCCACAGTTCACTCGTGACACCGTAAAAGGCAAAACACGAACCCGCCACGTTATCACAAAAAGCAAGCCACGTCAATATAGTCTCACTGTCAACTACACTTAACTTACTATTTTCACATCTCATTTTCCTATCTTTATATAAACCCTCCAGGCTCCTCTTTAATTTctttaccaccaccaacaacaaacatataaaccataaggaaaacagagaaagagagagATGGAGGACGACAATAGTAACAACAACAACAACAACAACGTGATCGCACCTTTCATCGTGAAAACATATCAGATGGTCAATGATCCTTCCACCGATTGGCTCATCACTTGGGGACCTGCTCACAACAGTTTCATAGTCGTTGATCCACTCGACTTCTCGCAACGAATCTTAC

HSFB1

HSFC1

Supplemental Figure 9. NAC019 Recognition Sites (CATGT) and Core Binding Sites (CACG) in the Promoter Regions of HSFB1 and HSFC1. NAC019 recognition sites and core binding sites in promoter regions of HSFB1 and HSFC1 are highlighted in pink. Upper case bases indicate exons and lowercase bases indicate introns or promoter regions. Lowercase bases in red indicate 5- UTR (untranslated region).



9


A

Supplemental Figure 10. Electrophoretic Mobility Shift Assay (EMSA) of Binding of NAC019 to cis Promoter Element in HSFA6b. (A) EMSA analysis showing MBP-NAC019 binding to the cis promoter elements in HSFA6b. (B) EMSA analysis showing MBP-NAC019, but not MBP-RCF2, binding to the cis promoter elements in HSFA6b. Competitor, non-biotin-labeled probe. Results in Supplemental Figure 10 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

Probe (20 fmol) + + + + + + MBP (µg) 0.3 0 0 0 0 0MBP-NAC019 (µg) 0 0.3 0.5 0.5 0.5 0.5Competitor 10x 100x 150x

Free probe

Bound probe

BProbe (20 fmol) + + + + + MBP (µg) 0.3 0 0 0 0 MBP-NAC019 (µg) 0 0 0.5 0 0.5 MBP-RCF2 (µg) 0.3 0.3

Free probe

Bound probe


10


HSFA1b HSFA7aHSFA6b

Fol

d ch

ange

A B

E

C

D

Supplemental Figure 11. RCF2 Is Enriched in Promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. (A)–(E) ChIP-qPCR analyses were performed with pooled samples from 15-d-old of rcf2-1plants expressing RCF2:RCF2-FLAG (Supplemental Figure 3A); these plants were treated at 37°C for 1 h to determine whether RCF2 is enriched in the promoter regions of HSFs where NAC019 binds. Regions of amplifications in (A)–(E) are specified (positions are relative to translation start site). Amplified regions B in (A)–(E) served as negative controls. Primers used for amplifications in (A)–(E) are listed in Supplemental Table 2. Error bars indicate the standard deviation (n = 6). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.05). Values in Supplemental Figure 11 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

ATG1 kb

A B

ATG1 kb

A B

HSFB1

Fol

d ch

ange

ATG1 kb

A B

HSFC1

ATG1 kb

A B

A B

A B A B A B 0

0.5

1

1.5

2

2.5

3No AntibodyAnti-FLAG

0

1

2

3


0

0.5

1

1.5

2

2.5


0

0.3

0.6

0.9

1.2

1.5

1.8No AntibodyAnti-FLAG

00.5

11.5

22.5

33.5

44.5

No AntibodyAnti-FLAG

A B

ATG1 kb

A B

a a aa a a a a a

aa

a a

a a a

b

b bb

-1717 -1580 1358 1547 -1859 -1719 1084 1293 -1323 -1175 908 1107

-1903 -1722 742 973 -2045 -1892 749 943


11

0

2

4

6

8

10

12

0 1

WT

rcf2-1

rcf2-1 +RCF2:RCF2(D161A) #7rcf2-1 +RCF2:RCF2(D161A) #8

HSP101

0

5

10

15

20

25

30

35

0 1

WT

rcf2-1

rcf2-1 +RCF2:RCF2(D161A) #7rcf2-1 +RCF2:RCF2(D161A) #8

HSFA7bA BR

elat

ive

mR

NA

leve

l (x

103 )

37C (h) 37C (h)

Rel

ativ

e m

RN

A le

vel (

x 10

3 )

aa a a aa a a

b b

c c c

c c c

Supplemental Figure 12. Expression Levels of HSFA7b and HSP101 in rcf2-1 Plants Expressing RCF2:RCF2(D161A). (A) and (B) Expression levels of HSFA7b and HSP101 in rcf2-1 plants expressing RCF2:RCF2(D161A)-FLAG under heat stress. RCF2 (D161A), the phosphatase inactive form of RCF2. One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.01). Results in Supplemental Figure 12 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.



12

0

5

10

15

20

25

WT #6 #8

35S:FLAG-RCF2 lines

0

5

10

15

20

25

30

35

WT #6 #14

35S:HA-NAC019 lines

Rel

ativ

e m

RN

A le

vel

Rel

ativ

e m

RN

A le

vel

A RCF2 NAC019

b

a

b b

a

b

B

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1

WT

nac019

nac019 + 35S:HA-NAC019 #1nac019 + 35S:HA-NAC019 #5

Rel

ativ

e m

RN

A le

vel

HSFA6b HSP18

0

1

2

3

4

5

0 1

WT

nac019

nac019 + 35S:HA-NAC019 #1nac019 + 35S:HA-NAC019 #5

0

5

10

15

20

25

30

35

0 1

WT

rcf2-1

rcf2-1 + 35S:FLAG-RCF2 #3rcf2-1 + 35S:FLAG-RCF2 #5

HSFA7b

Rel

ativ

e m

RN

A le

vel (

x 10

3 )0

2

4

6

8

10

12

0 1

WT

rcf2-1

rcf2-1 + 35S:FLAG-RCF2 #3rcf2-1 + 35S:FLAG-RCF2 #5

HSP101


37C (h)

37C (h)

C

D E F

37C (h) 37C (h)R

elat

ive

mR

NA

leve

l (x

103 )

Rel

ativ

e m

RN

A le

vel (

x 10

3 )

Supplemental Figure 13. Complementation of rcf2-1 with 35S:FLAG-RCF2 and Complementation of nac019 with 35S:HA-NAC019. (A) RCF2 expression level in wild-type plants expressing 35S:FLAG-RCF2. (B) NAC019 expression level in wild-type plants expressing 35S:HA-NAC019. (C) and (D) Expression of HSFA7b and HSP101 in rcf2-1 plants expressing 35S:FLAG-RCF2. (E) and (F) Expression of HSFA6b and HSP18 in nac019 plants expressing 35S:HA-NAC019. Error bars indicate the standard deviation (n = 6). One-way ANOVA (Tukey-Kramer test) was performed and statistically significant differences are indicated by different lowercase letters (p < 0.01). Values in Supplemental Figure 13 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

a a a a

a a a a a a a a

a a a a

b

c

bb

b

c

bbb

c

bb b

c

bb


13

Supplemental Figure 14 A AtNAC019 MGIQ-----ETDPLTQLSLPPGFRFYPTDEELMVQYLCRKAAGYDFSLQLIAEIDLYKFDPWVLPNKALFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKIISTEG-----QR 110 RcNAC019 MGVQ-----EMDPLTQLSLPPGFRFYPTDEELLVQYLCRKVAGHQFSLQIIGEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEG-----RK 110 VvNAC019 MGVP-----ETDPLSQLSLPPGFRFYPTDEELLVQYLCRKVAGQGFSLEIIGEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEG-----RK 110 PtNAC019 MGLQ-----ETDPLAQLSLPPGFRFHPTDEELLVQYLCKKVAGHHFSLQIIGEIDLYKFDPWFLPGKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEG-----RK 110 PpNAC019 MGVP-----ETDPLSQLSLPPGFRFYPTDEELLVQYLCRKVAGYQFSLQIIAEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKIITTEG-----RK 110 GmNAC019 MGVP-----EEDPLSQLSLPPGFRFYPTDEELLVQYLCRKVAGHHFSLPIIAEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKIITTEG-----RK 110 SlNAC019 MGVQ-----EMDPLTQLSLPPGFRFYPTDEELLVQYLCRKVAGHDFSLQIIAEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTDG-----RK 110 CsNAC019 MGVA-----EKDPLSELSLPPGFRFYPTDEELLVQYLCRKVAGHNFSLQIIGDIDLYKFDPWVLPSKAMFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKIISTQG-----KK 110 SbNAC019 MGLPVMRR-ERDAEAELNLPPGFRFHPTDDELVEHYLCRKAAGQRLPVPIIAEVDLYKFDPWDLPERALFGVREWYFFTPRDRKYPNGSRPNRAAGNGYWKATGADKPVAPRG-----RT 114 OsNAC019 MAAAVGGSGRRDAEAELNLPPGFRFHPTDEELVVHYLCRKVARQPLPVPIIAEVDLYKLDPWDLPEKALFGRKEWYFFTPRDRKYPNGSRPNRAAGRGYWKATGADKPVAPKGS---ART 117 ZmNAC019 MGQPVTRRRERDAEAELDLPPGFRFHPTDDELVEHYLCRKAAGQRLPVPIIAEVDLYRFDPWDLPERALFGAREWYFFTPRDRKYPNGSRPNRAAGDGYWKATGADKPVAPRAAAADART 120 AtNAC019 VGIKKALVFYIGKAPKGTKTNWIMHEYRLIEP----SRRNGSTKLDDWVLCRIYKKQSSAQKQVYDNGIANAREFSNNGTSSTTSSSSHFEDVLDSFHQEIDNRNFQFSNPNRISSLRPD 226 RcNAC019 VGIKKALVFYVGKAPKGTKTNWIMHEYRLLES----SRKNGSTKLDDWVLCRIYKKNSGAQK-PMTS-FPSSKEHSN--NGSSSSSSSHLDDVLDSLT-EIDDHFFALP---TAKTMQPD 218 VvNAC019 VGIKKALVFYVGKAPKGTKTNWIMHEYRLLEN----SRKNGSSKLDDWVLCRIYKKNSNSSK-PIAA-VLPSKAHS---NGSSSSSSSHLDDVLESLP-EIDDRFFSPNRMNSLRVSQPD 220 PtNAC019 VGIKKALVFYVGKAPKGTKTNWIMHEYRLLES----SRKSGSTKLDEWVLCRIYKKNSSAAQ-KSMS-SVSSKEYST--NGSCSSSSSHLEDVLDSLT-EIDDRLFALPRTNSLKQMQHE 221 PpNAC019 VGIKKALVFYVGKAPKGTKTNWIMHEYRLIEP----SRKNGSSKLDEWVLCRIYKKSSSSAAQKPMTTSVSSKEHSN-G-SSSSCSS-QLDDVLEWLP-DIDDRCFTLPRINSLKTLQQQ 222 GmNAC019 VGIKKALVFYIGKAPKGTKTNWIMHEYRLLDS----SRKNTGTKLDDWVLCRIYKKNSSAQK-AVQNGVVPSNEHTQYSNGSSSSSSSQLDDVLESLP-AIDERCFPMPRVNTLQQQQHE 224 SlNAC019 VGIKKALVFYIGKAPKGTKTNWIMHEYRLSEP----TTKTGSSRLDDWVLCRIYKKNSGGQK--SSCSDLQNKDISH---ASSSSSSSQFDDMLESLP-AIEDRYFSLPRVNSIRNFQQN 220 CsNAC019 VGIKKALVFYVGKAPKGTKTNWIMHEYRLIDT----SRKTGSTKLDDWVLCRIYKKNSSAQKLPRMTSSISSMECSN-GGSSSTCSSSHLDDVLESLP-EIKDGFFRLPRVNSLLTLQQD 224 SbNAC019 LGIKKALVFYAGKAPRGVKTDWIMHEYRLADAGRAAASKKGSLRLDDWVLCRLYNKKNEWEKMQLGKESAAGVGTAK--EEAMDMTTSHSHSHSQSHSHSWGETRTPESEIVDNDPFPEL 232 OsNAC019 VGIKKALVFYSGKAPRGVKTDWIMHEYRLADADRAPGGKKGSQKLDEWVLCRLYNKKNNWEKVKLEQQDVASVAAAA--PRNHHHQNGEVMDAAAADTMS-DSFQTHDSDIDNAS----- 229 ZmNAC019 LGIKKALVFYAGKAPRGVKTDWIMHEYRLADAGRRAK--KGSLRLDDWVLCRLYNKKNEWEKMRLGKGAAAGA-VKE--EEAMDMSTSHSQ-----ITHSWGETRTPESEIVDND----- 225 AtNAC019 LTEQKTGFHGLAD-TSNFDWASFAG--NVEHNNSVPELGMS--------HVVPNLEYNCG------------------------------YLKTEEEVESSHGFNNSG------------ 293 RcNAC019 ---DKININNLG--SGNFDWAT------LAGLNSVSELATA-----QQAQSQTQGLMNYTQNGDDLYVP------SFQQHIG------HVDIKMEEEVQSGVRTHRAD---SSRFLQQTS 307 VvNAC019 ---EKVNFHNLG--SGNFDWAT------LAGVSSLQELVSG-----VQSHAQPPAAVN---NSNEMYVP------SLPP-----------LIQAEEEVQSGLRTQRVDPVMNQGFFPQNS 304 PtNAC019 ---EKINLANLG--SGSFDWAT------LAGLNSLPELLQT-----QPGANYSNTNVNGVHVPSMPPLC------HADSSTG------RMGTSVEEEVQSGVRTQLRDG--NSGAFQQNS 311 PpNAC019 Q--EDSKLG-------------------LAGLNVVPELCPN------NQPQQSQGQMNVNYSNNDMYVPSIPPLCHVESPPE------RLAKTVDEEVQSGFRTQRVD----NSGFFQNS 305 GmNAC019 ---EKVNVQNLGE-GGLLDWTN------PSVLNSVVDFVSGNNNHNQLVQDQTQGMVNYN-ACNDLYVP------ALCHVGT------SVPQKMEEEVQSGVRNQRVQ---NNSWFLQND 318 SlNAC019 ---DKINLQQLS--SGNFDWAA------MAGLNSFPELRTG--NQVPTPGNQTPVLINTNQYHNHNDNLNNFNEFFANSTALNFHGEVKFEGGVDQEVESSVRAQRLNS-VNPGFFQENS 326 CsNAC019 H--ENLKFQNHLMGSTNFEWASTAAATSFEGYNSVAELAPL------AQSQAPSGLISS-----DMYIP-------AGQPPR---------STVEQEVQSGF--QRFH----NFGWLQ-- 307 SbNAC019 D--SFPAFQDPAA--------------AMMMVPKKEQVDDGS--AAANAAKSSDLFVDLS----------------------------------YDDIQGMYSG---------LDMLPPP 291 OsNAC019 -----AGLRHGGC--------------GGGGFGDVAPPRNG----FVTVKEDNDWFTGLN----------------------------------FDELQPPYMMNLQHM---QMQMVNPA 289 ZmNAC019 -----LPFPDPA-----------------MMVPKKERVDDG------GSARTSDLFVDLS----------------------------------YDDIQGMYSG---------LDMLPPA 274 AtNAC019 ---ELAQKGYGVDSFGYS----GQVGGFGFM-- 317 RcNAC019 SVLTQNLSNS-FDPYGFR-YSAQPGSGFGFRQ- 337 VvNAC019 NAFSQSFSNS-LDPFGFR-YPTQP-SGFGYRQ- 333 PtNAC019 GVMTPNFSSTLLDPYELR-YSTQPGSGYGFRQ- 342 PpNAC019 NVMTQNFCNP-TDPYGYSNRLGRSGLGFGGAEK 337 GmNAC019 --FTQGFQNS-VDTSGFK-YPVQP-VGFGFRN- 345 SlNAC019 TGFSSSYTNSVPDPFGIR-YPTQT-VNMGFTG- 356 CsNAC019 ----QNLSNS-GDGFGF---------------- 319 SbNAC019 G---EDFFSSLFASPRVKGNQPAGAAGLGLF-- 319 OsNAC019 APGHDGGYLQSISSPQMKMWQTI----LPPF-- 316 ZmNAC019 G---EDLYSSLFASPRVRGNQPTGPAGLGPF-- 302


14

B

C AtRCF2 MYSNNRVEVFHGDGRLGELEIYPSRELNQQQDDVMKQRKKKQREVMELAKMGIRISHFSQSGERCPPLAILTTISSCGLCFKLEASPSPA-------QESLSLFYSSCLRDNKTAVMLLGGEELHLVAMYSENIKNDRPC 133 GmRCF2 MYKS---VVYQGEVVVGEVDVYP-----EENNN-----------YKNFHVKEIRISHFSQPSERCPPLAVLHTVTSCGVCFKMESKTQQQ--------DGLFQLHSLCIRENKTAVMPLGGEEIHLVAMHSRNVDR--PC 111 PpRCF2 MYKS---VVYKGEELLGEVEIYP-----EENENK------NKNKNLVDELKEIRISYFSQSSERCPPVAVLHTISSHGVCFKMESKTSQS------QDTPLFLLHSSCVMENKTAVMPLGGEELHLVAMRSRNGDKRYPC 120 PtRCF2 MYKS---VVYKGDELLGEVEIYA-----QEQQQEEEENKNKKKRVIDEIVKEIRISHFSQTSERCPPLAVLHTITSIGVCFKMEESTSSSTTKISQQESPLHLLHSSCIQENKTAVMHLGGEELHLVAMPSRSNERQHPC 132 VvRCF2 MYKS---IVYEGDDVVGEVEIYP-----QNQG--------------LELMKEIRISHYSQPSERCPPLAVLHTITSCGVCFKMESSKAQS------QDTPLYLLHSTCIRENKTAVMSLGEEELHLVAMYSKKKDGQYPC 112 SlRCF2 MFKS-TVLLYEGERLVGEVEMYG------EKG-------------VVWGEKLIRISHYSPSSERCPPLAVLHTVTT-GLSFKLEPTKSKP----LTQDSPLTLLHSTCLRDNKTAVMSLGREELHLVAMQSKNIGGQCPC 115 CsRCF2 MYKS---VVYHGDELLGDVEIYP-----EEKNG-----------YKNIEVKEIRITHFSQPSERCPPLAVLHTIAASGICFKMESKTSQS------QDTPLNLLHSSCIMENKTAIMMFGVEELHLVAMFSRDLDKQYPC 115 SbRCF2 MFKS---MVYYGNTSIGEVEVWP-----KGEASA--------GLAVAAWAREIRVDRLSPPSERCPPLAVMHTVAVGARCLVMESRPPVAAD---VAPMPLVAMHAACLRDNKTAVVPLGDGELHLVAMTSRRNLTNHAC 121 OsRCF2 MIRS---MVYFGNTSIGEVEVWP-----SGDAS----------LAAAAWAREIRVDRLSPPSERCQPLAVMHTVAVGARCLVMESRLPKAAD---EPPQPLVAMHAACLKENKTAVVPLGEEELHLVAMTSRRNLTNHAC 119 ZmRCF2 MFKS---MVYYVNTSIGEVEVWP-----KGEASA--------GLTMAAWAREIRVERISPPSERCPPLAVMHTVAVGARCLVMESRPPVVAD---VVP-PLVVMHTACLRENKTAVVPLGDEELHLVAMTSRRNLTNHAC 120 RcRCF2 MYKS---VVYHGDELLGDVEIYP-----EEKNG-----------YKNIEVKEIRITHFSQPSERCPPLAVLHTIAASGICFKMESKTSQS------QDTPLNLLHSSCIMENKTAIMMFGVEELHLVAMFSRDLDKQYPC 115 AtRCF2 FWAFSVAPGIYDSCLVMLNLRCLGIVFDLDETLVVANTMRSFEDKIDGFQRRINNEMDPQRLAVIVAEMKRYQDDKNLLKQYIESDQVVENGEVIKVQSEIVPALSDNHQPLVRPLIRLQEKNIILTRINPMIRDTSVLV 273 GmRCF2 FWGFIVALGLYDSCLVMLNLRCLGIVFDLDETLIVANTMRSFEDRIDALQRKINSEVDPQRISGMQAEVKRYQDDKNILKQYAENDQVVDNGRVIKVQSEIVPALSDSHQPIVRPLIRLQDKNIILTRINPQIRDTSVLV 251 PpRCF2 FWGFSVAPGLYNSCLVMLNLRCLGIVFDLDETLIVANTMRSFEDRIEALQRKISSEVDPQRISGMLAEIKRYQDDKFILKQYAENDQVVENGRVIKTQSEAVPALSDNHQPIIRPLIRLHEKNIILTRINPQIRDTSVLV 260 PtRCF2 FWGFSVAPGLYDSCLVMLNLRCLGIVFDLDETLIVANTMRSFEDRIDALQRKISTEVDPQRILGMLSEVKRYHDDKNILKQYVENDQVVENGKVIKTQSEVVPALSDNHQPMVRPLIRLQEKNIILTRINPQIRDTSVLV 272 VvRCF2 FWGFNVALGLYSSCLVMLNLRCLGIVFDLDETLIVANTMRSFEDRIDALQRKINTEVDPQRISGMVAEV-------------------VENGKLFKTQPEIVPALSDNHQPIVRPLIRLQEKNIILTRINPLIRDTSVLV 233 SlRCF2 FWGFKVASGLYDSCLTMLNLRCLGIVFDLDETLIVANTMRSFEDRIEALQRKINSESDPQRASVMLAEVKRYQEDKIILKQYAENDQVVDNGKVIRSQSEVFPALSDNHQPIVRPLIRLQDRNIILTRINPMIRDTSVLV 255 CsRCF2 FWGFNVAMGLYNSCLDMLNLRCLGIVFDLDETLVVANTMRSFEDRIEALQRKISSEVDPQRANGMLAEVKRYQDDKIILKQYAENDQVIENGKVIKSQSEVVPALSDNHQPVVRPLIRLHEKNIILTRINPQIRDTSVLV 255 SbRCF2 FWGYKLPFGLYNSCLTMLNLRCLGIVFDLDETLIVANTSRSFEDRIDGLQRKLSNETDPQRRNGMLSEIKRYQDDKSILKQYIEGDQVYDDGKVYKAQPEIVPPLSDNQQPMTRPVIRLQDKNIILTRINPLIRDTSVLV 261 OsRCF2 FWGYKVPFGLYNSCLTMLNLRCLGIVFDLDETLIVANTTRSFEDRIDALQRKLSNETDPQRINGMIAEIKRYQDDKSILKQYIEGDQVYDDGKMYKVQPELVPPLSDNHQPMTRPVIRLQEKNIILTRINPLIRDTSVLV 259 ZmRCF2 FWGYKLPFGLYNSCLTMLNLRCLGIVFDLDETLIVANTSRSFEDRIDALQRKLSNETDPQRRNGMLSEIKRYQDDKSILKQYIEGDQVYDDGKVYKAQPEIVPPLSDNQQPMTRPVIRLQDKNIILTRINPLIRDTSVLV 260 RcRCF2 FWGFNVAMGLYNSCLDMLNLRCLGIVFDLDETLVVANTMRSFEDRIEALQRKISSEVDPQRANGMLAEVKRYQDDKIILKQYAENDQVIENGKVIKSQSEVVPALSDNHQPVVRPLIRLHEKNIILTRINPQIRDTSVLV 255


15

AtRCF2 RMRPSWEELRSYLTAKGRKRFEVYVCTMAERDYALEMWRLLDPEGNLINTNDLLARIVCVKSG--FKKSLFNVFLDGTCHPKMALVIDDRLKVWDEKDQPRVHVVPAFAPYYSPQAEAA-ATPVLCVARNVACGVRGGFF 410 GmRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPDSNLINSKELLGRIVCVKSG--LKKSLFNVFQDGLCHPKMALVIDDRLKVWDEKDQPRVHVVPAFAPYYAPQAEASNTIPVLCVARNVACNVRGGFF 389 PpRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPDSNLINSNKLLDRIVCVKSG--SRKSLFNVFQESLCHPKMALVIDDRLKVWDDRDQPRVHVVPAFAPYYAPQAEANNAVPVLCVARNVACNVRGGFF 398 PtRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPESNLINSKELLDRIVCVKSGKCLRKSLFNVFQDGICHPKMALVIDDRLKVWDERDQSRVHVVPAFAPYYAPQAEVNNAVPVLCVARNVACNVRGGFF 412 VvRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPESNLINSKELLDRIVCVKSG--SRKSLFNVFQDGICHPKMALVIDDRLKVWDEKDQPRVHVVPAFAPYYAPQAEANNAISVLCVARNVACNVRGGFF 371 SlRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPDSNLINSQELLDRIVCVKSG--LRKSLFNVFQDGNCHPKMALVIDDRLKVWDDKDQPRVHVVPAFAPYFAPQAEGNNSVPVLCVARNVACNVRGGFF 393 CsRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPDSNLINPKELLDRIVCVKSG--SRKSLFNVFQDGFCHPKMALVIDDRLKVWDEKDQPRVHVVPAFAPYYAPNAEGNNAIPVLCVARNVACNVRGGFF 393 SbRCF2 RLRPAWEDLRSYLIARGRKRFEVYVCTMAERDYALEMWRLLDPDSRLINSVQLHDRMVCVKSG--LKKSLLNVFHDGSCHPGMALVIDDRLKVWDEKDQLRVHVVPAFTPYYAPQAEANCSIPVLCVARNVACNVRGGFF 399 OsRCF2 RLRPAWEDLRSYLIARGRKRFEVYVCTMAERDYALEMWRLLDPDSRLINSVQLNDRMVCVKSG--LRKSLLNVFHDGSCHPGMALVIDDRLKVWDEKDQSRVHVVPAFTPYYAPQAEANCSIPVLCVARNVACNVRGGFF 397 ZmRCF2 CLRPAWEDLRSYLIARGRKRFEVYVCTMAERDYALEMWRLLDPDSRLINSVQLHDRMVCVKSG--LKKSLLNVFHDGSCHPGMALVIDDRLKVWDEKDQLRVHVVPAFTPYYAPQAEANCSIPVLCVARNVACNVRGGFF 398 RcRCF2 RLRPAWEDLRSYLTARGRKRFEVYVCTMAERDYALEMWRLLDPDSNLINPKELLDRIVCVKSG--SRKSLFNVFQDGFCHPKMALVIDDRLKVWDEKDQPRVHVVPAFAPYYAPNAEGNNAIPVLCVARNVACNVRGGFF 393 AtRCF2 RDFDDSLLPRIAEISYENDAEDIPSPPDVSHYLVSEDD-TSGLNGNKDPLSFDGMADTEVERRLKEAISASSAVLPAANIDPRIAA-PVQFPMASASSVSVPVPVQVVQQAIQPSAMAFPSIPFQQPQ---QPTSIAKHL 545 GmRCF2 KDFDDGLLQKIPQIAYEDDIKDIPSPPDVSNYLVSEDD-GSISNGHRDPFLFDGMADAEVERKLKDALSAASTIPVTTANLDPRLT-SLQYTMVPSGS--------VPPPTAQASMMPFPHVQFPQPA---TLVKPMGQA 516 PpRCF2 REFDDSLLQKIPEVFYEDDIKDVPS-PDVSNYLVSEDD-SSALNGNRDPLPFDGITDVEVERRMKEATPAASMVSSVFTSIDPRLA-PLQYTVPPSST--------LSLPTTQPSVMSFPSIQFPQAA---SLVKPLGHV 524 PtRCF2 KEFDEGLLQKIPEVAYEDDTDNIPSPPDVSNYLVSEDD-ASAVNGNRDQLSFDGMADAEVERQLKVFRCSLQYTIASSSSSMP----TSQPSMLASQQPMPAL--QPPKPPSQLSMTPFPNTQFPQVA---PSVKQLGQV 542 VvRCF2 KEFDEGLLQRIPEISYEDBIKDIRSAPDVSNYLVSEDD-ASVSNGNRDQPCFDGMADVEVERKLKDAISAPSTVTSLDPRLSP----PLQFAVAASSG-------LAPQPAAQGSIMPFSNKQFPQSA---SLIKPL--- 493 SlRCF2 KDFDEGLLQRISEVAYEDDIKQVPSAPDVSNYLISEDD-PSAVNGNKDSLGFDGMADSEVERRLKEAMLASTSVPSQMTNLDPRLVPALQYPVPPVIS----------QPSIQGPVVPFPTQHLPQVTS--VLKSSVTQI 520 CsRCF2 KEFDDILLQKISDISYEDDVNDIPSPPDVSNYLVSEDE-YSIANGNKDMPTFDGMPDMEVDRRMKDAFLASSTINSADPRVSS-----LQYTMASASCS-------VPLPPKQVTMPYFPNMPLPH-------VNSVAHV 513 SbRCF2 KDFDEGLLPRISNVHYEDEVNDISSAPDVGNYLITEDENAALVNGNRDSLPFDGMADAEVERRMKEAN--AQAFHQTAG--------NFVMPVAPAQN--------FVSSSVAPLAPPLGVMP-------PTFSQPVVQP 514 OsRCF2 KDFDEGLLPRISNVFYEDEINEIPSAPDVGNYLISEDENVAAVNGNRDPLAFDGMADAEVERRMKEASGNAQAFTTTAA--------NFVMPVLPGQN--------FVSSSVAPVAPSLGMVPLSNNQGPPPFTQPVAQL 521 ZmRCF2 KDFDEGLLPRISNVHYEDEVNEIS-APDVGNYLITDDENVALVNGNRDSLPFDGMADAEVERRMKEAN--AQSFHQTAG--------DFVMPVAPAQN--------FVSTSVASLAPPLGMMP-------SPFSQPVAPP 512 RcRCF2 KEFDDILLQKISDISYEDDVNDIPSPPDVSNYLVSEDE-YSIANGNKDMPTFDGMPDMEVDRRMKDAFLASSTINSADPRVSS-----LQYTMASASCS-------VPLPPKQVTMPYFPNMPLPH-------VNSVAHV 513 AtRCF2 VPSEPSLQSSPAREEGEVPESELDPDTRRRLLILQHGQDTRDPAPSEPSFPQRPP--VQAPPS-HVQS-RNGWFPVEEEMDPAQIRRA-VSKEYPLDSEMIHM-EKHRPRHPSFFSKIDNSTQSDRMLHE-NRRPPKESL 678 GmRCF2 APSEPSLHSSPAREEGEVPESELDPDTRRRLLILQHGQDTRDHASAEPPFPVRHP--VQTSAP-HVPSSRGVWFPAEEEIGSQPLNRV-VPKEFPVDSGPLGI-AKPRPHHPSFFSKVESSISSDRILHDSHQRLPKEMY 651 PpRCF2 GSAEPSLQSSPAREEGEVPESELDPDTRRRLLILQHGQDTRDQPPSEPPFPVRPP--MQASVP-RAQS-RPGWFPVEEEMSPRQLSRM-VPKDLPLDPETVQI-EKHRPHHSSFFPKVENSIPSDRILQE-NQRLPKEAF 657 PtRCF2 VPPEPSLQSSPAREEGEVPESELDPDTRRRLLILQHGHDSRDNAPSESPFPARPS--TQVSAP-RVQS-VGSWVPVEEEMSPRQLNR--TPREFPLDSDPMNI-EKHRTHHPSFFHKVESNIPSDRMIHE-NQRQPKEAT 674 VvRCF2 -APEPTMQSSPAREEGEVPESELDPDTRRRLLILQHGQDTREHASSDPPFPVRPP--IQVSVP-RVQS-RGSWFPADEEMSPRQLNRA-VPKEFPLDSDTMHI-EKHRPHHPSFFHKVESSASSDRILHE-NQRLSKEVL 625 SlRCF2 SPQDTSLQSSPAREEGEVPESELDPDTRRRLLILQHGQDTRDQVSSEPKFPIGTP--LQVSVPPRVQP--HGWFPAEEEVSPRQLNRPLPPKEFPLNPESMHI-NKHRPPHPPFLPKMETSMPSDRVFFE-NQRLPKEVI 654 CsRCF2 APNEPSLQSSPAREEGEVPESELDPDTRRRLLILQHGQDTRERLSSEPAFPARPPPLQQVAAP-RAQS-RGNWSPMEEEMSPRQLNRS-ARKDFPVDAEPMPMREKHRSNHPSFFAKVDNSILPDRIPHD-NQRLPKEAF 649 SbRCF2 GFSDS-LQGSPAREEGEVPESELDPDTRRRLLILQHGQDIRDP---TPPLPAIPP--VQVPVP-PVQP-HGNWFPTEDGLNPSNLNRG--SAGFTVESDPMLY-EKKQPPHPSFFHGGDSPMSSDRFGYQ-NQRFPSQLP 642 OsRCF2 SLSDP-LQGSPAREEGEVPESELDPDTRRRLLILQHGQDTRDP---TPPLPAVPP--VQVPVP-PVQP-HGNWFPVEDGMNPNNLNRG--SAGFPLESETMHY-DKKQLPHP-FFHGGENPISSDRFSYQ-NQRYPSQLP 648 ZmRCF2 GFSDS-LQGSPAREEGEVPESELDPDTRRRLLILQHGQDTRDP---TSPLPAIPP--VQVPVP-PVQP-HGNWFPTEDGINQSNLNRG--SAGFTVESDSIVY-EKKQPPHPSFFHGGDSPMPSDRFGYQ-NQRFPSQLP 640 RcRCF2 APNEPSLQSSPAREEGEVPESELDPDTRRRLLILQHGQDTRERLSSEPAFPARPPPLQQVAAP-RAQS-RGNWSPMEEEMSPRQLNRS-ARKDFPVDAEPMPMREKHRSNHPSFFAKVDNSILPDRIPHD-NQRLPKEAF 649 AtRCF2 R-RDEQLRSNNNLPDSHPFYGEDASWNQSSSRNSDLDFLPERS-VSATETSADVLHGIAIKCGAKVEYKPSLVSSTDLRFSVEAWLSNQKIGEGIGKSRREALHKAAEASIQNLADGYMRANGD-----PGPSHRDATPF 811 GmRCF2 H-RDDRPRLNHMLSSYRSFSGDDIPFSRSFSSHRDLDSESGHS-VLHADTPVAVLQEIALKCGTKVDFISSLVASTELQFSMEAWFSGKKIGHRVGRTRKEAQNKAAEDSIKHLADIYLSSAKDEPGSTYGDVS--GFPN 787 PpRCF2 H-RDDRLRFNHALSGYHSLSGEEIPLSRSSSSNRDVDFESGRA-ISNAETPAG------------------VLQEIAMKCGAKAWFAGEKIGEGSGKTRREAHYQAAEGSLKNLANIYLSRVKPDSVSVHGDMN--KFPN 775 PtRCF2 Y-RDDRMKLNHSTSNYPSFQG-ECPLSRSSS-NRDLDLESERA-FSSTETPVEVLQEIAMKCGTKVEFRPALIATSDLQFSIETWFVGEKVGEGTGKTRREAQRQAAEGSIKKLAGKIS----------YHDISYYSYPS 800 VvRCF2 H-RDDRLRLNHSLPGYHSFSGEEVPLGRSSS-NRDLDFESGRG-APYAETP----------------------AVGLLRNCNEVWNQGEKIGEGTGKTRREAQCQAAEASLMYLSYRYL----------HGDVN--RFPN 728 SlRCF2 P-RDDRMRFSQSQPSFR-PPGEDVSLGRSSSSNRVLDLDPGHY-DPYLDTPAGALQDIAFKCGVKVEFRSSFLSSPELQFCLEVLFAGEKVGEGIGRTRREAQRHAAEESLMYLADKYLSCIKADSSSTQGDGFR--FPN 789 CsRCF2 Y-RDDRMRVSRRPSSYPAFSGEEIPMNQSSSRSRDDDIESGR--SIWSETPVGALQEIAMKFGTKVEFKPGLVPSTDLQFSVEAWFVGEKIGEGIGHTRRDAQRQAAEGSIKNLANIYVS-------------------- 766 SbRCF2 HTEDHHMLQNHAPPKYRSFSGEELAARHVPSSQRNNQIESGRHFAQYAGTSAGILDGIALKCGSKVEYRSTLCDTAELQFSIEVWIVGEKVGEGIGRTRREAQHKAAEMSLRNLANKYLS------------SDPNKLTD 770 OsRCF2 HSEDHRVLQNHAPSRYRSFPGEELATRHVSSSQRNNQIVPGQHFARHAGSSAGILEEIAMKCGSKVEYRSALCDTADLQFSIEVWIVGEKVGEGIGRTRKEAQCQAAEISLRNLANKYLS------------SDPNKMTD 776 ZmRCF2 H-EDHPMMQNHAPPKYRSFSGEELASWHVPSSQRNNQIESGRHFAQYAGTSAGILEGIALKCGSKVEYKSALCDTAELQFSIEVWIVGEKVGEGIGRTRREAQRQAAEMSLRNLANKYLS------------SDPNKLSD 767 RcRCF2 Y-RDDRMRVSRRPSSYPAFSGEEIPMNQSSSRSRDDDIESGR--SIWSETPVGALQEIAMKFGTKVEFKPGLVPSTDLQFSVEAWFVGEKIGEGIGHTRRDAQRQAAEGSIKNLANIYVS-------------------- 766 AtRCF2 TNENISMGNANALNNQPFARDETALPVS----SRPTDPRLEGSMRHTGSITALRELCASEGLEMAFQSQR-QLPSDMVHRDELHAQVEIDGRVVGEGVGSTWDEARMQAAERALSSVRSMLGQPLHKRQGSPRSFG--GM 944 GmRCF2 VNDSGYMGIASSLGNQPLSKEDSASFSTAS-PSRVLDPRLDVSKRSMGSISSLKELCMMEGLDVNFLSAPAPVSTNSVQKDEVHAQVEIDGKVFGKGIGLTWDEAKMQAAEKALGSLRSKLGQSIQKRQSSPRPHQ--GF 924 PpRCF2 VNSNGFAGNLNSFGIQPFPKEESLSSSTSSEPSRPLDPRLEGSKKSMSSVSTLKELCMMEGLGVVFQPRP-PPSTNSVEKDEVHVQVEIDGEVLGKGIGLTWDEAKMQAAEKALGSLTSTL--YAQKRQGSPRSLQ--GM 910 PtRCF2 ANDNGFLGDMNSFGNQPLLKDENITYSATSEPSRLLDQRLEGSKKSMGSVTALKEFCMTEGLGVNFLAQT-PLSTNSIPGEEVHAQVEIDGQVLGKGIGLTWDEAKMQAAEKALGSLRTMFGQYTPKRQGSPRLMQ--GM 937 VvRCF2 ASDNNFMSDTNSFGYQSFPKEGSMSFSTASESSRLLDPRLESSKKSMGSISALKELCMMEGLGVEFLSQP-PLSSNSTQKEEICAQVEIDGQVLGKGTGSTWDDAKMQAAEKALGSLKSMLGQFSQKRQGSPRSLQ--GM 865 SlRCF2 ASDNGFVENMSPFG-----YQDRVSHSFASEPPRVLDPRLEVFKKSVGSVGALRELCAIEGLGLAFQTQP-QLSVNPGQKSEIYAQVEIDGQVFGKGIGPTWDDAKTQAAERALVALKSELAQFSHKRQGSPRSLQQQGF 923 CsRCF2 -------------------------------------------------------RCKAD-----------PSSAN---------------------------DMNKFPSDNGSG------------------------- 788 SbRCF2 MKENGFSGNRNVFGYSGNTRDDMLPLSSTSEESRFMKMENN-SRKTGGSVAALKELCTVEGYNLVFQE--RPSPADGLVGKESYAQVEVGGQILGKGVGLTWEEAKLQAADEALGTLRSMLGQLAHKRSGSPRSLAP--N 905 OsRCF2 MKENGFGSNTNIFGYPGNSRDDVLPIASTSEETRFVKMGENNSRKAGGSIAALKELCTAEGYNLVFQA--RTS-PDSSVGKEAYAEVEVCGQILGNGVGITWEDAKLQAADEALGMLRSMLGPLAQKRSSSPRSLAP--S 911 ZmRCF2 MKENDFSSNRNVFGYSGNTRDDMLPLSSTSEESRFMKMENNNSRKTGSSVAALKELCTVEGYNLVFQA--CPSSADGLVGKESYAQVQVGGQILGKGVGLTWEEAKLQAAAEALGTLRSMLGQLGHKRSGSPRSLAP--N 903 RcRCF2 -------------------------------------------------------RCKAD-----------PSSAN---------------------------DMNKFPSDNGSG------------------------- 788


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AtRCF2 SNKRLKPDFQR-SLQRMPSSGRYS------- 967 GmRCF2 SNKRLKQEYPR-PMQRMPSSARYPRNAPPIP 954 PpRCF2 SSKRMKQEFPQ-VLQRMPSSARYPKNAPPVP 940 PtRCF2 PNKRLKQEFPR-VLQRMPSSARYHKNASPVP 967 VvRCF2 G-KRLKSEFTR-GLQRTPSSGRYSKNTSPVP 894 SlRCF2 SNKRLKPEYSRGVQQRVPLSGRFPKNTSAMP 954 CsRCF2 --KRMKLDFHR-HLPKTK------------- 803 SbRCF2 FNKRFKPDFPR-TVQRVP-YGTYSRIEGHVP 934 OsRCF2 FDKRFKPDFPR-AVQRVP-YGRYSRIEGHVP 940 ZmRCF2 FNKRFKPDFPR-TVQRVP-YGTYSRIEGHVP 932 RcRCF2 --KRMKLDFHR-HLPKTK------------- 803

D

Supplemental Figure 14. Comparison of At-RCF2 and At-NAC019 with Their Close Homologs. (A) Comparison of amino acid sequences of AtNAC019 with its close homologs. The accession numbers for the aligned proteins are: AtNAC019 (Arabidopsis thaliana, NP_175697); RcNAC019 (Ricinus communis, XP_002533913); VvNAC019 (Vitis vinifera, XP_002284668 ); PtNAC019 (Populus trichocarpa, XP_002316917); PpNAC019 (Prunus persica, EMJ12761); GmNAC019 (Glycine max, NP_001238424); SlNAC019 (Solanum lycopersicum, XP_004244203); CsNAC019 (Cucumis sativus, XP_004147806); SbNAC019 (Sorghum bicolor, AFO85373); OsNAC019 (Oryza sativa Japonica Group, BAA89798); ZmNAC019 (Zea mays, ACL52720). (B) Phylogenetic tree of AtNAC019 and its close homologs. The protein identities are the same as defined in (A). (C)


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Comparison of amino acid sequences of AtRCF2 with its close homologs. The accession numbers for the aligned proteins are: AtRCF2 (Arabidopsis thaliana, NP_193898); VvRCF2 (Vitis vinifera, CAN72816); PtRCF2 (Populus trichocarpa, XP_002305017); PpRCF2 (Prunus persica, EMJ15747); GmRCF2 (Glycine max, XP_003529311); SlRCF2 (Solanum lycopersicum, XP_004232844); CsRCF2 (Cucumis sativus, XP_004134718); SbRCF2 (Sorghum bicolor, XP_002452510); OsRCF2 (Oryza sativa Japonica Group, NP_001047536); RcRCF2 (Ricinus communis, XP_002519032). (D) Phylogenetic tree of AtRCF2 and its close homologs. The protein identities are the same as defined in (C). Sequence alignments in (A) and (C) were performed with ClastalW program as a part of the Bioedit package (version 7.09) with default settings (Hall, 1999) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Identical or conserved amino acid residues are shaded in black or grey, respectively. Phylogenetic trees in (B) and (D) were generated with the Phylogeny.fr platform (http://www.phylogeny.fr/version2_cgi/advanced.cgi) as described (Dereeper et al., 2008). Scale bars in (B) and (D) indicate branch length.


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Supplemental Table 1. Genetic Analysis of the rcf2-1 Mutant (Wild Type [Female] X rcf2-1 [Male] Cross).

Generation Seedlings Tested Wild Type rcf2-1

F1 31 31 0

F2 520 395 125 0.256 0.50 < P < 0.70

2 P Value


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Supplemental Table 2. Primers Used in This Study.

Primer Name Primer Sequence (5' to 3') Purpose

RCF2 attB1 FGGGGACAAGTTTGTACAAAAAAGCAGGCTAGAGGAGAAGGAAGAACGACGGAAATC

RCF2 attB2 RGGGGACCACTTTGTACAAGAAAGCTGGGTCAGAGTATCTTCCCGAAGATGGCATCCGTTG

RCF2 OE attB1 FGGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTATAGTAATAATAGAGTAGAAGTGT

RCF2 OE attB2 RGGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAGAGTATCTTCCCGAAGATGGCATC

RCF2 RT F TTGGGATGAGAAGGATCAGCCGAGGGTACRCF2 RT R TACCTCACCTTCCTCTCTAGCAGGAGAACTCrcf2-1 dCAPs F ACTTACGGTGAAATGGTATTTGAACTTGGTTCCArcf2-1 dCAPs R1 TACTCCCAATCGCTAACAAGTATCACG

NAC19 OE attB1 FGGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGTATCCAAGAAACTGACCCGT

NAC19 OE attB2 RGGGGACCACTTTGTACAAGAAAGCTGGGTCTCACATAAACCCAAACCCACCAACTTG

NAC019 attB1 FGGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGCTAGAGAGTAGTCAAGCCAATCCA

NAC019 attB2 RGGGGACCACTTTGTACAAGAAAGCTGGGTCCATAAACCCAAACCCACCAACTTGC

RCF2 Split-LUC attB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTATAGTAATAATAGAGTAGAAGTGT

RCF2 Split-Luc attB2GGGGACCACTTTGTACAAGAAAGCTGGGTCAGAGTATCTTCCCGAAGATGGCATCCGT

NAC19 Split-LUC attB1GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGTATCCAAGAAACTGACCCGT

NAC019 Split-LUC attB2 GGGGACCACTTTGTACAAGAAAGCTGGGTCCATAAACCCAAACCCACCAACTTGC

RCF2 SpeI F AGTCACTAGTATGTATAGTAATAATAGAGTAGAAGTGRCF2 XhoI R AGTCCTCGAGAGAGTATCTTCCCGAAGATGGCANAC019 XbaI F AGTCTCTAGAATGGGTATCCAAGAAACTGACCCGTNAC019 SpeI R AGTCACTAGTCATAAACCCAAACCCACCAACSALK096295 LP ACCGGGTTTCCGATTTTACCCGACCGATGSALK096295 RP TCTTCAGGTAGCCACAGTTGTACTCGAG

NAC019 F TCAAGACCTAACCGGGTTGCCGGATC

NAC019 R TCTCTAGCATTCGCGATTCCATTATCG

Plasmid construction of RCF2:RCF2-FLAG for ChIP assay and rcf2-1 gene complementation

Plasmid construction of 35S:RCF2-FLAG

Plasmid construction of 35S:NAC019-HA

rcf2-1 mutation genotyping PCR primers followed by digeston with ScrFI (cut WT only)

RT-PCR analysis to detect mis-splicing of RCF2 transcript in rcf2-1, and for qRT-PCR

Plasmid construction of NAC019:NAC019-HA for ChIP assay and nac019 gene complementation

qRT-PCR analysis

Plasmid construction of RCF2-nLUC for Split-LUC assay

Plasmid construction of NAC019-cLUC for Split-LUC assay

Construction of pSPYNE-RCF2 for BiFC assay

Construction of pSPYCE-NAC019 for BiFC assay

Genotyping of nac019 mutant plants


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HSFA1b F TGAGCAGGAGAATCGTGGTGACAATGTGHSFA1b R CTCTACTTGAAGGGTTCCCATTGTCTACHSFA2 F GAAGGGCTTAACGAAACAGGGCCACCACHSFA2 R GATCCTTGCTGATTCACATTCTGCAAACCHSFA3 F CTTGGGGACTGACCGGAGCTAGCTTCGTAGHSFA3 R GCTAGTGCTACTGCAGCAAGTTTGGTTGHSFA4a F ATGATTCTTCATCCGATTCTATCGTCTCTTGGHSFA4a R TCATCATTCGCAAATTCCCATTGCTCAGGHSFA6a F AGAGGGTCTCAAAGAAACGCCACCAACGHSFA6a R TGTTCTTAAGAAGATGCCTCTCTCCCTTCAGHSFA6b F TCCATGGCTGAAGCAGCCATAAATGATCCHSFA6b R TGCTTGAAGAATCTGGGAAGGAGAGTTACAGHSFA7a F AACCCGTTTCTCCCGGAAGGCTGCGATCHSFA7a R TGAGAAATTGCTGTGTTTGAAATGACGAGGHSFA7b F ATGGACCCGTCGTCAAGCTCCAGAGCACGHSFA7b R TGCTTGAAGTATAGAGGCAGAATAGTGGCCGAGHSFB1 F ATACGACGGCGTAAATCGGTGATTGCHSFB1 R ACTCACTCTCTTCGTCAGACTCCACCHSFB2a F GACGCATCAAACAGTTGTTGCTCCTTCGTCHSFB2a R TCAGTGGGCTGAGATCCGACGTAATTCGACHSFB2b F CTCTCAGCCTGCTATGGCCGCGGCTGHSFB2b R GTGCAGTGGTGCAGCTCGTTGTCCTCTGHSFC1 F CTTCTCGCAACGAATCTTACCTGCTTATTTCHSFC1 R CTTGACCGTACATCCCCCGCGCGTGTTTCDREB2A F ATGGCAGTTTATGATCAGAGTGGAGATAGAAACDREB2A R TCATACAACCCTTCTTCGACCCTTTCGCAGGTACDREB2C F CTTCTTCGACTGCTGCCACTGCCACTGTGTDREB2C R TCTTTTCCTTTCAGCTCCTCTTTAATACACHSP18 F AGCAACGAACAATGTCTCTCATTCCAAGCHSP18 R AACCTTGACTTCTTCCTTCTTCAGGCCTGHSP26.5 F ACTGTCTCCTAGTTTGATGGGTCAATCTTGTGHSP26.5 R TGAAGTAGCCATGATCGTCGGTGTTACGHSP70B F AGCTATTGGTATCGATCTCGGCACTACHSP70B R AAGCCTCAGCGACTTCCTTCATCTTCACHSP101 F ACGAGACAATTGCTACAGCTCATGAGCHSP101 R AGAAGACCCATAATCAACTGGTCAACAGCAt5g60910 qPCR F GCTTCTCAAAAAGATTAAGGAGAGGGAGAt5g60910 qPCR R AGCCGGAAGCAGAGAGTTTGGTTCCGT

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis of Agmous-like 8

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis

qRT-PCR analysis


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At4g11370 qPCR F CGATCTGACCCGACACGCTCTCTCTAt4g11370 qPCR R GGACATTTCATCTTGTTGTAGTCAACGATCTUB8 F ATAACCGTTTCAAATTCTCTCTCTCTUB8 R TGCAAATCGTTCTCTCCTTG

HSFA1b ChIPA F GCATGGCTATTTATCCAACCTGGAG

HSFA1b ChIPA R GCATTCATTCTTTCCCTCACATACTTGTCTCT

HSFA6b ChIPA F AGCCGATAATGCTTGCACTAAGACAAC

HSFA6b ChIPA R GTTTGATACGTATAAACACAGTGGGTCC

HSFA7a ChIPA F CTGTCTCTGGTACTACAGACCAGATG

HSFA7a ChIPA R CTCTTCCTAGAAGCTGTAGACTTCGTAGCC

HSFB1 ChIPA F CAGTAAATGGTTCCGTTTAAGCTGTTAG

HSFB1 ChIPA R TCTCCACATAGAAAAGTCGGAATGAAC

HSFC1 ChIPA F GATTCTACGCAACGTGTCGAATCAGTACTTG

HSFC1 ChIPA R GATGGCAAGTTATTAGTCCGAGCCGTTG

HSFC1 ChIPB F ACGGCTCGGACTAATAACTTGCCATCTAAG

HSFC1 ChIPB R GTTGTCCATAGCGAAATAAAGGAGAACAAG

HSFA1b ChIPB F GTTCCCGAATCCGTACCATCGCCGA

HSFA1b ChIPB R TGCTCCACGAAACGACCTCATTGG

HSFA6b ChIPB F GGTTCATTAAAGAGGAGTTTCCTGC

HSFA6b ChIPB R CTAATGGTTGTGGATAGCTCAATG

HSFA7a ChIPB F TGCTCCACCTCCATTTCTGACCAAG

HSFA7a ChIPB R GAATGCAAATCCCAGACGACAAAACTTG

ChIP-qPCR of HSFC1 in plants expressing NAC019:NAC019-HA , amplify region from -1917 to -1749 upstream of translation starting site ATG

ChIP-qPCR of HSFA1b in plants expressing NAC019:NAC019-HA , amplify region from 8 to 142 downstream of translation starting site ATG, as negative control ChIP-qPCR of HSFA6b in plants expressing NAC019:NAC019-HA , amplify region from 14 to 185 downstream of translation starting site ATG, as negative control ChIP-qPCR of HSFA7a in plants expressing NAC019:NAC019-HA , amplify region from 72 to 185 downstream of translation starting site ATG, as negative control

ChIP-qPCR of HSFA1b in plants expressing NAC019:NAC019-HA or RCF2:RCF2-FLAG , amplify region from -1717 to -1580 upstream of translation starting site ATG

ChIP-qPCR of HSFA6b in plants expressing NAC019:NAC019-HA or RCF2:RCF2-FLAG , amplify region from -1859 to -1719 upstream of translation starting site ATG

ChIP-qPCR of HSFA7a in plants expressing NAC019:NAC019-HA or RCF2:RCF2-FLAG , amplify region from -1323 to -1175 upstream of translation starting site ATG

ChIP-qPCR of HSFB1 in plants expressing NAC019:NAC019-HA or RCF2:RCF2-FLAG , amplify region from -1903 to -1722 upstream of translation starting site ATG

ChIP-qPCR of HSFC1 in plants expressing NAC019:NAC019-HA or RCF2:RCF2-FLAG , amplify region from -2045 to -1892 upstream of translation starting site ATG

Reference gene for qRT-PCR analysis

qRT-PCR analysis of RING-H2 figure A1A


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HSFB1 ChIPB F GCTGTGACGGCGGCGCAAAGATCAG

HSFB1 ChIPB R CCTTCTTCGTTCCATGAAACGACGTCG

HSFC1 ChIPC F CACCTTTCATCGTGAAAACATATCAGATG

HSFC1 ChIPC R GTAAGATTCGTTGCGAGAAGTCGAG

HSFA1b ChIPB F NEW ACCGCCAGATTGTTAGATATCAGCCGTCG

HSFA1b ChIPB R NEW GTTACTCCAGAAACTCTACTTGAAGGGT

HSFA6b ChIPB F NEW GTTTGCAAACGAAGGGTTTCTTAGAGGGCA

HSFA6b ChIPB R NEW AGCTCCATCATCAACACTTGCTTGTCTCG

HSFA7a ChIPB F NEW ACGAGCTTCGCAGAGAGAAGCAAGTGC

HSFA7a ChIPB R NEW CGAAGGACTCTGCATTGCTCTAGCCAG

HSFB1 ChIPB F NEW CTAGTGACGTTCTTGACGGGTCATCTG

HSFB1 ChIPB R NEW CACTCACCACATAATTCTTTTCATCCCGGTCC

HSFC1 ChIPB F NEW CAACACCGTCGCCGTCATCAACGGAGAATC

HSFC1 ChIPB R NEW CTCCGTTAACACTCTCCGGCAAAGAAAACG

RCF2 NotI F AGTCGCGGCCGCATGTATAGTAATAATAGAGTAGAAGTG

RCF2 EcoRI R AGTCGAATTCTTAAGAGTATCTTCCCGAAGATGGCANAC019 NdeI F AGTCCATATGATGGGTATCCAAGAAACTGACCCGTNAC019 SalI R AGTCGTCGACCATAAACCCAAACCCACCAACNAC019 XbaI F AGTCTCTAGAATGGGTATCCAAGAAACTGACCCGTNAC019 SalI R AGTCGTCGACTCACATAAACCCAAACCCACCAAC

RCF2 FLAG SpeI FAGTCACTAGTATGGACTACAAAGACGATGACGACAAAATGTATAGTAATAATAGAG

RCF2 XhoI R AGTCCTCGAGTTAAGAGTATCTTCCCGAAGATGGCAHSFA6b XhoI F AGTCCTCGAGAGTCATGTACGTCAAGTCGTGAAGGHSFA6b SpeI R AGTCACTAGTGTGGAGTTTTGGGTGTGAAGATGAAG

ChIP-qPCR of HSFC1 in plants expressing NAC019:NAC019-HA , amplify region from 44 to 169 downstream of translation starting site ATG, as negative control

ChIP-qPCR of HSFA1b in plants expressing RCF2:RCF2-FLAG , amplify region from 1358 to 1547 downstream of translation starting site ATG, as negative control

ChIP-qPCR of HSFA6b in plants expressing RCF2:RCF2-FLAG , amplify region from 1084 to 1293 downstream of translation starting site ATG, as negative control

ChIP-qPCR of HSFA7a in plants expressing RCF2:RCF2-FLAG , amplify region from 908 to 1077 downstream of translation starting site ATG, as negative control

ChIP-qPCR of HSFB1 in plants expressing RCF2:RCF2-FLAG , amplify region from 742 to 973 downstream of translation starting site ATG, as negative control

ChIP-qPCR of HSFC1 in plants expressing RCF2:RCF2-FLAG , amplify region from 749 to 943 downstream of translation starting site ATG, as negative control

Plasmid construction of MBP-NAC019

Plasmid construction of pGreenII 62SK-NAC019 for dual luciferase reporter assay

Plasmid construction of HSFA6b:LUC fordual luciferase reporter assay

Plasmid construction of pGreenII 62SK-FLAG-RCF2 for dual luciferase reporter assay

Plasmid construction of MBP-RCF2

ChIP-qPCR of HSFB1 in plants expressing NAC019:NAC019-HA , amplify region from 4 to 110 downstream of translation starting site ATG, as negative control


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RCF2 D161A F GGTATTGTCTTTGCTCTTGATGAAACCCTTG

RCF2 D161A R CATCAAGAGCAAAGACAATACCCAGACATC

HSFA6b FCGCATGTTTATAAATGTAGCCTTGGCCACGCATGTTTATAAATGTAGCCTTGGCCACGCATGTTTATAAATGTAGCCTTGGCCACGCA

Biotin-labeled (5'-end) or unlabled sense probe for EMSAs

HSFA6b RTGCGTGGCCAAGGCTACATTTATAAACATGCGTGGCCAAGGCTACATTTATAAACATGCGTGGCCAAGGCTACATTTATAAACATGCG

Biotin-labeled (5'-end) or unlabled antisense probe for EMSAs

Site-directed mutagenesis of pDONR221-RCF2 which serves as an entry clone for construction of pEarleyGate202-RCF2(D161A), or a template for construction of pGreenII 62SK-FlAG-RCF2(D161A) with the primer pair RCF2 FLAG SpeI F and RCF2 XhoI R; site-directed mutagenesis of pDONRzeo-RCF2 (genomic fragment used for the construction of pearleyGate302-RCF2) which serves as an entry clone for plasmid construction of pEarleyGate302-RCF2(D161A).


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Supplemental References

Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F.,

Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., and Gascuel, O. (2008). Phylogeny.fr:

robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36 (Web Server issue):

W465–W469.

Hall, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analyses

program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95–98.

Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O., D'Angelo, C.,

Bornberg-Bauer, E., Kudla, J., and Harter, K. (2007). The AtGenExpress global stress

expression data set: protocols, evaluation and model data analysis of UV-B light, drought and

cold stress responses. Plant J. 50: 347-363.


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Supplemental Figures 9-22-2013 - The Plant Cell · Supplemental Figure 1. ... F7J7 T6K22 F18E5 F17L22 T8O5 F1N20 T10I14 F7K2 chromosome 4 SM229_155,3 FIZ4 ... 35 01 WT rcf2-1 RCF2:RCF2-FLAG

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