supporting information (si) appendix - PNAS

SUPPORTING INFORMATION (SI) APPENDIX LOW PHOTOSYNTHETIC EFFICIENCY 1 is i required i d for f lightli ht regulated photosystem II biogenesis in Arabidopsis Honglei Jina,1, Mei Fua,1, Zhikun Duanb,1, Sujuan Duana, Mengshu Lia, Xiaoxiao Donga, Bing Liua, Dongru Fenga, Jinfa Wanga, Lianwei Pengb and Hong-Bin Wanga,* a

State Key Laboratory of Biocontrol, Biocontrol Guangdong Provincial Key Laboratory of Plant Resources Resources,

School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People’s Republic of China. b

College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234,

China. 1 These

authors contributed equally to this work.

*Address correspondence to [email protected] (Hong-Bin Wang) Supplemental figures Figure S1-S22 Supplemental tables Table S1-S2 SI Materials and Methods

B

A lpe1-1 (SALK_059367) ATG

TAG

1

3 2

5 4

6

lpe1-2 (SALK_030882) lpe1-3 (SALK_110539)

D

C

0.8

8 Col‐0 6

lpe1 1 lpe1‐1

4

lpe1‐2 lpe1‐3

2 0

1

3 Time (weeks)

5

FW/Line (g line-1)

Rosette siz ze (cm)

10

0.6 0.4 0.2 0

1

3 Time (weeks)

Fig. S1. Phenotypic characterization of lpe1 mutants. (A) Schematic diagram of the LPE1 gene predicted from DNA sequence analysis. Exons (thick lines) and 5'UTR (thin lines) are indicated. The positions of the T-DNA insertions corresponding to lpe1-1, lpe1-2, and lpe1-3 are shown. The ATG start and TAG stop codons are marked. (B) PCR analysis of genomic DNA from wild-type plants and lpe1 mutants to confirm homozygosity of the mutants. 1 and 2: amplification for wild type and lpe1-1 (SALK_059367); 3 and 4: amplification for wild type and lpe1-2 (SALK_030882); 5 and 6: amplification for wild type and lpe1-3 (SALK_110539). (C) Rosette diameters of wild-type and lpe1 mutant plants measured after 1, 3, and 5 weeks of growth. (D) Fresh weight of wild-type and lpe1 plants measured after 1, 3, and 5 weeks of growth. FW: fresh weight. Each data point represents at least 20 independent plants.

5

A

Col-0

lpe1-1 lpe1-2 lpe1-3

B

Col-0

lpe1-1 lpe1-2 lpe1-3

D

Col-0

lpe1-2 lpe1-3 lpe1-1 p p p

PSII‐LHCII PSII dimer/PSI monomer PSI monomer/CF1 PSII core monomer CP43‐less PSII core monomer LHCII trimer

Unassembled protein

BN-PAGE

C

Col-0

lpe1-1 p lpe1-2 p lpe1-3 p

ATPB

PSII‐LHCII PSII dimer/PSI monomer PSI monomer/CF1 PSII core monomer CP43‐less PSII core monomer LHCII trimer

Unassembled protein Unassembled protein

PsaB

Cytf

Fig. S2. Immunological analysis of photosystem complexes separated using BN-PAGE. ((A)) Representative p unstained BN-PAGE g gel. Thylakoid y membranes from wild-type yp ((Col-0)) and lpe1 p mutant p plants were solubilized with 2% DM and separated using native PAGE. (B) A representative immunoblot showing a BN-PAGE gel probed with anti-ATPB antiserum. (C) A representative immunoblot showing a BN-PAGE gel probed with anti-PsaB antiserum. (D) A representative immunoblot showing a BN-PAGE gel probed with anti-Cytf antiserum. The samples in each lane contained equal amounts of chlorophyll. Three additional independent biological replicates of each experiment were performed with similar results.

Fig. S3. Quantitative analysis of proteins immunodetected in Figure 2 E using Phoretix 1D Software (Phoretix International, UK). The values (mean ± SE; n = 3 independent biological replicates) are given as the ratios between protein levels in wild-type (Col-0) plants and lpe1 mutants. (Student’s t-test; *: P< 0.05; **: P< 0.01.)

B

A

C

ST

D

GT ST

GT

g S4. Chloroplast p p mutants and wild-type yp plants. p Fig. structure of lpe1 Transmission electron microscopy images showing chloroplasts from (A) wild-type (Col-0) plants and (B) lpe1-3 mutant plants. Close-up views of (C) wild-type plants and (D) lpe1-3 mutant plants. GT: grana thylakoids; ST: stroma thylakoids. Scale bars: 1 μm. Three independent biological replicates were performed; similar results were obtained.

Motif

Motif

Motif

Motif

Motif

HMM Motif sequence

index name

length start

end

score

1

P

38

146

183

11.2

LQVFCAMIKGFGKDKRLKPAVAVVDWLKRKKSESGGVI

2

P

35

184

218

14 7 14.7

GPNLFIYNSLLGAMRGFGEAEKILKDMEEEGIVPN

3

P

35

219

253

23.6

IVTYNTLMVIYMEEGEFLKALGILDLTKEKGFEPN

4

P

35

254

288

00 0.0

PITYSTALLVYRRMEDGMGALEFFVELREKYAKRE

5

P

35

311

345

0.0

ICYQVMRRWLVKDDNWTTRVLKLLNAMDSAGVRPS

6

Pi

35

346

380

00 0.0

REEHERLIWACTREEHYIVGKELYKRIRERFSEIS

7

P

42

381

422

18.2

LSVCNHLIWLMGKAKKWWAALEIYEDLLDEGPEPNNLSYELV

8

P

35

423

457

19 1 19.1

VSHFNILLSAASKRGIWRWGVRLLNKMEDKGLKPQ

9

P

35

458

492

0.0

RRHWNAVLVACSKASETTAAIQIFKAMVDNGEKPT

10 0

P

35

493 93

527 5

28.8 88

VISYGALLSALEKGKLYDEAFRVWNHMIKVGIEPN S G S G G

11

P

35

528

562

10.5

LYAYTTMASVLTGQQKFNLLDTLLKEMASKGIEPS

12

P

35

563

597

0.0

VVTFNAVISGCARNGLSGVAYEWFHRMKSENVEPN

13

P

35

598

632

7.7

EITYEMLIEALANDAKPRLAYELHVKAQNEGLKLS

Fig. S5. Sequences of 13 predicted PPR motifs in LPE1. The PPR motifs of LPE1 were analyzed using information in the database established by Cheng et al. [Plant J. 2016 Feb;85(4):532-47]. LPE1 is predicted to contain 13 PPR motifs. The name, length, start, end, HMM score, and amino acid sequence of each PPR motif are shown.

Treatment CK NaCl Na2CO3 CaCl2 Urea LPE1LPE1 FLAG PsbO D1

Fig. S6. Association analyses of LPE1 with membrane. The thylakoid membranes from LPE1-FLAG transgenic plants were sonicated in the presence of 1 M NaCl, 200 mM Na2CO3, 1 M CaCl2, or 6 M urea for 30 minutes at 4°C. PsbO (the 33kD luminal protein of PSII) and D1 (the PSII core protein) were used as markers. Membranes that had not been subjected to any salt treatment were used as controls (CK). All experiments were repeated three times with similar results results.

30

[R RNA (Col-0)]

[RNA A (LPE1-FLAG G)]

25 20 15 10 5 0

bH psbI bI psbA bA psbB bB psbC bC psbD bD psbE bE psbF bF psbH

psbJ bJ psbK bK psbL bL psbM bM psbN bN psbT bT psaB B

Fig. S7. Identification of LPE1 RNA targets by RIP analysis. RNAs associated with LPE1 were isolated following immunoprecipitation (IP) of LPE1 from LPE1-FLAG transgenic g plants using p g the anti-FLAG antibody. y All p plastid-encoded PSIIrelated genes were analyzed. Quantification of the association between each mRNA and LPE1 was determined using quantitative RT-PCR. Five additional independent biological replicates were performed with similar results.

kDa 170 130 95 72

His‐LPE1

55 42 34

26

Fig. S8. Expression and purification of LPE1. BL21 cells were harvested after incubation overnight with isopropylthio-β-D-galactoside. Expressed His-LPE1 proteins were purified on a Ni-NTA agarose resin matrix. Samples g SDS-PAGE and stained with Coomassie brilliant blue. were resolved using

5

2 1

AccD1 ATPA ATPB ATPE ATPF ATPH ATPI CCSA CEMA clpP1 MatK NdhA NdhB NdhC NdhD NdhE NdhF NdhG NdhH NdhI NdhJ NdhK petA petB petD petG petI petN PsaA PsaB PsaC PsaJ PsaJ PsbA PsbB PsbC PsbD PsbE PsbF

Log2(lpe1-3/Col-0)

4 3

0 -1 -2 -3 -4 -5 4

Log2(lpe1 1-3/Col-0)

3 2

PsbH PsbI PsbJ PsbK PsbL PsbM PsbN PsbT RbcL Rpl14 Rpl15 Rpl16 Rpl2 Rpl20 Rpl22 Rpl23 Rpl32 Rpl33 Rpl36 Rps19 RpoA RpoB RpoC1 RpoC2 RpS11 RpS12 RpS14 RpS18 RpS2 RpS3 RpS4 RpS7 RpS8 16S‐rRNA 23S‐rRNA rpoC1 rpoC2 rpoC3 rpoC4 rpoC5

5

1 0 -1 -2 -3 -4 -5 5

Fig. S9. Levels of Plastid Transcripts in lpe1-3 Mutants. The levels of mRNA and rRNA transcripts from lpe1-3 and wild-type plants were measured. The graph d i t th depicts the llog2 ratio ti off transcript t i t levels l l in i the th mutant t t compared d with ith those th in i wild-type ild t plants. l t The Th mRNA level of each gene was normalized first with respect to ACTIN 2 (At3g18780) and then to wildtype expression level. Five independent biological replicates were performed. A Student’s t-test analysis did not detect significant differences in transcript levels between lpe1 and wild wild-type type plants. plants

GL

GL

HL

gg g D1 aggregate

MW (KD)

D1 dimer

40

D1 monoer

32

Degraded D1 fragment

6.5 Anti-D1

HL

CBB

Fig. S10. Analysis of different forms of D1 protein under high light in wild-type plants and lpe1-3 mutants. Th l k id were isolated Thylakoids i l t d ffrom wild-type ild t and d mutant t t plants l t grown under d standard t d d plant l t growth lighting (GL) conditions or after 3h high light (HL) treatment. Samples containing equal amounts of chlorophyll were subjected to SDS-PAGE on a Tricine gel, followed by immunoblot analysis with an antibody against the D1 C-terminus. The different forms of D1 protein are indicated on the right right-hand hand side. MW: molecular mass. The gel was stained with Coomassie brilliant blue (CBB) to quantify the amount of total protein. Three independent biological replicates were performed; similar results were obtained.

Fig. S11. Confirmation of Anti-HCF173 Antibody. A 520-aminoacid peptide (corresponding to amino acids 79–598 of HCF173) was expressed in Escherichia coli BL21 (DE3) and used to raise antibodies against HCF173 in rabbits. Affinity-purified anti-HCF173 polyclonal antibodies were produced. An equal quantity (0.5 μg) of chloroplast proteins from wild-type and hcf173 mutant plants was loaded in each lane, except the first (0.25 μg of protein). Three additional independent biological replicates of the experiment were performed; similar results were obtained.

A

B

RNase inhibitor

-

+

+

RNase inhibitor

-

+

+

RNase

+

-

-

RNase

+

-

-

psbA mRNA

-

-

+

psbA mRNA

-

-

+

120 [RNA (L LPE1-FLAG G)] [RNA A (Col-0)]

100 80

Anti-HCF173 psbA psbD psbE

IP:FLAG Anti-FLAG Anti-HCF173

60 40 20

Input

Anti-FLAG Anti-Actin

0 Fig. S12. The effect of psbA mRNA on the interaction between LPE1 and HCF173. (A) RNAs associated with LPE1 were isolated following RNA immunoprecipitation (RIP) of LPE1 from LPE1-FLAG transgenic plants using the anti-FLAG antibody. RNase (20 μg/mL) treatment was used to digest RNA, RNA or psbA mRNA transcribed in vitro was added to increase the abundances of psbA mRNA in RIP assays. (B) The effect of different amounts of psbA mRNA on the interaction between LPE1 and HCF173. After RIP assays from (A), proteins associated with LPE1 were isolated and identified by western blot assays. Similar results were obtained from four independent biological replicates.

Col-0

lpe1-3

HCF173

CBB Staining

Fig. S13. Immunological Analysis of HCF173 Abundance in Wild-Type and lpe1 Mutant Plants. Total proteins extracted from wild-type (Col-0) plants and lpe1-3 mutants were separated using 12% SDS-ureaPAGE. Gels were electroblotted onto PVDF membranes, and probed with antisera against HCF173. Samples were loaded on an equal loading basis and stained with Coomassie brilliant blue (CBB) to show protein levels. Similar res results lts were ere obtained from fo fourr independent biological replicates replicates.

B

C 1.2

LPE1-FLAG

0.8 0.6 0.4

IP

0.2 0

Input

D1

1 [RNA (Col-0))]

1 Anti-FLAG A

Relative expres ssion of o HCF173 ge ene

1.2

[RN NA (LPE1-FLA AG)]

A

0.8

psbA psbD psbE

0.6 0.4 0.2 02 0

Actin

Fig. g S14. The effect of HCF173 deficiency y on the association of LPE1 with p psbA mRNA. (A) qRT-PCR analysis of the suppression of HCF173 gene expression in VIGS-HCF173 transgenic plants. Relative transcript levels of HCF173 in VIGS-HCF173 plants were normalized to those in VIGS-GFP transgenic plants. (B) Western blot analysis of proteins present in crude leaf extracts of VIGS-GFP and VIGS-HCF173 transgenic plants, and RIP analysis using the anti-FLAG, anti-D1, and anti-Actin antibodies. (C) qRT-PCR analysis of the association of psbA mRNA with LPE1. Three additional independent biological replicates were performed, with similar results. Plants used in all experiments (A-C) were transgenic for VIGS vectors expressing GFP and HCF173, which were introduced in the LPE1-FLAG transgenic background.

A

Illumination time after etiolation

48 h

Col-0 C llpe1-3 0

1

0.5

40

Col-0 lpe1-3

0.4 0.3

30

ETR

ΦPSII

24 h

lpe e1-3

Image Fv/Fm

B

8h

Col--0

0h

0.2

10

0.1 0

20

0

8

24

48

Illumination time after etiolation (h)

0

0

8

24

48

Illumination time after etiolation (h)

Fig. S15. PSII activity during light-induced greening of etiolated wild-type and lpe1-3 Arabidopsis seedlings. (A) Images and false-color images representing Fv/Fm during light-induced greening of etiolated wild-type and lpe1-3 Arabidopsis seedlings. After growth in the dark for 5 days, etiolated seedlings were illuminated for 0, 8, 24, or 48 h, and images and false-color images representing Fv/Fm were captured. (B) PSII quantum yyield (ΦPSII) ( ) and electron transport p rate ((ETR)) of light-induced g greening g g of etiolated seedlings. g After g growth in the dark for 5 days, etiolated seedlings of the wild type and lpe1-3 mutant were illuminated for 0, 8, 24, or 48 h as in (A), and ΦPSII and ETR were captured. All experiments were repeated at least three times with similar results. Each data point in the quantitative analysis represents at least three independent biological replicates.

Illumination time after etiolation 0

8

24

48

h

psbA psbB psbD psbO HCF173 Lhcb1 Actin Fig. S16. Analysis of transcription of PSII-related genes during light-induced greening of etiolated wild-type Arabidopsis seedlings. After growth in the dark for 5 days, etiolated seedlings were illuminated for 0, 8, 24, or 48 h Total RNA of light h. light-induced induced greening of etiolated wild-type wild type Arabidopsis seedlings from Fig. 6A was isolated and the transcripts of photosytem II-related genes were analyzed by RT-PCR. The experiments were repeated for three times, and similar results were obtained.

Relative A Abundance of LPE E1 mRNA

2 1.5 1 0.5 0

0

8

24

48

h

Illumination time after etiolation

Fig. S17. Analysis of LPE1 gene transcription during light-induced greening i off etiolated ti l t d wild-type ild t seedlings. dli Total RNA was isolated from etiolated wild-type (Col-0) seedlings during lightinduced greening and reverse-transcribed for real-time quantitative PCR analysis.

A

Illumination time after etiolation

0

8

24

48

h

D1 LPE1-FLAG LPE1 FLAG Actin

Relative Abundance 1 mRNA of LPE1

B

2 1.5 1 0.5 0

0

8

24

48

h

Illumination time after etiolation

Fig. S18. Analysis of the abundance of LPE1 protein and LPE1 mRNA during light induced greening of etiolated LPE1-FLAG light-induced LPE1 FLAG transgenic seedlings seedlings. (A) Western blot analysis of LPE1 and D1 proteins during light-induced greening of etiolated wild-type (Col-0) seedlings. (B) Real-time quantitative PCR analysis of LPE1 genes during light-induced greening of etiolated LPE1-FLAG transgenic seedlings. Total RNA was isolated from etiolated wild-type wild type seedlings during lightlight induced greening as described in (A) and reverse-transcribed for real-time quantitative PCR analysis.

A

B 30 LPE1 HCF173

20

psbA mRNA

10

0 0 8 24 48 Illumination time after etiolation (h)

Rela ative abunda ances of IP

Relative abundan nces of inpu ut

30

LPE1 HCF173 20

psbA mRNA

10

0

0

8 24 48 Illumination time after etiolation (h)

Fig. S19. Quantification of association of LPE1 with HCF173 and psbA mRNA. The levels of proteins immunodetected of input (A) and IP (B) as shown in Fig. 6C were analyzed using Phoretix 1D Software S f (Phoretix ( International, UK). ) The values (mean ( ± SE; S n = 3 independent biological replicates) are given as the ratio to the amount of protein at 0 h illumination after. The levels of psbA mRNA were quantified using quantitative RT-PCR. Means ± SE (n = 3 independent biological replicates) are shown.

Relative Ab bundance of psbA mRNA

105 104 103 102

CK DTNB DTT

101 100

Figure S20. Relative abundance of psbA mRNA in DTNB-treated, DTTtreated, and untreated 35S::LPE1:FLAG transgenic plants in Fig. 6E. The mRNA level of each gene was normalized first with respect to ACTIN 2 (At3g18780) expression and then against expression in wild-type plants. Five independent biological replicates were performed.

Populus trichocarpa

91 60

Ricinus communis

57

Vitis vinifera

29

Glycine max

48

Solanum lycopersicum

98

Arabidopsis thalina Cucumis sativus Fragaria vesca

64

Oryza sativa Z mays Zea

100

Hordeum vulgare

63 100

Aegilops tauschii Physcomitrella patens

0.1

Fig. S21. Phylogenetic Analysis of LPE1 Protein and Its Homologs from Other Land Plants. Phylogenetic analyses of LPE1 from plants were performed using the MEGA program (www.megasoftware.net). The phylogenetic tree was generated using g MEGA5. Full-length g amino acid sequences q of p proteins homologous g to Arabidopsis thaliana LPE1 from Physcomitrella patens, Populus trichocarpa, Vitis vinifera, Cucumis sativus, Ricinus communis, Glycine max, Fragaria vesca, Solanum lycopersicum, Oryza sativa, Zea mays, Hordeum vulgare, and Aegilops tauschii were selected to generate a bootstrap neighbor-joining phylogenetic unrooted tree. Percentages over 50% % ffrom 1000 bootstrap replicates are shown. Scale bar: 0.1 amino acid substitutions.

Fig. S22. Alignment Analysis of LPE1 Proteins from Other Land Plants. The amino acid sequence of the At3g44610 protein was compared with homologous sequences from other land plants, including Physcomitrella patens, Populus trichocarpa, Vitis vinifera, Cucumis sativus, Ricinus comm nis Glycine communis, Gl cine max, ma Fragaria vesca, esca Solanum Solan m lycopersicum, l copersic m Oryza Or a sativa, sati a Zea mays, ma s Hordeum Horde m vulgare, lgare and Aegilops tauschii. Conserved amino acids are indicated by a black background. The sequences were aligned usingClustalW. The conserved cysteine (C) are indicated in red triangles.

Table S1 Chlorophyll Contents and Chlorophyll Fluorescence Parameters in Wild-Type (Col-0) and lpe1 Mutants Col 0 Col-0

lpe1 1 lpe1-1

lpe1 2 lpe1-2

lpe1 3 lpe1-3

Chl a(mg/g FW)

1.443±0.027

1.082±0.047*

0.945±0.006**

0.699±0.040**

Chl b(mg/g FW)

0.4776±0.010

0.360±0.016*

0.321±0.008**

0.278±0.103**

Chl a/b

3 023±0 114 3.023±0.114

3 009±0 134 3.009±0.134

2 948±0 017* 2.948±0.017

2 520±0 193** 2.520±0.193

Fv/Fm

0.834±0.006

0.741±0.039**

0.611±0.026**

0.482±0.059**

F0

0.088±0.007

0.151±0.015**

0.257±0.012**

0.358±0.072**

Fm

0 534±0 041 0.534±0.041

0 610±0 004* 0.610±0.004

0 640±0 015* 0.640±0.015

0 618±0 045* 0.618±0.045

qN

0.687±0.008

0.746±0.008*

0.792±0.010**

0.820±0.013**

Y(NO)

0.283±0.006

0.317±0.029**

0.368±0.028**

0.395±0.023**

qI q

0.583±0.014 ±

0.756±0.001** ±

0.785±0.003** ±

1.270±0.063** ±

qE

0.912±0.018

0.829±0.122*

0.551±0.048**

0.339±0.014**

Measurements of chlorophyll fluorescence parameters were made on plants following 30 minutes of dark adaption. For Y(NO), qN, qE, and qI measurements, actinic light intensity was 500 μmol photons m-2 s-1. Chl a: chlorophyll a; Chl b: chlorophyll b; FW: fresh weight; F0: minimal fluorescence; Fm: maximal fluorescence; Fv/Fm: maximum quantum yield of PSII; qN: non-photochemical quenching; Y (NO), yield of non-regulatable non-photochemical quenching. qE: energy-dependent quenching; qI: photoinhibitory quenching. Data are presented as means ± SE ((n = 3). p ) An asterisk indicates a significant g difference between the mutant and wild type yp ((WT)) using g the Student’s t-test; *:p< 0.05; **:p< 0.01.

Table S2. A List of Primers Used in This Study Primer

Sequence

LPE1 Mutants Identification LPE1-1-F LPE1-1-R LPE1-2-F LPE1-2-R LPE1-3-F LPE1-3-R

5'-TGGACTACTTTGCACACGATG-3' 5'-ACTACCGAAGCAAGCCTGTTC-3' 5'-TGGACTACTTTGCACACGATG-3' 5'-ATCCTACCCCAAACGATGATC-3' 5'-CTGTTTCTTGCTGGAAACCTG-3' 5'-ATCTTGCTGAGTGCAGCTAGC-3'

Real Time-PCR LPE1-F LPE1-R ACTIN-F ACTIN-R HCF173-F HCF173-R Lhcb1-F Lhcb1-R

5'-GTATTGAACCGAGCGTTGTGAC-3' 5'-AGCCTCAATCAACATCTCGTAAGTT-3' 5'-GGTAACATTGTGCTCAGT GGTG-3' 5'-CTCGGCCTTGGAGATCCACATC-3' 5'-GCGACTGATGCGAGGTTTC-3' 5'-TCCTTAGTGGTTATGGTTCCG-3' 5'-CGTGACCATGCGTCGTACCGTC-3' 5'-CCT CAG GGAATGTGCATCCG-3'

His-tag Construct LPE1-F LPE1-R

5'-ATGGCTGATATCGGATCCGAATTCATGTGTGAGCCAAAGAGAAG -3' 5'-GAGTGCGGCCGCAAGCTTGTCGACAGGTCTATTCTTTTTGTCTG -3'

GFP Assay LPE1-F LPE1-R

5'-GCTCTAGAATGCAAGCTTTAAGCATTTTG -3' 5'-CAGGTACCAGGTCTATTCTTTTTGTCTGGT-3'

BiFC Assay LPE1-F LPE1-R HCF173-YN-F HCF173-YN-R HCF244-YN-F HCF244-YN-R

5'-GCTCTAGAATGCAAGCTTTAAGCATTTTG -3' 5'-CAGGTACCAGGTCTATTCTTTTTGTCTGGT-3' 5'-CTCAGGCCTGGCGCGCCACTAGTGGATCCATGGTGGGTAGTATTGTTGG-3' 5'-GGGAGCGGTACCCTCGAGGTCGACTGTGTTCTTCTCAAGTA-3' 5'-CTCAGGCCTGGCGCGCCACTAGTGGATCCATGGCTTCGCTCAGGCTCCC-3' 5'-GGGAGCGGTACCCTCGAGGTCGACGAAGTAGATGTCTGATTGCT-3'

Y2H HCF173-BD-F HCF173-BD-R LPE1-AD-F LPE1-AD-R

5'-ATCTCAGAGGAGGACCTGCATATGATGGTGGGTAGTATTGTTGG-3' 5'-CGGCCGCTGCAGGTCGACGGATCCTGTGTTCTTCTCAAGTACAGA-3' 5'-GGGTGGGCATCGATACGGAATTCATGCAAGCTTTAAGCATTTTG-3' 5'-TACGATTCATCTGCAGGGATCCAGGTCTATTCTTTTTGTCTGG-3'

VIGS HCF173-V-F HCF173-V-R

5'-AACTTGAAAAATCTCACTTTCGTT-3' 5'-CCACCATTTTTGAACTCTGCTA-3'

RIP Assay psbA-F psbA-R psbB-F

5'-GAGCAGCAATGAATGCGATA-3' 5'- CCTATGGGGTCGCTTCTGTA-3' 5'-CGTGCGACTTTGAAATCTGA-3'

psbB-R psbC-F psbC-R psbD-F psbD-R psbE-F psbE-R psbF-F psbF-R petA-F petA-R psaA-F psaA-R ATPB-F ATPB-R psbH-F psbH-R psbI-F psbI-R psbJ-F psbJ-R psbK-F psbK-R psbL-F psbL-R psbM-F psbM-R psbN-F psbN-R psbT-F psbT-R

5'- TAGCACCATGCCAAATGTGT-3' 5'-ACTTCCCCACCTAGCCACTT-3' 5'- AGCCCAAAACTGCAGAAGAA-3' 5'- CACAAATCTTTGGGGTTGCT-3' 5'- CCATCCAAGCACGAATACCT-3' 5'- TGTCTGGAAGCACAGGAGAA-3' 5'- AACCGGTGCTGACGAATAAC-3' 5'-GGACCTATCCAATTTTTACAGTGC-3' 5'- GTTGGATGAACTGCATTGCT-3' 5'- CAGAGGGCGAATCCATTAAA-3' 5'- GCCAAAACAACCGATCCTAA-3' 5'- GCCAAGAAATCCTGAATGGA-3' 5'- CATCTTGGAACCAAGCCAAT-3' 5'-CCGTTTCGTACAAGCAGGAT-3' 5'- CGGGGTCAGTCAAATCATCT-3' 5'- TCTAGATCTGGTCCAAGAAGCA-3' 5'- CATTGCAACACCCATCAAAG-3' 5'- TTTCTCTCTTCATATTTGGATTCCT-3' 5'- TTCTTCACGTCCCGGATTAC-3' 5'- CTGGAAGGATTCCTCTTTGG-3' 5'- CAGGGATGAACCTAATCCTGA-3' 5'- AGGCCTACGCCTTTTTGAAT-3' 5'- CGAAAACTTACAGCGGCTTG-3' 5'- CAATCAAATCCGAACGAACA-3' 5'- GAAATAATTCGAAAATAAAACAGCAA-3' 5'- TGCACTCTTCATTCTCGTTCC-3' 5'- TCATTTTGACTAACGGTTTTTACG-3' 5'- GGAAACAGCAACCCTAGTCG-3' 5'- CGTGTTCCTCGAATGGATCT-3' 5'- GGAAGCATTGGTTTATACATTTCTCT-3' 5'- AAATTTTAGGTGGTTCCCGAAA-3'

SI Materials and Methods Plant Materials and Growth Conditions All the T-DNA and transgenic Arabidopsis thaliana lines used in this study were in the Col-0 background. The lpe1-1, lpe1-2, and lpe1-3 mutants were obtained from the Arabidopsis Biological Resource Center (stock numbers SALK_059367, SALK_030882, and SALK_110539). A. thaliana plants were grown in soil in a growth chamber (100 μmol photons m-2 s-1; 12 h light/12 h dark photoperiod; 21°C and 60% relative humidity). Plants used in the chlorophyll fluorescence assays and protein analyses were 3 and 4 weeks old, respectively. To study the effects of high light, plants were placed in a high light growth chamber (1200–1500 photons μmol m-2 s-1) while maintaining the 12 h light/12 h dark cycle.

Analysis of Chlorophyll and Chlorophyll Fluorescence Chlorophyll was extracted from 3-week-old plants using 80% acetone in 2.5 mM HEPES-KOH, pH7.5; the chlorophyll content was determined as previously described (1). Chlorophyll fluorescence parameters were measured using the MAXI version of the IMAGING-PAM M-Series chlorophyll fluorescence system (Heinz-Walz Instruments) as previously described (2).

Transmission Electron Microscopy Transmission electron microscopy was performed as described by Yao and Greenberg (2006). Fully expanded leaves from the lpe1-3 and wild type plants were collected 4 weeks after infection. Micrographs were taken using a transmission electron microscope (JEM1400; JEOL).

Isolation of Thylakoid Membranes Thylakoid membranes were prepared as previously described (3). Isolated thylakoid membranes were quantified based on total chlorophyll. Total proteins

from leaf thylakoid membrane were extracted. Protein concentrations were determined using the Bio-Rad detergent-compatible colorimetric protein assay according to the manufacturer’s protocol (Bio-Rad).

RT-PCR and Quantitative Real-Time RT-PCR Total RNA was extracted from Arabidopsis rosette leaves using an RNAeasy Plant Mini Kit (QIAGEN). RNA samples were reverse-transcribed into first-strand cDNA using a PrimeScript RT Reagent Kit (Takara). Quantitative real-time RT-PCR was carried out using gene-specific primers and SYBR Premix ExTaq reagent (Takara) and a real-time RT-PCR system (RoChe-LC480), following the manufacturer’s instructions. Reactions were performed in triplicate for each sample, and expression levels were normalized against ACTIN and UBQ4. The primers for quantitative real-time RT-PCR analysis of plastid genes were as described previously (4).

BN-SDS-PAGE and Immunoblot Analyses BN-PAGE and two-dimensional analysis and immunodetection of proteins on a PVDF membrane were performed as described (2). For quantification of thylakoid proteins, gels were loaded on an equivalent chlorophyll basis, in amounts ensuring immunodetection was in the linear range. Other than anti-His and anti-Actin (which were raised in mice), all other primary antibodies

and

antisera

were

raised

in

rabbits.

Antisera

against

photosynthetic proteins were purchased from Agrisera. D1, AS05084; D2, AS06146; CP43, AS111787; CP47, AS10939; PsbE, AS06112; PsbF, AS06113; PsbO, AS05092; Cytf, AS08306; PsaA, AS06172; PsaB, AS10695; PsaD, AS09461; LHCa1, AS01005; LHCb1, AS01004; ATPB, AS05085.

In Vivo Labeling of Chloroplast Proteins In vivo protein labeling was performed as described previously (5). Primary

leaves of 12-d-old young seedlings were pre-incubated in buffer containing 20 mg/ml cycloheximide for 30 minutes and radiolabeled with 1 m Ci/mL [35S]-Met in the presence of cycloheximide for 20 minutes. After pulse labeling, thylakoid proteins were separated by SDS-urea-PAGE, and the labeled proteins were visualized by autoradiography.

RNA Gel Blot and Polysome Association Analyses Total RNA was extracted from Arabidopsis leaves using Trizol reagent (Invitrogen). RT-PCR analysis was performed with a Scientific Revert Aid First Strand cDNA Synthesis Kit (Thermo). RNA gel-blot analysis was performed using the DIG Easy Hybrid system (Roche). Polysome association analysis was performed as described previously (6). The signals were visualized with a Lumino Graph WSE-6100 (ATTO). RL 6,000 RNA markers (Takara) were used as molecular markers.

Subcellular Localization of GFP Fusions and BiFC Subcellular localization of GFP fusion proteins and BiFC were performed as previously described (7).

Chloroplast Fractionation and Immunolocalization Studies Separation of thylakoid and stroma phases and salt washing of thylakoids were performed as described previously (8).

Analysis of D1 Protein Accumulation under High Light Four-week-old plants were illuminated at 1,200 μmol photons m-2 s-1. To analyze the accumulation and degradation of D1 proteins, proteins were extracted from leaves ground under liquid nitrogen and used for SDS-PAGE and subsequent immunoblotting with the C-terminal D1 antibody.

Immunoprecipitation

Immunoprecipitation of LPE1 with HCF173 was performed as described (9) with minor modifications. Chloroplast proteins were solubilized with 2% (w/v) DM in 20% glycerol (w/v), 25 mM BisTris-HCl, pH 7.0, and 1mM PMSF for 20 minutes at 4°C. After centrifugation, the supernatant was diluted in an equal volume of the same buffer without DM and preincubated with affinity-purified anti-FLAG antibody at 4°C for 2 h. Preincubated thylakoid membrane proteins (200 mL, 0.5 mg chlorophyll/mL) were incubated at 4°C overnight with Protein A/G Plus agarose in Pierce spin columns. After incubation overnight with constant rotation at 4°C, the resin was washed five times with ice-cold PBS buffer (pH 7.8), and bound proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE, and subjected to immunoblot analysis.

RNA Immunoprecipitation (RIP) RIP assays were performed as described (10) with minor revisions. Leaf tissue samples were collected from 4-week-old Arabidopsis plants, and 2 g of tissue was ground to a fine powder under liquid nitrogen using a mortar and pestle. Samples were homogenized in 12.5 mL/g lysis buffer (50 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 100 mM KCl, 0.1% Nonidet P-40, 1 μg/mL leupeptin, 1 μg/mL aprotonin, 0.5 mM phenylmethylsulfonyl fluoride, one tablet of Complete proteinase inhibitor (Roche), and 50 units/mL RNase OUT; Invitrogen). To test the effect of redox state, DTT or DTNB was added to the lysis buffer and samples were incubated for 1 h. Cell debris was pelleted by centrifugation for 5 minutes at 12,000 rcf at 4°C. Clarified lysates were incubated with 4 μg/mL anti-FLAG antibody for 15 minutes at 4°C and then with 100 μL of Protein A agarose (Roche) per mL for 30 minutes at 4°C. Beads were washed six times for 10 minutes with lysis buffer at 4°C and then divided for protein and RNA analyses. RNAs were recovered by incubating the beads in 0.5 volumes of proteinase K buffer (0.1 M Tris-HCl, pH 7.4, 10 mM EDTA, 300 mM NaCl, 2% SDS, and 1 μg/μL proteinase K; Roche) for 15 minutes at 65°C; extracted with saturated phenol, phenol:chloroform:isoamyl alcohol, and

chloroform; and precipitated with ethanol. For RT-PCR and quantitative RT-PCR assays, 1 μg of total RNA was used for the input fraction, and 20% of the RNA immunoprecipitate was used for immunoprecipitation.

EMSA Assays The mature form of wild-type LPE1 without the putative plastid transit peptide was expressed in Escherichia coli as a His-tagged fusion protein, and purified from soluble extracts. For labeling of the synthetic first 82 nucleotides of the 5' UTR of psbA mRNA oligonucleotides (5'-UUCAUAACAAGCUCUCA AUUAUCUACUUAGAGAAUUUGUGCGCUUGGAGUCCCUGAUUAUUAAA UAAACCAAGGAUUUUACC-3'), the DIG Northern Starter Kit (Roche) was employed. EMSA was performed as described previously (11). The purified recombinant LPE1 was incubated with RNA probes in binding buffer [40mM Tris-HCl, pH 8.0; 30 mM KCl, 1 mM MgCl2; 0.01% NP-40 (w/v); 1 mM DTT; 5% glycerol; 10 μg/mL BSA], for 60 minutes at 30°C. The method for testing the effect of redox was as described previously (12) with minor revision; DTT (5 mM, 50 mM, 100 mM) or DTNB (0.15 mM, 1.5 mM, 3 mM) was added to binding buffer. The RNA–protein complexes were separated on 10% native polyacrylamide gels. After electrophoresis, the RNA was transferred onto a nylon membrane. The signals from the labeled RNA were detected using a Gene Image CDP-Star detection kit DIG Northern Starter Kit (Roche).

Generation of Anti-HCF173 Polyclonal Antibodies A 520-amino-acid peptide (corresponding to amino acids 79-598 of HCF173) was expressed in Escherichia coli BL21 (DE3) and used to immunize rabbits. Anti-HCF173 polyclonal antibodies were confirmed in Fig. S9.

VIGS Assay Plasmids pTRV1 and pTRV2 based on Tobacco rattle virus were used for

VIGS. To construct pTRV2-HCF173 vectors, HCF173 cDNAs were PCR amplified using primers described in Supplemental Table S1 and cloned into the pTRV2 vector. The pTRV2-GFP vector was utilized as a negative control. The pTRV1 and pTRV2 derivatives were introduced into Arabidopsis plants by Agrobacterium-mediated transformation as described previously (13).

Determination of the Redox State of LPE1 Protein in Vivo The in vivo redox states of proteins were determined using protein extracts prepared from LPE1-FLAG transgenic plants exposed to light for different length of time using extraction buffer without reducing agents, with minor modifications of the method described previously (13), For the preparation of protein extracts, samples were frozen in liquid nitrogen. Extracts were precipitated with 10% (w/v) TCA and washed twice with 100% acetone before resuspending the protein pellet in 1% SDS and 50 mM Tris-HCl buffer. Each sample was incubated with AMS (Life Technologies) for 20 min at room temperature. AMS-treated samples were separated, blotted, and probed with CHLM antibodies.

Accession Numbers Amino acid sequence data for LPE1 and its homologs in other species can be found in the GenBank databases under the following accession numbers: Arabidopsis thaliana, NP_190245; Populus trichocarpa, XP_002324000; Vitis vinifera,

XP_002268821;

Cucumis

sativus,

XP_011651578;

Ricinus

communis, XP_002526948; Glycine max, XP_003548551; Fragaria vesca, XP_004308618;

Solanum

lycopersicum,

XP_004231824;

Zea

mays,

XP_008647641; Oryza sativa, XP_015618214; Hordeum vulgare, BAJ85989; Aegilops tauschii, EMT16957; Selaginella moellendorffii, XP_002961146; Physcomitrella patens, XP_001752777.

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10. 11. 12. 13.

 

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