Supplemental Data. Hoffmann et al. (2013). Plant Cell 10 ......Jul 08, 2013 · 35S:pfp5...

* 20 * 40 * 60 * 80 * 100AtER-ANT1 : ---------------------------------------------------------------------------------MALIGKSERFSADFVMGGAA : 20OsER-ANT1 : -------------------------------------------------------------------MPSAAAGDGKGKRRLVGMPPARAAAADFAMGGAA : 34Os02g07189 : MAEQANQPTVLQKFGGQFHLGSSFSEGVRARNI-CPSVSSYDRRFTTRSYMTQGLV------N--GGINVPMMSSSPIFANAPAEKGGKNFMIDFLMGGVS : 92Os05g03027 : MADDLGPPTVLQKIHGQSMMFSKISPYS-----LMKNPALYNANTSYSVPLKSYNG------MDGNNGFSSVTSVSPVFASAPKEKGLSGFMIDFMMGGVS : 90AtAAC1 : MVDQVQHPTIAQKAAGQFMRSSV-SKDVQ---VGYQRPSMYQRHATYGNYSNAAFQ------FPPTS-RMLATTASPVFVQTPGEKGFTNFALDFLMGGVS : 90AtAAC2 : MVEQTQHPTILQKVSGQLLSSSV-SQDIRGYASASKRPATYQKHAAYGNYSNAAFQ------YPLVAASQIATTTSPVFVQAPGEKGFTNFAIDFMMGGVS : 94AtAAC3 : -MDGSKHPSVFQKLHGQSYLINRLSPSVQARGYCVSG-----------AYVNGGLQSLLQPTSHGVGSSLIPHGSFPVLAHAPSEKTGTGFLIDFLMGGVS : 89

********************* f DF MGG* 120 * 140 * 160 * 180 * 200

AtER-ANT1 : AIVAKSAAAPIERVKLLLQNQGEMIKTGHLIRPYTGLGNCFTRIYREEGVLSFWRGNQANVIRYFPTQASNFAFKGYFKNLLGCSKEKDGYLKWFAGNVAS : 121OsER-ANT1 : AVVAKTGAAPVERVKLLLQNQAEMLRRGSLTRPYRGIADAFGRVLREEGVAALWRGNQANVIRYFPTQAFNFAFKGYFKSIFGYDKEKDGKWKWLAGNVAS : 135Os02g07189 : AAVSKTAAAPIERVKLLIQNQDEMIKAGRLSEPYKGIGDCFGRTIKDEGFASLWRGNTANVIRYFPTQALNFAFKDYFKRLFNFKKDKDGYWKWFGGNLAS : 193Os05g03027 : AAVSKTAAAPIERIKLLIQNQDEMIKSGRLSHPYKGIADCFGRTIKDEGVIALWRGNTANVIRYFPTQALNFAFKDHFKRMFNFKKDKDGYWKWFAGNLAS : 191AtAAC1 : AAVSKTAAAPIERVKLLIQNQDEMIKAGRLSEPYKGIGDCFGRTIKDEGFGSLWRGNTANVIRYFPTQALNFAFKDYFKRLFNFKKDRDGYWKWFAGNLAS : 191AtAAC2 : AAVSKTAAAPIERVKLLIQNQDEMLKAGRLTEPYKGIRDCFGRTIRDEGIGSLWRGNTANVIRYFPTQALNFAFKDYFKRLFNFKKDKDGYWKWFAGNLAS : 195AtAAC3 : AAVSKTAAAPIERVKLLIQNQDEMIKAGRLSEPYKGISDCFARTVKDEGMLALWRGNTANVIRYFPTQALNFAFKDYFKRLFNFKKEKDGYWKWFAGNLAS : 190

A V K3aAAP6ER6KLL6QNQ EM64 G L PY G6 1cF R 4 EG lWRGN ANVIRYFPTQA NFAFK yFK 6f K 4DGywKWfaGN6AS* 220 * 240 * 260 * 280 * 300

AtER-ANT1 : GSAAGATTSLFLYHLDYARTRLGTDAKECSVN-GKRQFKGMIDVYRKTLSSDGIKGLYRGFGVSIVGITLYRGMYFGMYDTIKPIVLVGSLEGNFLASFLL : 221OsER-ANT1 : GSAAGATTSSLLYHLDYARTRLATDAIES-QG-SKRQFSGLLDVYKKTLKTDGIRGLYRGFSVSIVGITLYRGLYFGIYDTMKPLILVGPLQENFFASFAL : 234Os02g07189 : GGAAGASSLFFVYSLDYARTRLANDAKAA-KGGGERQFNGLVDVYRKTLKSDGIAGLYRGFNISCVGIIVYRGLYFGMYDSLKPVVLTGSLQDNFFASFAL : 293Os05g03027 : GGAAGACSLFFVYSLDYARTRLANDAKAAKKG-GGRQFNGLVDVYRKTLASDGIAGLYRGFNISCVGIIVYRGLYFGMYDSLKPVVLVGNLQDNFLASFLL : 291AtAAC1 : GGAAGASSLLFVYSLDYARTRLANDAKAAKKGGGGRQFDGLVDVYRKTLKTDGIAGLYRGFNISCVGIIVYRGLYFGLYDSVKPVLLTGDLQDSFFASFAL : 292AtAAC2 : GGAAGASSLLFVYSLDYARTRLANDSKSAKKGGGERQFNGLVDVYKKTLKSDGIAGLYRGFNISCAGIIVYRGLYFGLYDSVKPVLLTGDLQDSFFASFAL : 296AtAAC3 : GGAAGASSLLFVYSLDYARTRLANDAKAAKKG-GQRQFNGMVDVYKKTIASDGIVGLYRGFNISCVGIVVYRGLYFGLYDSLKPVVLVDGLQDSFLASFLL : 290

G AAGA 3 f6Y LDYARTRLa Dak g g RQF G66DVY4KT6 3DGI GLYRGF 6S vGI 6YRG6YFG6YD36KP66L g L2 F ASF L* 320 * 340 * 360 * 380 *

AtER-ANT1 : GWSITTSAGVIAYPFDTLRRRMMLTSGQPVKYRNTIHALREILKSEGFYALYRGVTANMLLGVAGAGVLAGYDQLHQIAYKHWVQ---------- : 306OsER-ANT1 : GWAITTFSGACAYPFDTLRRRMMLTSGQPLKYKNAFHAAKQIVSTEGFFTLFRGVGANILSGMAGAGVLAGYDQLHRFAGQHGYNFESKMKGALK : 329Os02g07189 : GWLITNGAGLASYPIDTVRRRMMMTSGEAVKYKSSMDAFSQILKNEGAKSLFKGAGANILRAIAGAGVLSGYDQLQILFFGKKYGSGGA------ : 382Os05g03027 : GWGITIGAGLASYPIDTVRRRMMMTSGEAVKYNSSLDAFKQIVAKEGAKSLFKGAGANILRAVAGAGVLAGYDKLQVVVFGKKYGSGGG------ : 380AtAAC1 : GWVITNGAGLASYPIDTVRRRMMMTSGEAVKYKSSLDAFKQILKNEGAKSLFKGAGANILRAVAGAGVLSGYDKLQLIVFGKKYGSGGA------ : 381AtAAC2 : GWLITNGAGLASYPIDTVRRRMMMTSGEAVKYKSSFDAFSQIVKKEGAKSLFKGAGANILRAVAGAGVLAGYDKLQLIVFGKKYGSGGA------ : 385AtAAC3 : GWGITIGAGLASYPIDTVRRRMMMTSGEAVKYKSSLQAFSQIVKNEGAKSLFKGAGANILRAVAGAGVLAGYDKLQLIVLGKKYGSGGG------ : 379

GW IT aG YP DT6RRRMM6TSG2 6KY A 2I6 EG L54G gAN6L 6AGAGVL GYD L y

H1 H2

H6

METS

METS

H1

NCS

H3

H4

METS

H3

H5

H5

Supplemental Figure 1. Alignment of the predicted amino acid sequence of rice ER-ANT1 and related MCF carriers from Arabidopsis and rice.

Identical or similar amino acid residues among the selected MCF members are shaded in black,conserved residues are shaded in gray, and residues conserved in three proteins are shaded in light gray.Solid black bars indicate the six membrane-spanning regions (H1 to H6) characteristic for MCF proteins.The conserved mitochondrial energy transfer signatures (METS=P-x-[DE]-x-[LIVAT]-[RK]-x-[LRH]-[LIVMFY]-[QGAIVM]) following each odd numbered membrane-spanning domain and the conservednucleotide carrier signature (NCS) are marked with white bars. Important amino acid residues for thefunction of mitochondrial AACs are marked with triangles; black triangles represent amino acid residuesalso conserved in rice ER-ANT1, and the white triangle indicates one residue not conserved in rice ER-ANT1 and Arabidopsis ER-ANT1.

Supplemental Data. Hoffmann et al. (2013). Plant Cell 10.1105/tpc.113.113605

1

At-ER-ANT1Os-ER-ANT1

Os02g0718900Os05g0302700

At-AAC1At-AAC2At-AAC3


Os02g0718900Os05g0302700



Os02g0718900Os05g0302700



Os02g0718900Os05g0302700


Supplemental Figure 2. Heterologous expression of rice ER-ANT1 in E.coli cells andsubstrate dependency of adenine nucleotide uptake.

IPTG-induced E.coli cells harboring the plasmid encoding ER-ANT1 from rice wereincubated for 15 min with increasing substrate concentrations. A, Concentration dependentuptake of [32P]-ATP. B, Concentration dependent uptake of [32P]-ADP. The insets showdouble-reciprocal plots of the import data, indicating a KM value of 8.9 µM and a vmax of0.296 nmol mg-1 protein for ATP (A) and a KM value of 12.7 µM and a vmax of 0.217 nmolmg-1 protein for ADP (B). The values represent means of 5 independent experiments.Standard errors were less than 5% of the mean values.


2

A B C

Supplemental Figure 3. Molecular characterization of 35S:Os-er-ant1 plants byPCR.

A, PCR reaction to control the absence of Arabidopsis er-ant1 cDNA in 35S:Os-er-ant1 plants, using the primer combination TLP-4 and TRP-4. B, Verification ofthe T-DNA insertion in 35S:Os-er-ant1 plants and homozygous er-ant1-2 plants(control), using the Arabidopsis er-ant1-specific primer TLP-4 and the T-DNA-specific primer LBb1 and gDNA as template. C, Detection of Os-er-ant1 cDNA inhomozygous 35S:Os-er-ant1 plants using the gene-specific primer combinationCH33 and CH40. Used primers are listed in Supplemental Table 3.


3

CaMV35S Os-er-ant1 OCS-Term. bar Pnos pAg7 LR

EcoRI HindIII

Supplemental Figure 4. Complementation of er-ant1-2 knockout plants with the closest orthologfrom rice, Os-ER-ANT1.

er-ant1-2 knockout plants (SALK_023441) were transformed with the plant transformation vectorpGPTV-bar (Becker et al., 1992) carrying the er-ant1 cDNA from rice (Os11g0661300) under thecontrol of the 35S-promoter. A, Schematic presentation of the construct used for thecomplementation of er-ant1-2 plants. R, right T-DNA border; L, left T-DNA border; pnos, nopalinesynthase promoter; bar, BASTA®-resistance; pAg7, gene 7 terminator. B, Transcript levels of Os-er-ant1 in 35S:Os-ER-ANT1 plants analyzed by qRT-PCR. The elongation factor EF1-α gene wasapplied as a control (n.d., not detectable). Used primers are listed in Supplemental Table 3. C,Growth phenotypes of 6-week-old wildtype and 35S:Os-er-ant1 plants under ambient CO2 conditions.D, Growth phenotypes of 8-week-old wildtype and er-ant1 knockout mutants under ambient CO2conditions.

A

B

35S:Os-er-ant13 13Col-0C

D

35S:Os-er-ant1


4

Col-0 er-ant1-1 er-ant1-2

35S:pfp5 35S:slr0293

C

Supplemental Figure 5. Overexpression of pfp5 and slr0293 in er-ant1 knockout plants.

er-ant1 knockout plants were transformed with the plant transformation vector pMDC32 (Curtis &Grossniklaus, 2003), carrying the cDNA of the P-proteins from Flaveria pringlei (pfp5) or Synechocystissp. PCC6803 (slr0293) under the control of the 35S-promoter. A and B, Schematic presentation of theconstructs used for the overexpression of pfp5 and slr0293. R, right T-DNA border; L, left T-DNAborder; CaMV 35S, 35S-promoter; HygR, hygromycin-resistance; ms, mitochondrial signal sequence;Term., terminator. C, Transcript levels of pfp5 and slr0293 in wildtype and overexpressor linesanalyzed by qRT-PCR. The elongation factor EF1-α gene was applied as a control (n.d., notdetectable). Used primers are listed in the Supplemental Table 3. D, Immuno-detection and SDS-PAGE to compare the P-protein levels between wildtype and transgenic plants. 15 µg of total proteinwere loaded. a) wildtype, b) er-ant1-2, c) 35S:pfp5, d) 35S:slr0293. E and F, Growth phenotypes of 6-week-old 35S:pfp5 plants and 35S:slr0293 plants under ambient CO2 conditions.

Anti P-protein

kDa

E F

D

70

55

100

130

170

a b c d

a b c d

A B


5

Supplemental Figure 6. Further evidence for enhanced ROS accumulation in wild-type and er-ant1 knockoutplants by NBT staining and determination of H2O2 in leaf extracts.

A, NBT staining for superoxide detection in leaves from plants grown under ambient CO2 conditions. B, NBTstaining in leaves from plants grown under 2% CO2. The experiment was conducted as described in the legendto figure 5. C, Chemiluminescence-assay to detect H2O2 contents in leaf extracts of wildtype and er-ant1knockout plants grown under ambient CO2 conditions. D, H2O2 levels in leaf extracts from plants grown under2% CO2. The assay was based on the ferricyanide-catalyzed oxidation of luminol as described in theSupplemental Materials and Methods section. Data represent means of 5 individual samples ± SE. Asterisksindicate the significance level between wild-type and er-ant1 knockout plants according to Student`s t-test (*p <0.05, ** p < 0.01).

er-ant1-1

Col-0

er-ant1-2

er-ant1-1

Col-0

ambient CO2

control

er-ant1-1

er-ant1-2

control

er-ant1-1

Col-0

er-ant1-2er-ant1-2

2% CO2

Col-0

A B

C D


6


Supplemental Figure 7. Transcript levels of ER chaperone genes in response to ER stress.

Leaf discs of 5-week-old wildtype and er-ant1 knockout mutants were incubated in 50 mM MES,pH 5.7 supplemented with 5 µg ml-1 tunicamycin or DMSO as a control for three hours. Transcriptlevels of the genes coding for the molecular binding proteins BiP1 and 2 (A), BiP3 (B) andcalreticulin (CRT; C) were analyzed by qRT-PCR using the elongation factor EF1-α gene ascontrol. Data represent means of 3 individual samples ± SE. Used primers are listed inSupplemental Table 3.

7


8

Supplemental Table 1

Selected metabolites of wildtype and er-ant1 knockout plants under ambient CO2 conditions. Leaf samples were harvested after 8 hours of illumination, and metabolites were determined by HPLC, IC and by mass spectrometry as described in the Supplemental Materials and Methods section. The data represent mean values of at least 2 individual replicates, ± SE. Asterisks indicate the significance level between wildtype and er-ant1 knockout plants according to Student`s t-test (**p < 0.01; ***p < 0.001). ns, not significant.

Phosphorylated Intermediates [pmol g-1 FW] Col-0 er-ant1-1 er-ant1-2

UDP-Glucose 113.1 ± 4.8 173.1 ± 9.4 ns 180.2 ± 6.7 ns

ADP-Glucose 21.7 ± 1.1 35.1 ± 1.2 ns 31.8 ± 2.2 ns

Glu-6-P/Fru-6-P 55.9 ± 5.8 62.9 ± 3.7 ns 60.1 ± 2.3 ns

3-PGA 56.8 ± 5.2 97.3 ± 7.5 ns 92.4 ± 5.8 ns

Carbohydrates [µmol g-1 FW] Col-0 er-ant1-1 er-ant1-2

Glucose 1.37 ± 0.033 1.03 ± 0.036 ** 1.06 ± 0.1 ns

Fructose 1.05 ± 0.12 0.66 ± 0.096 ns 0.72 ± 0.048 ns

Sucrose 1.44 ± 0.138 0.55 ± 0.045 ** 0.50 ± 0.0078 **

Starch (C6-units) 2.22 ± 0.099 0.125 ± 0.006 *** 0.46 ± 0.008 ***

Carboxylates [µg g-1 FW] Col-0 er-ant1-1 er-ant1-2

Citrate 1901.5 ± 49.3 4233.1 ± 24.2 *** 4514.7 ± 42.0 ***

Malate 648.3 ± 13.9 1017.1 ± 136.8 ns 1271.2 ± 44.6 ***

Fumarate 550.5 ± 7.3 333.8 ± 17.8 *** 439.9 ± 50.7 ns

Pyruvate 0.02 ± 0.0021 0.038 ± 0.002 ns 0.036 ± 0.0013 ns

Amino acids [µmol g-1 FW] Col-0 er-ant1-1 er-ant1-2

Alanine 5.34 ± 0.031 0.68 ± 0.018 *** 0.71 ± 0.019 ***

Arginine 0.37 ± 0.002 1.04 ± 0.020 *** 1.10 ± 0.021***

Asparagine 2.63 ± 0.159 1.09 ± 0.046 ** 1.16 ± 0.102 **

Aspartate 4.74 ± 0.311 1.44 ± 0.018 *** 1.46 ± 0.028 **

Glutamate 9.88 ± 0.258 5.79 ± 0.079 *** 5.7 ± 0.017 ***

Glutamine 1.91 ± 0.096 1.24 ± 0.073 ns 1.25 ± 0.070 ns

Glycin e 1.2 ± 0.207 31.8 ± 1.351*** 28.9 ± 0.626 ***

Histidine 0.74 ± 0.065 0.50 ± 0.012 ns 0.51 ± 0.03 ns

Methionine 0.13 ± 0.024 0.17 ± 0.004 ns 0.17 ± 0.002 ns

Proline 0.22 ± 0.01 0.04 ± 0.001ns 0.04 ± 0.002 ns

Serine 3.84 ± 0.006 3.93 ± 0.065 ns 3.87 ± 0.063 ns

Threonine 1.2 7 ± 0.015 3.18 ± 0.050 ns 3.27 ± 0.043 ns

Total 32.84 ± 1.195 52.59 ± 1.762 ns 49.06 ± 1.023 ns


9


Comparison between the amino acid sequences of Arabidopsis ER-ANT1 and related MCF carriers from Arabidopsis and rice.

Identity to Similarity to Protein Code At-ER-ANT1(%) At-ER-ANT1 (%)

Arabidopsis ER-ANT1 At5g17400 - -

rice ER-ANT1 Os11g0661300 63 76

putative rice AAC1/2 Os02g0718900 48 61

putative rice AAC3 Os05g0302700 47 61

Arabidopsis AAC1 At3g08580 47 61




10


Primers used for screening, cloning and for qRT-PCR studies.

Cloning and heterologous expression of Os-ER-ANT1 in E. coli cellsSK1 5’- GAA TCC GCC GTC GCA TAT GCC ATC CG -3’ SK2 5’- TCC AAA CTT CAA CCA AGG ATA TGC CTC -3’

Screening and cloning of transgenic plants

CH6 PFp5_fwd_AscI 5’- GGG CGC GCC ATG GAC CGT GCA -3’ CH7 PFp5_rev_PacI 5’- GGC TTA ATT AAA GCA GTA GCC TCC GC -3’ CH10 PFp5_ rev_BsiWI 5’- GGG CGT ACG AAC TTG TGA ACC AAA -3’ CH12 Slr0293_fwd_BsiWI 5’- CGT ACG ATG CCC AAC CTA GAG CCC -3’ CH13 Slr0293_rev_PacI 5’- GGG TTA ATT AAC TAT CCT TCC TTA TAA GCC -3’ TLP-4 5’- CAG GAG TGC TTG CGG GAT ATG -3’ TRP-4 5’- TGG TTT GGG TAA TTG CTT TAC CAG -3’ LBb1 5’- GCG TGG ACC GCT TGC TGC AAC T -3’ CH33 5’- CCC CAC TCA GGC TTT TAA CTT CGC ATT -3’ CH34 5’- GCA GCC CAC TGA ACT GAC GTT TGC TT -3’

qRT-PCR

CH8 PFp5_fwd 5’- GGG CTT GTA AGG GAG ACT CCA TAT T -3’ CH9 PFp5_rev 5’- GCC TGT TCG GTA GGA GCA AAA GGA -3’ CH14 Slr0293_fwd 5’- CGA GCT TTC CCG CCG ATT TAA CCC -3’ CH15 Slr0293_rev 5’- CCA GCG GGG GCA AAG GGG TGA -3’ EF1α_fwd 5’- CTT CAC CTC CCA GGT CAT C -3’ EF1α_rev 5’- TGG GCT TGG TTG GAG TCA TC -3’ BiP-1/2_fwd 5’- CGA TCA TCT TCT TCG GAT G -3’ BiP-1/2_rev 5’- GCC CGA TCA ATC TCT TAA C -3’ BiP-3_fwd 5’- ACA CAG CGA AGA TGA CGA G -3’ BiP-3_rev 5’- GGT TCG CTC CGG GTT CTT G -3’ CRT-1/2_fwd 5’- ACT GGT AGC CTT TAC TCT G -3’ CRT-1/2_rev 5’- GCA GTC CAC TCA CCA TCT TC -3’


11

Supplemental Methods

Construction of the Sequence Alignment

Multiple alignments of protein sequences were performed with the program ClustalW

(Thompson et al., 1994).

Heterologous Expression of rice ER-ANT1 in Escherichia coli

To construct E.coli plasmids expressing rice ER-ANT1 with an N-terminal His-tag, the

coding region of the rice er-ant1 ortholog (Os11g0661300) was amplified by PCR on

first strand cDNA from Oryza sativa leaf tissue using the primers SK1 and SK2 (see

Supplemental Table 3) and subcloned into an EcoRV linearized pBluescript vector

(Stratagene, La Jolla, USA). The complete coding region was then excised from this

vector using NdeI/BamHI and inserted into the IPTG-inducible expression vector

pET16b (Merck Biosciences/Novagen, Darmstadt, Germany) using the same

restriction sites.

For heterologous expression of rice ER-ANT1 and uptake experiments with [ 32P]-ATP

or [32P]-ADP , the E.coli strain BL21 (DE3) was used as described earlier (Haferkamp

et al., 2002; Leroch et al., 2008).

Generation of Mutants

To generate a plant transformation construct for rice ER-ANT1 overexpression in er-

ant1-1 Arabidopsis plants, the complete coding region of the rice er-ant1 ortholog

(Os11g0661300) was amplified by PCR on first strand cDNA from Oryza sativa leaf

tissue using the primers SK1 and SK2 (see Supplemental Table 3) and subcloned

into the SmaI-cut vector pA35S (Höfte et al. 1991). The complete gene cassette,

including the 35S-promoter, the er-ant1 rice cDNA and the terminator region, was cut


12

out of the pA35S-construct by digestion with EcoRI and HindIIII and inserted into the

plant transformation vector pGPTV-bar (Becker et al., 1992) using the same

restriction sites. Insertion of the resulting plasmid into er-ant1-2 plants was performed

by Agrobacterium tumefaciens-mediated transformation using the floral-dip method

(Clough & Bent, 1998). The molecular characterization of er-ant1-2 knockout plants

and 35S:Os-er-ant1 expressing plants was performed with genomic DNA and cDNA

isolated from 4-week-old plants using the primers listed in Supplemental Table 3.

For the generation of mutants overexpressing a gene encoding the GDC-P-protein

from either the C3 plant Flaveria pringlei (pfp5; Bauwe et al., 1995) or the gene

slr0293 from the cyanobacterium Synechocystis sp. PCC6803 (Hasse et al., 2007)

two different plant transformation constructs were cloned in which the cDNA

sequences of the genes pfp5 or slr0293 were expressed under the control of the

35S-promoter. First, the complete coding region of pfp5 was amplified with the

primers CH6 and CH7 (see Supplemental Table 3) and subcloned into the SmaI-cut

vector pBSK (Stratagene, Heidelberg, Germany). The resulting construct was cut

with AscI and PacI (New England Biolabs, Frankfurt/M., Germany) and ligated into

the plant transformation vector pMDC32 (Curtis & Grossniklaus, 2003), using the

same restriction sites. Insertion of the resulting plasmid into er-ant1-1 plants

(SALK_043626) was performed by Agrobacterium tumefaciens-mediated

transformation.

For the overexpression of the gene slr0293 from Synechocystis sp. PCC6803, the

coding region of slr0293 was amplified with the primers CH12 and CH13 (see

Supplemental Table 3) and subcloned into the SmaI cut vector pBSK. Due to the lack

of a signal peptide in the corresponding cyanobacterial protein, the predicted

mitochondrial signal sequence of pfp5 (198 bp) was amplified with the primers CH6

and CH10 and inserted into a second SmaI cut pBSK vector. Both constructs were


13

subsequently cut with the enzymes BsiWI and EcoRV and fused together in a single

pBSK vector. The complete construct, including the coding region of the

mitochondrial signal sequence and slr0293, was digested with AscI and PacI and

inserted into the vector pMDC32, using the same restriction sites. Overexpression

plants were generated by Agrobacterium tumefaciens-mediated transformation of er-

ant1-2 knockout plants (SALK_023441).

Detection of H2O2 in Arabidopsis Leaf Extracts

H2O2 contents in Arabidopsis leaf extracts H2O2 were determined by a

chemiluminescence assay based on the ferricyanide-catalyzed oxidation of luminol

(Queval et al., 2008). Leaves from 4-weeks-old plants grown under ambient air or 2%

CO2 were harvested and homogenized in liquid nitrogen. 5 mg ml -1 leaf tissue were

extracted in 0.2 M HCl, neutralized with 50 mM NaH 2PO4 and adjusted to a pH of 6.0

with 0.2 M NaOH. Neutralized leaf extracts were treated with 2 U ml -1 ascorbate

oxidase for 5 min to remove antioxidants, incubated with a Dowex ion exchange resin

(1 x 8, Bio-Rad, Munich, Germany) and centrifuged for 5 min at 20.000x g. The

chemiluminescence assay was performed in a Tecan Infinite-200 plate reader by

mixing 5-15 µl of the supernatant with 11 µl of 2.2 mM luminol and 0.05 M K 2PO4

buffer (pH 8.0) to a final volume of 150 µl. Reactions were started by injecting 100 µl

of 2.8 mM ferricyanide (K3Fe(CN)6), and the resulting luminescence signal was

integrated over 2 s. A calibration curve of standard H 2O2 solutions was used to

calculate leaf contents.


14

Quantification of Neutral Sugars, Carbonic Acids and Phosphorylated

Intermediates

For the extraction of soluble sugars, samples were prepared according to the amino

acid preparations described above. Starch was quantified as glucose (C6) units after

hydrolysis as described previously (Reinhold et al., 2007). Sugar quantification was

performed by ion exchange chromatography according to Hassler et al. (2012). For

the extraction of carbonic acids, plant material was ground under liquid N 2 and each

50 mg aliquot was mixed with 500 µl H2O, heated for 15 min at 95°C and centrifuged

for 15 min at 18.000xg. Carbonic acids in the supernatant were quantified by ion

chromatography on a 761 IC Compact device (Metrohm, Zofingen, Switzerland),

using a C4150 Metrosep organic acids 250 x 7.8 column (mobile phase: 0.35 mM

H2SO4/10 mM lithium chloride, flow rate: 0.6 ml min -1, pressure: 5.0 MPa,

temperature: 21°C). Phosphorylated intermediates were quantified by mass

spectrometry analyses in the lab of Dr. Mohammad Hajirezaei (IPK Gatersleben,

Germany).

Monitoring the Unfolded Protein Response (UPR)

To monitor ER stress response in wild-type and er-ant1 plants, leaf discs from 6-

week-old plants were incubated for 6 hours in 50 mM morpholino-ethane sulphonic

acid (MES), pH 5.7 (KOH) supplemented with 5 µg ml -1 tunicamycin (Sigma)

dissolved in DMSO (Iwata & Koizumi, 2005). Controls were treated with the appro-

priate amount of solely DMSO. Isolation of RNA and quantitative RT-PCR were per-

formed as described above.


15

Supplemental References

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analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-

cleavage system from Flaveria pringlei. Eur. J. Biochem. 234: 116-124.

Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992). New plant binary

vectors with selectable markers located proximal to the left T-DNA border. Plant

Mol.Biol. 20: 1195-1197.

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735-743.

Curtis, M.D., and Grossniklaus, U. (2003). A gateway cloning vector set for high-

throughput functional analysis of genes in planta . Plant Physiol. 133: 462-469.

Hasse, D., Mikkat, S., Thrun, H.A., Hagemann, M., and Bauwe, H. (2007).

Properties of recombinant glycine decarboxylase P- and H-protein subunits from the

cyanobacterium Synechocystis sp. strain PCC 6803. FEBS Lett. 581: 1297-1301.

Hassler, S., Lemke, L., Jung, B., Möhlmann, T., Krüger, F., Schumacher, K.,

Espen, L., Martinoia, E., and Neuhaus, H.E. (2012). Lack of the Golgi phosphate

transporter PHT4;6 causes strong developmental defects, constitutively activated

disease resistance mechanisms and altered intracellular phosphate

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Höfte, H., Faye, L., Dickinson, C., Herman, E.M., and Chrispeels, M.J. (1991).

The protein-body proteins phytohemagglutinin and tonoplast intrinsic protein are

targeted to vacuoles in leaves of transgenic tobacco. Planta 184: 431-443.


16

Iwata, Y., and Koizumi, N. (2005). An Arabidopsis transcription factor, AtbZIP60,

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