rapid single-step affinity purification of ha-tagged plant ...dec 09, 2019 · moreover,...

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Short title: Affinity purification of plant mitochondria 1

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Corresponding Author: 3

Andreas P.M. Weber, 1Institute of Plant Biochemistry, Cluster of Excellence on Plant Science 4

(CEPLAS), Heinrich Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany 5

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Rapid single-step affinity purification of HA-tagged plant mitochondria 7

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Franziska Kuhnert1, Anja Stefanski2, Nina Overbeck2, Leonie Drews3, Andreas S. Reichert3, 9

Kai Stühler2, Andreas P.M. Weber1 10

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1Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Heinrich 12

Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany 13

2Molecular Proteomics Laboratory, Biomedical Research Center, Heinrich Heine University, 14

Universitätsstrasse 1, 40225 Düsseldorf, Germany 15

3Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich-Heine University, 16

Universitätsstrasse 1, 40225 Düsseldorf, Germany 17

One-sentence summary: Triple hemagglutinin-tagging of a mitochondrial outer membrane 18

protein enables single-step affinity purification of intact plant mitochondria in less than 20 19

minutes. 20

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Author contributions: F.K. generated transgenic lines harboring affinity-tagged 22

mitochondria, isolated mitochondria, analyzed the data, and drafted the manuscript; A.S., 23

N.O., and K.S. performed the proteomics analysis and analyzed the data; L.D. and A.S.R. 24

assisted with the Seahorse Analyzer measurements; A.P.M.W. conceived and supervised the 25

experiments and contributed to the writing of the manuscript. 26

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Plant Physiology Preview. Published on December 9, 2019, as DOI:10.1104/pp.19.00732

Copyright 2019 by the American Society of Plant Biologists

www.plantphysiol.orgon October 18, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

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Funding: This work was supported by the Deutsche Forschungsgemeinschaft (CRC 1208 28

and funding under Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111“). 29

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Email address of the author for contact: [email protected] 32


mailto:[email protected]


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ABSTRACT 33

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. 34

However, isolating mitochondria from plant cells for physiological and biochemical analyses is 35

a lengthy and tedious process. Established isolation protocols require multiple centrifugation 36

steps and substantial amounts of starting material. To overcome these limitations, we tagged 37

mitochondria in Arabidopsis thaliana with a triple haemagglutinin-tag for rapid purification via 38

a single affinity purification step. This protocol yields a substantial quantity of highly pure 39

mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for 40

enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic 41

profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant 42

deficient in the mitochondrial glutamate transporter À BOUT DE SOUFFLE (BOU) and 43

identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 44

controls. Our work sets the stage for the development of advanced mitochondria isolation 45

protocols for distinct cell types. 46

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INTRODUCTION 48

In all eukaryotic organisms, mitochondria are the major source of ATP, which is produced via 49

the oxidative phosphorylation (OXPHOS) pathway, thus playing a vital role in cellular energy 50

metabolism. Mitochondria also participate in amino acid metabolism as well as in 51

photorespiration in photosynthetic eukaryotes. Analysis of the biochemical and physiological 52

functions of mitochondria frequently requires the isolation of intact mitochondria. Mitochondria 53

can be isolated from leaf tissue in less than 1 h by differential centrifugation. This method 54

yields mitochondria with good integrity and appropriate enzyme activity; however, 55

mitochondria are frequently contaminated with plastids and peroxisomes. Hence, in many 56

cases, a combination of differential centrifugation and Percoll density gradient is used (Millar 57

et al., 2001; Werhahn et al., 2001; Keech et al., 2005). Whereas this produces a pure fraction 58

of respiratory active mitochondria with low plastid and peroxisome contamination, such 59

procedures generally take several hours and require up to 50 g of starting material for 60

producing sufficient yields (Keech et al., 2005). Moreover, traditional protocols are not practical 61

for the isolation of mitochondria from mutants with severely impaired growth or from less 62

abundant tissue types such as flowers and for the analysis of mitochondrial metabolites. 63

Furthermore, media used for the isolation of mitochondria typically contain high concentrations 64

of sugars and other metabolites that can potentially interfere with MS-based metabolite 65

analyses. 66

Recently, Chen and coworkers reported a method for the rapid isolation of mitochondria from 67

human HeLa cell cultures via co-immunopurification (co-IP) (Chen et al., 2016). The authors 68

generated transgenic HeLa cell lines expressing a triple hemagglutinin (HA)-tagged enhanced 69

green fluorescent protein (eGFP) fused to the outer mitochondrial membrane (OMM) 70

localization sequence of OMP25 (3HA-eGFP-OMP25). Because the epitope-tag was 71

displayed on the surface of mitochondria, these transfected cell lines could be used to rapidly 72

enrich mitochondria after cell homogenization. The HA-tagged mitochondria were captured 73

and pulled down using magnetic beads coated with an anti-HA-tag antibody. Given the small 74

size (1-µm diameter) and non-porous behavior of anti-HA-tag beads, these beads performed 75

better than the porous agarose matrix for the enrichment of mitochondria. Thus, the authors 76

established a method that ensures a high yield of pure mitochondria in approximately 12 min. 77

The isolated mitochondria showed high purity, integrity, and functionality. Additionally, the 78

authors developed a simple potassium-based buffer system that maintains mitochondrial 79

intactness and is compatible with downstream analyses, such as metabolite analysis by 80

LC/MS (Chen et al., 2016). 81



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Photorespiration plays a crucial role in photosynthesis by detoxifying 2-phosphoglycolate, 82

which is produced by the oxygenation of Rubisco and acts as an inhibitor of several plastidial 83

enzymes (Ogren and Bowes, 1971; Kelly and Latzko, 1976; Husic et al., 1987). Plants reclaim 84

2-phosphoglycolate in the complex pathway of photorespiration, yielding 3-phosphoglycerate, 85

which is returned to the Calvin Benson cycle. The photorespiratory pathway includes several 86

enzymatic steps that occur in four subcellular compartments: plastids, peroxisomes, 87

mitochondria, and the cytosol (Eisenhut et al., 2019). Knockout mutants of genes encoding 88

enzymes and transporters involved in photorespiration often show a photorespiratory 89

phenotype, characterized by chlorotic leaves and growth inhibition under ambient carbon 90

dioxide (CO2) conditions, which can be rescued in a CO2-enriched environment (Peterhansel 91

et al., 2010). A key step in photorespiration is the conversion of two glycine molecules into 92

one serine residue in the mitochondrial matrix, accompanied by the release of CO2 and 93

ammonia. This step is catalyzed by the glycine decarboxylase (GDC) multienzyme system, 94

comprising the P-protein (GLDP), H-protein (GDCH), L-protein (GDCL), and T-protein (GLDT), 95

in combination with serine hydroxymethyltransferase (SHM) (Voll et al., 2006; Engel et al., 96

2007). In green tissues, these proteins constitute up to 50% of the total protein content of the 97

mitochondrial matrix, indicating the importance of glycine oxidation in mitochondria (Oliver et 98

al., 1990). 99

The Arabidopsis thaliana bou-2 mutant was previously identified as lacking the mitochondrial 100

glutamate transporter À BOUT DE SOUFFLE (BOU), which is involved in photorespiration 101

(Eisenhut et al., 2013; Porcelli et al., 2018). Plants lacking the inner mitochondrial membrane 102

(IMM) protein BOU show a pronounced photorespiratory phenotype under ambient CO2 103

conditions, significantly elevated CO2 compensation point, and highly reduced GDC activity in 104

the isolated mitochondria (Eisenhut et al., 2013). Because BOU is co-expressed with genes 105

encoding components of the GDC complex, and the bou-2 mutant shows a similar metabolic 106

phenotype as the shm1 mutant, it was hypothesized that BOU transports a metabolite 107

necessary for the proper functioning of GDC (Voll et al., 2006; Eisenhut et al., 2013). Recently, 108

it was demonstrated that heterologously expressed BOU functions as a glutamate transporter 109

(Porcelli et al., 2018). Glutamate is neither a substrate nor a product of the reaction catalyzed 110

by GDC. Besides of its role in amino acid and nitrogen metabolism, glutamate is necessary 111

for the glutamylation of tetrahydrofolate (THF), a cofactor of GLDT and SHM (Suh et al., 2001). 112

Glutamylation of THF increases its stability. Moreover, THF-dependent enzymes generally 113

prefer polyglutamylated folates over monoglutamylated folates as a substrate (Suh et al., 114

2001). However, because (1) BOU is not the only glutamate transporter in mitochondria, and 115

(2) glutamylation of folates is not restricted to mitochondria, the exact physiological function of 116

BOU remains unclear (Hanson and Gregory, 2011; Monné et al., 2018). Notably, a 117



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glutamate/glutamine shuttle across the mitochondrial membrane was previously suggested to 118

support the reclamation of ammonia released during photorespiration (Linka and Weber, 119

2005). 120

Building on the previous work of Chen and coworkers, we developed an affinity-tagging 121

strategy for the rapid isolation of mitochondria from Arabidopsis. We generated transgenic 122

Arabidopsis lines carrying an HA-tagged TRANSLOCASE OF THE OMM 5 (TOM5) and 123

isolated highly pure and intact mitochondria from these lines in less than 25 min. The isolated 124

mitochondria were successfully subjected to proteomics and enzyme activity analyses. 125

Moreover, we applied the isolation strategy to the bou-2 mutant, revealing differential protein 126

abundance and enzyme activities. 127

RESULTS 128

Identification of TOM5 a suitable anchor peptide and generation of affinity-tagged 129

Arabidopsis lines 130

Recently, Chen and colleagues reported a rapid protocol for the isolation of intact mitochondria 131

from transgenic HeLa cells expressing the mitochondrial fusion protein 3HA-eGFP-OMP25 132

using co-IP. The Arabidopsis genome does not encode an ortholog of OMP25. Therefore, we 133

screened the available Arabidopsis mitochondrial proteome data, including the OMM proteins 134

with known topology and function, and identified TOM5 as a potential candidate for our 135

mitochondria affinity-tagging approach. Together with TOM6, TOM7, TOM20, TOM22/9, and 136

TOM40, TOM5 forms the protein import apparatus of plant mitochondria (Werhahn et al., 137

2003). In yeast (Saccharomyces cerevisiae), TOM5 is an integral protein of the OMM. It has 138

a negatively-charged N-terminal domain, which faces toward the cytosol (Dietmeier et al., 139

1997) and can be fused to GFP without altering the subcellular localization of TOM5 (Horie et 140

al., 2003). The protein import machinery is well conserved among eukaryotes. The predicted 141

N-terminal cytosolic domain of Arabidopsis TOM5 is necessary for the recognition of 142

cytosolically synthesized mitochondrial preproteins (Wiedemann et al., 2004). Therefore, we 143

generated an N-terminal translational fusion of the Arabidopsis TOM5 gene with triple HA-144

tagged synthetic GFP (sGFP) gene under the control of the Arabidopsis UBIQUITIN10 145

promoter (UB10p) (UB10p-3HA-sGFP-TOM5; Supplemental Fig. S1). The construct was 146

used to stably transform Arabidopsis ecotype Columbia (Col-0) and bou-2 mutant. Expression 147

and localization of the fusion protein was verified in root and leaf tissues of 10-day-old 148

Arabidopsis seedlings via confocal laser scanning microscopy. Arabidopsis lines expressing 149

the 3HA-sGFP-TOM5 protein in leaf and root mitochondria of Col-0 (tagged Col-0) and bou-150

2 (tagged bou-2) seedlings were identified based on the co-localization of the fluorescent 151



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signal of GFP signal with that of the mitochondrial marker CMXRos (Fig. 1A–D). CMXRos is 152

a lipophilic cationic dye that accumulates in the mitochondria because of the negative 153

membrane potential; thus, it solely stains mitochondria with an intact respiratory chain 154

(Pendergrass et al., 2004). Because the fluorescent signals of the 3HA-sGFP-TOM5 protein 155

(green) fully overlapped with those of the MitoTracker (red), we conclude that overexpression 156

of the UB10p-3HA-sGFP-TOM5 construct in Col-0 and bou-2 does not affect mitochondrial 157

intactness. Notably, tagged Col-0 or bou-2 lines showed no apparent phenotypic differences 158

compared with non-tagged Col-0 or bou-2 plants (control), respectively, under our culture 159

conditions (Supplemental Fig. S2). 160

Affinity-based purification of intact mitochondria 161

Tagged Col-0 and bou-2 lines were used for the isolation of intact HA-tagged mitochondria via 162

co-IP using magnetic anti-HA beads. We chose HA as the epitope-tag for purification because 163

it has a high affinity for its cognate antibody, and Chen and coworkers previously 164

demonstrated that the size and non-porous behavior of the anti-HA beads yields a high 165

amount of mitochondria (Chen et al., 2016). Additionally, we used the LC/MS-compatible 166

buffer containing KCl and KH2PO4 (KPBS) developed by Chen et al. (2016). Our purification 167

procedure included five steps: homogenization of plant material in KPBS (1 min), filtration of 168

the homogenate (1 min), two centrifugation steps (5 min and 9 min), and co-IP (7 min, including 169

washing steps). Altogether, mitochondria were purified from plant material in less than 25 min 170

(Fig. 2). If the first low-speed centrifugation step used to remove contaminating chloroplasts 171

and cell debris is omitted, the isolation time can be reduced to 18 min. The purified 172

mitochondria were verified by immunoblot analyses using known organelle-specific protein 173

markers. Mitochondria were enriched via co-IP only from lines harboring the mitochondrial 174

3HA-sGFP-TOM5 protein (tagged line), as demonstrated by immunoblot analyses with 175

antibodies directed against different mitochondrial marker proteins including isocitrate 176

dehydrogenase (IDH; mitochondrial matrix), alternative oxidase 1/2 (AOX1/2; IMM), and 177

voltage-dependent anion channel 1 (VDAC1; OMM). No enrichment of mitochondria was 178

observed in control Col-0 lines, indicating that the beads bind specifically to the HA-tag on 179

mitochondria in tagged lines (Fig. 3, Supplemental Fig. S3A). Comparison with classical 180

mitochondria isolation protocols using differential centrifugation and density gradient 181

purification revealed that the mitochondrial fraction enriched using our affinity-tagging method 182

showed significantly less contamination with proteins from plastids, peroxisomes, the 183

endoplasmic reticulum (ER), nuclei, and the cytosol (Fig. 3, Supplemental Fig. S3B). The 184

following proteins were used as markers for different organelles: Rubisco large subunit (RbcL; 185

plastid), catalase (Cat; peroxisome), lumenal-binding protein 2 (BiP2; ER), histone H3 186

(nucleus), and heat shock cognate protein 70 (HSC70; cytosol). Contamination with 187



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thylakoids, which are a major contaminant of leaf mitochondria, was assessed by determining 188

the chlorophyll-to-protein ratio of isolated mitochondria. Rapidly enriched mitochondria show 189

minor contamination by thylakoids (0.18 ± 0.08 µg Chl mg-1 protein). The here presented 190

chlorophyll-to-protein ratio is two-fold lower compared to classical mitochondria isolations 191

(0.34 ± 0.08 µg Chl mg-1 protein; Keech et al., 2005). 192

The intactness of mitochondria isolated from HA-tagged lines via co-IP was assessed based 193

on respiratory measurements with the Seahorse Analyzer, the latency of malate 194

dehydrogenase (MDH) activity, and mitochondrial staining with the mitochondrial marker 195

CMXRos (Fig. 4A-C). Mitochondria isolated with our affinity-tagging approach showed 196

respiratory coupling with succinate as substrate. Respiration is inhibited by the respiratory 197

chain inhibitor Antimycin A. Addition of the complex III inhibitor lead to a significant reduction 198

of the oxygen consumption rate in the mitochondria but not the control samples (OCR; Fig. 199

4A). Intactness and activity was further verified by latency experiments with MDH. Activity of 200

MDH was only present in detergent-treated samples containing enriched mitochondria but not 201

the control or non-treated samples indicating intactness of the OMM and IMM (Fig. 4B). 202

Moreover, affinity-purified mitochondria retained the mitochondrial marker CMXRos that solely 203

stains mitochondria with an active respiratory chain (Fig 4C; Pendergrass et al., 2004). 204

Typically, we used 5–10 g of Arabidopsis seedlings grown on agar plates for the isolation of 205

mitochondria, and this yielded 400–700 µg of total mitochondrial protein. However, 206

mitochondria could also be isolated from 1 g of starting material, yielding 50–200 µg of total 207

mitochondrial protein. This is advantageous for very young seedlings or mutants with severely 208

impaired growth. Isolation from less than 1 g of starting material may also result in an 209

appropriate yield of mitochondria, but this was not tested in the current study. We found that 210

recovery of mitochondria with our method was 7.1 ± 3.3%. 211

Taken together, our data indicate that mitochondria can be rapidly isolated via co-IP using a 212

simple LC/MS-compatible buffer. 213

Enzyme assays using mitochondria affinity-purified from tagged Col-0 and bou-2 214

mutant lines 215

We used our rapid mitochondria isolation method to study the effect of the mitochondrial 216

carrier protein BOU on mitochondrial metabolism in Arabidopsis. 217

To assess the effect of the bou-2 mutant allele on mitochondrial metabolism, we rapidly 218

isolated mitochondria from 10-day-old tagged Col-0 and bou-2 seedlings grown under 219

elevated CO2 conditions (3,000 ppm) and seedlings sampled five days after shift to ambient 220

CO2 conditions (380 ppm CO2), with three independent biological replicates included for each 221



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treatment. Mitochondria were lysed and used to measure the activity of MDH, aspartate 222

aminotransferase (AspAT), glutamate dehydrogenase (GluDH), alanine aminotransferase 223

(AlaAT), γ-aminobutyric acid transaminase (GABA-T), and formate dehydrogenase (FDH). 224

Enzyme activities were calculated from the initial slopes. Enzyme activities in tagged bou-2 225

mutant lines were compared with those in tagged Col-0 lines, which were set to 100% for both 226

experimental conditions. 227

The activities of MDH and FDH were not affected in tagged bou-2 mutant lines under any of 228

the conditions tested (Fig. 5A, B). The activities of AspAT and GABA-T in tagged mutant lines 229

were similar to those in tagged Col-0 lines, when mitochondria were isolated from seedlings 230

grown under elevated CO2 conditions, but were significantly reduced in tagged mutant lines 231

after the shift to ambient CO2 conditions (Fig. 5C, D). The activity of AlaAT was significantly 232

reduced (Fig. 5E), whereas that of GluDH was significantly increased in tagged mutant lines 233

under elevated CO2 conditions; however, enzyme activity of the latter reverted back to the 234

levels in tagged Col-0 when seedlings were shifted to ambient CO2 conditions (Fig. 5F). 235

Together, our results suggest a possible involvement of BOU in mitochondrial amino acid and 236

nitrogen metabolism. 237

In addition, we found that the activity of MDH was strongly reduced in 4-week-old tagged bou-238

2 mutant plants (Supplemental Fig. S4). However, no change was observed in MDH activity 239

in 10-day-old tagged bou-2 plants, suggesting pleiotropic effects in older leaf tissues due to 240

accumulating photorespiratory intermediates. 241

Proteomic analysis of mitochondria affinity-purified from tagged Col-0 and bou-2 242

mutant lines 243

Mitochondria were isolated from 10-day-old tagged Col-0 and bou-2 seedlings grown under 244

elevated CO2 conditions, with four independent biological replicates included. Proteome 245

analysis of the isolated mitochondria revealed 15,688 peptides belonging to 1,240 proteins 246

present in at least three of the four replicates (Supplemental Table S1, Supplemental Table 247

S2). 248

Subcellular localization of the quantified proteins was annotated using the SUBAcon database 249

(Hooper et al., 2017). Summing up the label-free quantitation (LFQ) intensities of the spectra 250

showed that 80% of the identified proteins in our study resulted from proteins localized or 251

predicted to be localized to the mitochondria, 11.5% from plastid-localized proteins, and 5.7% 252

from proteins with no clear subcellular localization (designated as ambiguous). If we apply the 253

evaluation on all quantified peptides in this study, more than 90% of all identified peptides 254

were assigned to mitochondria-localized proteins. Contamination of the mitochondrial proteins 255



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by proteins from peroxisomes, the ER, the Golgi, vacuoles, endomembranes, plasma 256

membranes, nuclei, and the cytosol was less than 1% each (Supplemental Table S1, 257

Supplemental Table S2). These results indicate high purity of the rapidly isolated 258

mitochondria, which was comparable with the purity of classically isolated mitochondria 259

(Klodmann et al., 2011; Senkler et al., 2017). 260

Next, we compared our proteome data with previous proteomic analyses and quantified 261

proteins and some of the subunits of known mitochondrial complexes (Supplemental Table 262

S1, Supplemental Table S2). The OXPHOS pathway of mitochondria consists of five protein 263

complexes (I–V) located in the IMM. Complexes I to IV represent oxidoreductases, which 264

comprise the respiratory chain that regenerates oxidized forms of cofactors involved in 265

mitochondrial metabolism, thereby creating an electron flow. This leads to the simultaneous 266

export of protons into the intermembrane space (IMS). The built-up proton gradient is used by 267

Complex V to generate ATP. Complex I is the largest complex involved in the OXPHOS 268

pathway and comprises at least 47 protein subunits that form the so-called membrane and 269

peripheral arms (Peters et al., 2013; Meyer et al., 2019). Except NDUA1, NDUB2, Nad4L, and 270

Nad6 (At3g08610, At1g76200, AtMg00650, and AtMg00270, respectively), we could identify 271

all Complex I subunits in our proteomic data set, including eight previously proposed assembly 272

factors (Meyer et al., 2019), five γ-carbonic anhydrases, and five additional proteins proposed 273

to form a matrix-exposed domain attached to Complex I (Sunderhaus et al., 2006). In addition 274

to its function in the OXPHOS pathway, Complex II also participates in the tricarboxylic acid 275

cycle (TCA). Six out of eight subunits of Complex II (Millar et al., 2004) and the assembly 276

factor SDHAF2 were identified in our data set. Additionally, peptides of all proteins and 277

isoforms of Complex III as well as eight of its assembly factors (Meyer et al., 2019) were 278

identified in our data set. The cytochrome c oxidase complex (Complex IV) consists of 16 279

proposed subunits in Arabidopsis (Mansilla et al., 2018). Of these, 9 subunits and 11 assembly 280

factors of Complex IV were identified in our proteomic data set. Complex V consists of 15 281

subunits, of which 13 were identified in our proteomic data set; Atp6 (AtMg00410 and 282

AtMg011701) and Atp9 (AtMg01080) were the only two subunits that could not be identified. 283

In addition, we found three of the five proposed assembly factors (Meyer et al., 2019). 284

Furthermore, we identified three proteins involved in the assembly of OXPHOS 285

supercomplexes as well as alternative NAD(P)H dehydrogenases and alternative oxidases 286

that have previously been defined as alternative pathways (Meyer et al., 2019). 287

Except for the abovementioned subunits of the SDH complex, all proteins of the TCA cycle, 288

including the pyruvate dehydrogenase complex, and the GDC multienzyme system were 289

identified in the rapidly isolated mitochondria. Additionally, our proteomic data set contained a 290

number of pentatricopeptide and tetratricopeptide repeat proteins involved in RNA metabolism 291



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as well as heat shock proteins and ribosomal proteins involved in protein control and turnover 292

(Supplemental Table S1, Supplemental Table S2). 293

In Arabidopsis, the majority of mitochondrial proteins are encoded by nuclear genes, 294

translated in the cytosol, and then imported into the mitochondria. The import and sorting of 295

nuclear-encoded mitochondrial preproteins requires functional TOM and sorting and assembly 296

machinery (SAM) in the OMM, mitochondrial IMS import and assembly (MIA) machinery in the 297

IMS, and translocase of the IMM (TIM) in the IMM (Murcha et al., 2014). In this study, we 298

identified all proteins necessary for functional protein import into mitochondria in our proteome 299

data set, except for the OMM protein TOM6, IMS protein ERV1, IMM protein PRAT5, and 300

matrix proteins MGE1 and ZIM17. Additionally, we identified plant-specific import components, 301

including OM64, PRAT3, and PRAT4, in the OMM (Murcha et al., 2015), and plant 302

homologues to proteins of the mitochondrial contact site and cristae organizing system 303

(MICOS), which connects the IMM to OMM (van der Laan et al., 2016). Other notable OMM 304

proteins identified in our proteomic data set included GTPases MIRO1 and MIRO2, lipid 305

biosynthesis protein PECT, and β-barrel proteins VDAC1–4 (Supplemental Table S1, 306

Supplemental Table S2). Recently, it was shown that the cytosolic protein GAPC interacts with 307

VDAC (Schneider et al., 2018); we also identified GAPC in our proteomic data set. 308

Overall, we conclude that mitochondria isolated using our rapid isolation method are suitable 309

for proteomic analyses. 310

Differential analysis of the mitochondrial proteome of tagged Col-0 and bou-2 lines 311

To assess the effect of bou-2 mutation on the mitochondrial proteome, we performed 312

comparative proteomic analysis of tagged Col-0 and bou-2 lines. A total of 47 proteins showed 313

significantly increased abundance in the mutant, of which five were localized to the 314

mitochondria. Additionally, 44 proteins showed significantly decreased abundance in the 315

tagged bou-2 samples, of which 22 were predicted to be localized to the mitochondria (Table 316

1); among these proteins, BOU was the least abundant. The bou-2 line is a GABI-Kat line that 317

carries a T-DNA insertion in the second exon of the BOU gene (Kleinboelting et al., 2012; 318

Eisenhut et al., 2013). We identified two peptides of BOU in at least three of the four replicates 319

of tagged bou-2 samples. Both peptides were translated from the first exon of the gene. 320

Because the T-DNA was inserted in the second exon of the gene, it is possible that the first 321

exon was translated. However, a functional protein is not synthesized in the knockout mutant 322

(Eisenhut et al., 2013). Among the mitochondrial proteins showing significantly reduced 323

abundance in tagged bou-2 seedlings, we identified six proteins of the OXPHOS pathway 324

(three Complex I proteins and one protein each of Complex II, III, and V), two proteins involved 325

in protein translocation, two proteins involved in metabolite transport, two proteins involved in 326



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lipid metabolism, three proteins involved in protein turnover/synthesis, one protein involved in 327

the TCA cycle, and six proteins (including FDH) involved in other processes. Among the 328

proteins with significantly increased abundance in tagged bou-2 mutant, we identified two 329

proteins involved in RNA/DNA metabolism, one protein involved in THF metabolism, one 330

MIRO-related GTPase, and one LETM1-like protein (Table 1). 331

Previously, Eisenhut and colleagues showed that GDC activity is reduced in mitochondria 332

isolated from 4-week-old bou-2 mutant plants (Eisenhut et al., 2013). The authors showed that 333

the bou-2 mutant accumulated higher amounts of glycine than the wild type and exhibited 334

differential amount and status of the P-protein. Immunoblot analysis showed no differences in 335

the levels of other GDC proteins in the bou-2 mutant compared with the wild type (Eisenhut et 336

al., 2013). In our proteomic data set, none of the proteins of the GDC complex or SHMT 337

showed significant differences between tagged Col-0 and bou-2 seedlings (Table 2). However, 338

the amounts of GLDP1, GLDP2, GDCH1, GDCL1, GDCL2, and GLDT were slightly reduced, 339

whereas those of SHM1, SHM2, and GDCH3 were increased in the tagged mutant compared 340

with tagged Col-0. Among these proteins, the strongest reduction was detected in the amount 341

of GDCH1. However, differences in protein levels between tagged Col-0 and bou-2 seedlings 342

were not statistically significant. Only one peptide related to GDCH2 was detected in our data 343

set; however, because it was detected in only one of the four replicates, it is not listed 344

Supplemental Table S1. 345

In this study, we showed that tagged mitochondria of the bou-2 mutant displayed reduced 346

AlaAT activity, increased GluDH activity, and no change in MDH, AspAT, GABA-T, and FDH 347

activities under elevated CO2 conditions compared with tagged Col-0 (Fig. 5A–F). Except for 348

FDH, none of the assayed enzymes showed significantly altered amounts in our proteomic 349

data set (Table 2). The amount of FDH was significantly reduced in tagged bou-2 samples; 350

however, its activity was not altered in the tagged mutant under elevated CO2 conditions 351

compared to tagged Col-0, indicating post-translational modification of FDH. The level of 352

AlaAT was slightly increased in the tagged mutant but showed only 60% activity compared 353

with tagged Col-0 under both elevated and ambient CO2 conditions. The activities of MDH, 354

AspAT, and GABA-T did not differ between tagged Col-0 and tagged bou-2 mutant under 355

elevated CO2 conditions, although these proteins were more abundant in tagged mutant 356

samples. The activity of GluDH was significantly increased in the tagged mutant compared 357

with tagged Col-0 under elevated CO2 conditions, which may be associated with the increased 358

amount of protein detected in the tagged bou-2 seedlings in our proteomic data set. However, 359

this increase was not statistically significant. 360



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Overall, we conclude that differences in the activities of MDH, AspAT, GluDH, AlaAT, GABA-361

T, and AlaAT measured in this study and that of GDC measured in a previous study most 362

likely do not relate to changes in protein abundance in mitochondria of tagged Col-0 vs. tagged 363

bou-2 mutant but instead might be caused by metabolic impairment or post-translational 364

modifications. 365

DISCUSSION 366

Recently, analyses of mitochondrial proteome content, complexome composition, post-367

translational modifications, energy metabolism, OXPHOS complex formation and function, 368

protein translocation, and metabolite shuttles have been conducted to further our 369

understanding of mitochondrial metabolism in Arabidopsis (König et al., 2014; Fromm et al., 370

2016; De Col et al., 2017; Rao et al., 2017; Senkler et al., 2017; Porcelli et al., 2018; Hu et al., 371

2019; Kolli et al., 2019; Meyer et al., 2019; Nickel et al., 2019). Many of these analyses 372

required the isolation of intact mitochondria. Here, we report a procedure for the rapid isolation 373

of HA-tagged mitochondria from tagged Arabidopsis lines via co-IP. Mitochondria isolated 374

using this method showed high enrichment of mitochondrial marker proteins with only minor 375

contamination, as demonstrated by immunoblot and quantitative proteomic analyses (Fig. 3, 376

Supplemental Table S1, Supplemental Table S2). 377

The method reported here enables the isolation of intact mitochondria from Arabidopsis 378

seedlings in less than 25 min (Fig. 2, Fig. 3, Fig. 4). Moreover, by omitting the first low-speed 379

centrifugation step, the method duration could be shortened to 18 min, although the resulting 380

mitochondrial fraction contained a higher level of other contaminating cellular components. 381

The mitochondrial fraction used for proteomic analyses in this study was obtained using the 382

slightly longer protocol that results in lower contamination with non-mitochondrial proteins. 383

Nevertheless, this isolation method is significantly faster than the standard isolation 384

procedures that generally take up to several hours. To date, we have been able to successfully 385

isolate mitochondria from whole seedlings, leaves, and roots (data not shown). Moreover, this 386

method could be used for the rapid isolation of mitochondria from other plant tissues such as 387

flowers and developing seeds because (1) the expression of the affinity tag is driven by the 388

ubiquitous UB10p promoter (Supplemental Fig. S1) and (2) only a small amount of starting 389

material (as low as 1 g) is needed. Additionally, this method is advantageous for the isolation 390

of mitochondria from very young tissues or mutants with growth defects or reduced biomass 391

accumulation. The rapid isolation method is superior to the standard isolation protocols with 392

respect to the yield of mitochondria; we were able to isolate 200 µg of total mitochondrial 393

protein from 1 g of starting material and up to 700 µg total mitochondrial protein from 10 g of 394

whole Arabidopsis seedlings. By contrast, the standard isolation protocols yield only 1.2 mg 395



14

mitochondria from 50 g of leaves (Keech et al., 2005). Generally, a higher yield is expected 396

using our isolation method, as the HA-tag has high affinity for its cognate antibody. However, 397

yield strongly depends on the amount of beads per gram tissue and the extract-to-bead ratio. 398

Further optimization of both may result in stable amounts of isolated mitochondria and even 399

higher yields. 400

The purity of affinity-purified mitochondria was similar to that of mitochondria isolated using 401

standard protocols including density gradients (Senkler et al., 2017). Immunoblot analysis 402

revealed only minor contamination with proteins of the ER or peroxisomes in affinity-purified 403

mitochondria (Fig. 3). These results were corroborated by quantitative proteomic analysis. 404

Less than 3% of all quantified proteins were assigned to peroxisomes, the ER, the Golgi, 405

vacuoles, endomembranes, plasma membranes, nuclei, and the cytosol. The highest 406

contamination was due to plastid-localized proteins (Supplemental Table S1, Supplemental 407

Table S2). However, we cannot exclude the possibility that some of the contaminants bound 408

non-specifically to the beads, despite extensive washing. Approximately 80% of the identified 409

proteins and 90% of the identified peptides showed mitochondrial localization (Supplemental 410

Table S1, Supplemental Table S2). Thus, we conclude that the purity of mitochondria isolated 411

using the rapid affinity purification method is comparable with that of mitochondria isolated 412

using traditional methods. Isolated mitochondria show respiratory coupling that is comparable 413

with previous isolations using traditional methods (Keech et al., 2005). Consistent with this 414

result, we could show that the mitochondrial marker CMXRos, a dye that solely stains 415

mitochondria with an intact respiratory chain, was retained in the mitochondria after affinity 416

purification. We could not efficiently elute the mitochondria from the magnetic beads with HA 417

peptide. However, mitochondria could be efficiently eluted using SDS-PAGE or detergent lysis 418

buffer. Elution of intact mitochondria might be achievable by integrating a protease cleavage 419

site between the HA-tag and GFP; this will be explored in future studies. 420

In our data set, we were able to identify 96% and 99% of the proteins detected by Klodmann 421

et al. (2011) and Senkler et al. (2017), respectively, in previous analyses of mitochondrial 422

proteomes and their complexome composition (Supplemental Table S1, Supplemental Table 423

S2). On the basis of a recent review on the composition and function of OXPHOS in 424

mitochondria (Meyer et al., 2019), we were able to identify 86% of all the predicted subunits 425

and assembly factors. To date, all of the proteins reviewed in Meyer et al. (2019) have not 426

been confirmed, and it might require an in-depth analysis or membrane enrichment to confirm 427

their presence in mitochondria in proteomic studies. Previously, it was reported that the 428

subunit ND4L is difficult to detect in proteomic studies because of its hydrophobic nature 429

(Peters et al., 2013). Consistent with this observation, we also could not identify this protein in 430

our proteome or in the list of quantified peptides (Supplemental Table S1, Supplemental Table 431



15

S2). However, we were able to detect all proteins of the TCA cycle (except two subunits of 432

Complex II), all GDC proteins, the majority of the proteins and subunits of the TIM/TOM protein 433

import apparatus, metabolite transporters of amino acids, dicarboxylic acids, cofactors, ions 434

and energy equivalents (e.g., BOU, BAC, UCP, DCT, SAMC, NDT, APC, AAC, and PHT), as 435

well as many proteins involved in protein synthesis/turnover and DNA/RNA metabolism 436

(Supplemental Table S1, Supplemental Table S2). Thus, our rapidly isolated mitochondria 437

showed good purity, integrity, and functionality. 438

Among all of the identified proteins, 20% could not be assigned to mitochondria. These 439

proteins included components of PSI and PSII; these could be clearly categorized as 440

contamination. However, we also identified proteins with unknown function and no clear 441

prediction of localization. In addition, we detected proteins such as GAPC, which was 442

previously shown to interact with the OMM protein VDAC (Schneider et al., 2018). Therefore, 443

we predict that some of the proteins classified as contaminants might represent novel 444

mitochondrial proteins, for example, as part of OMM protein complexes in the cytosol or as 445

components of complexes at organellar contact sites. However, this needs to be evaluated in 446

more detail in the future. 447

Application of the novel method to a mutant lacking the mitochondrial glutamate transporter 448

BOU resulted in the detection of surprisingly few changes in protein abundances compared 449

with tagged Col-0. Only 22 mitochondrial proteins showed a significant reduction in abundance 450

in the tagged bou-2 mutant, whereas five proteins were significantly increased in abundance. 451

However, only changes in a mitochondrial folylpolyglutamate synthetase (FGPS) and in FDH, 452

which contributes to the production of CO2 in the mitochondria by oxidizing formate, might be 453

connected to photorespiration. Formate is released from 10-formyl-THF by 10-formyl 454

deformylase, an enzyme involved in the maintenance of the THF pool in mitochondrial matrix 455

(Collakova et al., 2008). Reduced GDC activity in bou-2 might result in a lower production of 456

formate and finally a reduced abundance of FDH. FGPS is involved in vitamin B9 metabolism 457

by catalyzing the glutamylation of THF, a cofactor of GLDT and SHM (Hanson and Gregory, 458

2011). No significant changes were detected in the abundance of any of the proteins of the 459

GDC multienzyme system. SHM1, SHM2, and GDCH3 were slightly more abundant in the 460

mutant than tagged Col-0, whereas the others seem to be slightly reduced; however, this trend 461

did not meet the significance threshold (Table 2). The observed changes were not pronounced 462

and did not explain why the GDC activity was reduced to approximately 15% of the Col-0 level 463

in the bou-2 mutant. A possible explanation could be that Eisenhut and coworkers used 4-464

week-old rosettes (Eisenhut et al., 2013), whereas we used 10-day-old seedlings in the current 465

study. This possibility is supported by the observation that no difference in MDH activity could 466

be detected between 10-day-old mutant and tagged Col-0 seedlings (Fig. 5A). However, in 4-467



16

week-old leaves, the MDH activity was significantly reduced in the mutant compared with 468

tagged Col-0 (Supplemental Fig. S2). In contrast to GDC, the abundance of FDH was 469

significantly reduced in the mutant, whereas its activity was unaltered compared with tagged 470

Col-0 (Table 1, Fig. 5B). Previously, FDH was identified in a lysine acetylome study of 471

Arabidopsis mitochondria from 10-day-old Col-0 seedlings (König et al., 2014). This might 472

indicate that FDH activity is more likely regulated by post-translational modifications than by 473

protein abundance. 474

The BOU protein was recently assigned the function of a glutamate transporter (Porcelli et al., 475

2018). Glutamate is indirectly linked to photorespiration, as it is needed for the 476

polyglutamylation of THF, which increases its stability and promotes the activity of THF-477

dependent enzymes (Hanson and Gregory, 2011). However, in addition to BOU, 478

mitochondria-localized uncoupling proteins 1 and 2 show glutamate uptake activity in 479

Arabidopsis (Monné et al., 2018). This raises the question why knockout of the BOU gene 480

leads to a photorespiratory phenotype in young tissues, if BOU is not the only glutamate 481

transporter of mitochondria. Additionally, polyglutamylation is not restricted to mitochondria, 482

as Arabidopsis contains three isoforms of FGPS localized to mitochondria, plastids, and the 483

cytosol (Hanson and Gregory, 2011). Folate transporters prefer monoglutamylated forms of 484

THF, whereas enzymes generally prefer polyglutamylated forms (Suh et al., 2001). These 485

data indicate that BOU performs other functions, in addition to glutamate transport in vivo. We 486

exclude its function as a mitochondrial glutamate/glutamine shuttle, as BOU shows no 487

glutamine uptake activity (Porcelli et al., 2018). However, it is possible that BOU is involved in 488

folate metabolism, as folate biosynthesis occurs only in mitochondria (Hanson and Gregory, 489

2011). This possibility, however, needs to be investigated in future studies. 490

CONCLUSIONS 491

Our experiments show that, in addition to the analysis of protein-protein interactions, affinity-492

tagging is a powerful tool for the isolation of functional organelles from Arabidopsis. The 493

mitochondria isolated using this method showed high purity and integrity. Future studies will 494

be required to efficiently elute mitochondria from magnetic beads and to determine the 495

applicability of this method for metabolite analyses. To conduct metabolite analyses, it is 496

encouraging that the LC/MS-compatible buffer system developed previously for mammalian 497

mitochondria (Chen et al., 2016) can also be used for the isolation of mitochondria from plant 498

tissues. Expressing the affinity tag under the control of cell-specific promoters will allow the 499

isolation of mitochondria from specific cell types, such as meristems or guard cells (Yang et 500

al., 2008; Schürholz et al., 2018). The use of cell-specific promoters in our construct will help 501

unravel the complex role of mitochondria in various cell types. We expect that similar tagging 502



17

strategies will be applicable to other plant cell organelles, such as plastids and peroxisomes. 503

Moreover, simultaneous expression of several different affinity tags will facilitate the affinity 504

purification of different organelles from a single extract. 505

MATERIALS AND METHODS 506

Plant growth conditions 507

Arabidopsis ecotype Col-0 and bou-2 mutant (GABI-Kat line number 079D12; http://www.gabi-508

kat.de/db/lineid.php) (Kleinboelting et al., 2012; Eisenhut et al., 2013) were used in this study. 509

Seeds were sterilized by washing with 70% (v/v) ethanol supplemented with 1% (v/v) Triton 510

X-100 twice for 10 min each, followed by washing with 100% ethanol twice for 10 min each. 511

Seeds were grown on half-strength Murashige and Skoog (1/2 MS) medium (pH 5.7) 512

supplemented with 0.8% (w/v) agar. Seeds were subjected to cold stratification for 2 days at 513

4°C. After germination, seedlings were grown for 10 days under a 12-h light/12-h dark 514

photoperiod under 100 µmol m-2 s-1 light intensity and 3,000 ppm CO2-enriched atmosphere, 515

unless otherwise stated. 516

Measurement of dry weight 517

10-day-old Arabidopsis Col-0, tagged Col-0, bou-2, and tagged bou-2 were collected in 1.5-518

ml microcentrifuge tubes, dried for 3 days at 70°C, and then weighed. 519

4-week-old Arabidopsis Col-0 and tagged Col-0 rosettes grown at ambient CO2 conditions 520

were collected in paper bags, dried for 5 days at 70°C, and then weighed. 521

Construction of transgenic lines 522

The HA epitope-tag was amplified with a start codon from the Gateway binary vector pGWB15 523

(GenBank accession AB289778). The coding sequence (CDS) of sGFP, minus the start and 524

stop codons but including a linker peptide (GGSG) at the 5' and 3' ends, was amplified from 525

the Gateway binary vector pGWB4 (GenBank accession AB289767). The CDS of TOM5 526

(At5g08040; minus the start codon) was amplified from Arabidopsis cDNA. Starting from the 527

5'-end to the 3'-end, the amplified 3HA-tag, sGFP, and TOM5 were cloned into the pUTKan 528

vector (Krebs et al., 2012) under the control of the Arabidopsis UB10p using restriction 529

endonucleases. The construct was introduced into Agrobacterium tumefaciens, strain 530

GV3101::pMP90 (Koncz and Schell, 1986), which was then introduced into Col-0 and bou-2 531

plants via Agrobacterium-mediated transformation using the floral dip method, as described 532

previously (Clough and Bent, 1998). Homozygotes of the T3 generation were used for 533

analyses. 534



18

The generated vector pUTKan-3HA-sGFP-TOM5 has been deposited on Addgene (ID 535

130674). 536

Confocal laser scanning microscopy 537

The expression of 3HA-sGFP-TOM5 was verified via confocal laser scanning microscopy 538

using the Zeiss LSM 78 Confocal Microscope and Zeiss ZEN software. The Col-0 and bou-2 539

seedlings regenerated from independent transformation events were incubated with 200 nM 540

MitoTracker Red CMXRos (Molecular Probes) in 1/2 MS supplemented with 3% (w/v) sucrose 541

for 15 min. Images were captured using the following excitation/emission wavelengths: sGFP 542

(488 nm/490–550 nm) and MitoTracker Red CMXRos (561 nm/580–625 nm). Pictures were 543

processed using the ImageJ software (https://imagej.nih.gov/ij/). 544

Rapid isolation of mitochondria using co-IP 545

Epitope-tagged mitochondria were rapidly isolated using co-IP, as described previously (Chen 546

et al., 2016). Briefly, 1–10 g of Arabidopsis seedlings were harvested and homogenized in 40 547

ml KPBS (10 mM KH2PO4 [pH 7.25] and 136 mM KCl) for twice 5 s at low speed and 15 s at 548

high speed in a Warren blender. The resulting homogenate was filtered through three layers 549

of miracloth supported by a nylon mesh (pore size 40 µm) and centrifuged at 2,500 g for 5 550

min. The pellet containing cell debris and chloroplasts was discarded. The supernatant was 551

subsequently centrifuged at 20,000 g for 9 min. The pellet representing the crude 552

mitochondrial fraction was resuspended in 1 ml KPBS using a fine paintbrush and 553

homogenized with three strokes using a Potter-Elvehjem (B. Braun, Melsungen). Crude 554

mitochondria were incubated with 50–250 µl pre-washed magnetic anti-HA beads 555

(ThermoFisher Scientific) on an end-over-end rotator for 5 min in 1.5-ml microcentrifuge tubes. 556

Magnetic beads were pre-washed according to the manufacturer’s instructions using 557

phosphate-buffered saline (PBS) supplemented with 0.5% (v/v) Triton X-114. Magnetic beads 558

were separated using a magnetic stand (DYNALTM, DynaMag-2 Magnet, InvitrogenTM) and 559

washed at least three times with each 1 ml KPBS. The purified mitochondria were lysed using 560

50–250 µl mitochondria lysis buffer (50 mM TES/KOH [pH 7.5], 2 mM EDTA, 5 mM MgCl2, 561

10% [v/v] glycerol, and 0.1% [v/v] Triton X-100) for enzyme activity assays and immunoblot 562

analysis or directly frozen in liquid nitrogen for proteome analyses. All steps were carried out 563

at 4°C. The amount of protein recovered after lysis was determined using the Quick StartTM 564

Bradford Protein Assay Kit (Bio-Rad), with bovine serum albumin (BSA) as the standard. 565

Chlorophyll content of rapidly enriched mitochondria was measured as previously described 566

(Porra, 2002). 567



19

Isolation of mitochondria using the traditional approach 568

Mitochondria were isolated from 10-day-old Col-0 seedlings via differential centrifugation and 569

Percoll gradient purification, as described previously (Kühn et al., 2015). 570

Immunoblot analysis 571

25 µg of total leaf extract and 6.45 µg of isolated mitochondria fractions were heated at 96°C 572

in SDS-PAGE loading buffer for 10 min and separated on 12% SDS-polyacrylamide gels 573

(Laemmli, 1970). Proteins were transferred to 0.2-µm polyvinylidene difluoride membranes 574

(PVDF; BioRad) or 0.45-µm nitrocellulose membranes (ThermoScientific) using standard 575

protocols. Protein transfer was verified by staining the membranes with Ponceau S red (Sigma 576

Aldrich). Membranes were blocked according to the manufacturer’s instructions for 1 h, 577

washed with Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST), and subsequently 578

incubated with either a primary antibody or a single-step antibody overnight at 4°C. Antibodies 579

against marker proteins were diluted as follows: anti-AOX (1:1,000), anti-IDH (1:5,000), anti-580

VDAC1 (1:5,000), anti-HA-HRP (1:5,000; Miltenyi Biotec), anti-RbcL (1:7,500), anti-Cat 581

(1:1,000), anti-BiP2 (1:2,000), anti-Histone H3 (1:5,000), and anti-HSC70 (1:3,000). 582

Membranes were washed with TBST twice for 10 min each and incubated with the secondary 583

goat anti-Rabbit-HRP antibody (1:5,000; Merck Millipore) at room temperature for 1 h or at 584

4°C overnight. Subsequently, membranes were washed with TBST five times for 5 min each 585

and visualized using a chemiluminescence detection system (Immobilon Western HRP 586

Substrate, Merck Millipore). All steps were carried out with PBS, when using anti-Cat antibody. 587

Antibodies were purchased from Agrisera if not stated otherwise. 588

Seahorse XFe96 Extracellular Flux Analyzer 589

Mitochondria were isolated from 10-day-old tagged Col-0 as described above. Purified 590

mitochondria bound to magnetic anti-HA beads were resuspended in mitochondria assay 591

buffer for Seahorse Analyzer measurement (70 mM sucrose, 220 mM mannitol, 10 mM 592

KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% [w/v] fatty acid-free BSA, pH 7.2 at 593

37°C) to a concentration of 0.2 µg/µl. Each 20-µl mitochondria suspension (4 µg total 594

mitochondrial protein) per well was analyzed in a cell culture plate for Seahorse Analyzer using 595

an Agilent Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies). Mitochondria 596

were attached to the bottom of the plate using a magnetic stand. Subsequently, mitochondria 597

assay buffer for Seahorse Analyzer measurement including succinate as substrate (final 598

concentration 10 mM) was carefully added. Assay protocol was adapted from the Agilent 599

Seahorse XF Cell Mito Stress Test manual. First basal respiration with succinate as substrate 600

was measured in the presence or absence of Antimycin A (final concentration 4 µM; Sigma 601

Aldrich), following injection of Antimycin A (to a final concentration of 4 µM) to inhibit the 602



20

respiratory chain. Cartridge hydration overnight was performed according to the 603

manufacturer’s instruction. As control isolations were performed on 10-day-old Col-0 604

seedlings. 605

Recovery 606

Mitochondrial recovery was determined based on cytochrome c oxidase activity in 607

homogenized extract and isolated mitochondria via co-IP, as described previously (Lai et al., 608

2019). Cytochrome c oxidase activity was measured by following the oxidation of cytochrome 609

c at 550 nm (Spinazzi et al., 2012). The assay mixture contained 50 mM potassium phosphate 610

buffer (pH 7.0), 40 µM fully reduced cytochrome c and 0.1–0.5 µg total mitochondrial protein 611

or 5–10 µg total extract protein. 612

Staining of intact mitochondria 613

In mitochondria staining experiments purified mitochondria bound to 100 µl magnetic anti-HA 614

beads were incubated for 15 min in 1 ml KPBS supplemented with 200 nM MitoTracker Red 615

CMXRos in the dark on an end-over-end rotator. Magnetic beads were washed three times 616

with KPBS. Mitochondria were lysed using 1 ml mitochondria lysis buffer. Released 617

fluorescence was measured using a fluorescence spectrophotometer (SynergyH1, BioTek) 618

with the following excitation/emission settings: 570 nm/600–650 nm. 619

Latency of MDH activity 620

To assess latency of MDH activity purified mitochondria bound to 100 µl magnetic anti-HA 621

beads were first eluted in 100 µl KPBS followed by elution in 100 µl KPBS supplemented with 622

0.1% (v/v) Triton X-114. MDH activity in both elution fractions was measured as described 623

previously (Tomaz et al., 2010). 624

Enzyme assays 625

Activities of mitochondrial enzymes were measured using a plate-reader spectrophotometer 626

(SynergyH1, BioTek), based on the absorbance at 340 nm. Mitochondria were enriched using 627

150 µl magnetic anti-HA beads and lysed in 200–250 µl mitochondrial lysis buffer. The activity 628

of MDH was measured based on the oxidation of NADH to NAD+ at 340 nm, as described 629

previously (Tomaz et al., 2010). The reaction mixture contained 50 mM KH2PO4 (pH 7.5), 0.2 630

mM NADH, 5 mM EDTA, 10 mM MgCl2, 2 mM OAA (Tomaz et al., 2010), and 0.1–0.4 µg total 631

mitochondrial protein. AspAT activity was measured in a reaction coupled with MDH, as 632

described previously (Wilkie and Warren, 1998), with 0.3–1 µg total mitochondrial protein per 633

assay. No external pyridoxal-5’-phosphate was added to the reaction mixture. GluDH activity 634

was measured as described previously (Turano et al., 1996), based on the reduction of NAD+ 635



21

to NADH at 340 nm. To determine the amination activity, 0.5–2 µg total mitochondrial protein 636

was used per assay. GABA-T activity was measured in a reaction coupled with succinate-637

semialdehyde dehydrogenase (SSADH), as described previously (Clark et al., 2009). The 638

assay buffer contained 50 mM TAPS (pH 9), 0.2 mM NAD+, 0.625 mM EDTA, 8 mM GABA, 2 639

mM pyruvate, 1 U/ml SSADH, and 2–5 µg total mitochondrial protein. The NAD+-dependent 640

SSADH was purified from Escherichia coli, as described previously (Clark et al., 2009). The 641

recombinant purified protein catalyzed the production of NADH with a specific activity of 1.6 642

U/mg protein. AlaAT activity was measured in reaction coupled with lactate dehydrogenase, 643

as described previously (Miyashita et al., 2007), with 1–5 µg total mitochondrial protein. FDH 644

activity was measured based on the reduction of NAD+ to NADH at 340 nm and 30°C, with 1–645

5 µg total mitochondrial protein. The assay buffer contained 100 mM potassium phosphate 646

buffer (pH 7.5), 1 mM NAD+, and 50 mM sodium formate. 647

Sample preparation for LC/MS analysis 648

To elute proteins from magnetic beads, 30 µl of Laemmli buffer was added to the reaction 649

mixture, and samples were incubated at 95°C for 10 min. Subsequently, 20 µl of protein 650

sample was loaded on an SDS-polyacrylamide gel for in-gel digestion. The isolated gel pieces 651

were reduced using 50 µl of 10 mM DTT, then alkylated using 50 µl of 50 mM iodoacetamide, 652

and finally digested using 6 µl of trypsin (200 ng) in 100 mM ammonium bicarbonate. The 653

peptides were resolved in 15 µl of 0.1% (v/v) trifluoracetic acid and subjected to LC/MS 654

analysis. 655

LC/MS analysis 656

The LC/MS analysis was performed on a Q Exactive Plus mass spectrometer (Thermo 657

Scientific, Bremen, Germany) connected to an Ultimate 3000 Rapid Separation LC system 658

(Dionex; Thermo Scientific, Idstein, Germany) and equipped with an Acclaim PepMap 100 659

C18 column (75 µm inner diameter 25 cm length 2 mm particle size; Thermo Scientific, 660

Bremen, Germany). The length of the isocratic LC gradient was 120 min. The mass 661

spectrometer was operated in positive mode and coupled with a nano electrospray ionization 662

source. Capillary temperature was set at 250°C, and source voltage was set at 1.4 kV. The 663

survey scans were conducted at a mass to charge (m/z) of 200–2000 and a resolution of 664

70,000. The automatic gain control was set at 3,000,000, and the maximum fill time was set 665

at 50 ms. The ten most intensive peptide ions were isolated and fragmented by high-energy 666

collision dissociation (HCD). 667



22

Computational MS data analysis 668

Peptide and protein identification and quantification was performed using MaxQuant version 669

1.5.5.1 (MPI for Biochemistry, Planegg, Germany) with default parameters. The identified 670

Arabidopsis peptides and proteins were queried against a specific proteome database 671

(UP0000006548, downloaded 12/11/17) from UniProt. The oxidation and acetylation of 672

methionine residues at the N-termini of proteins were set as variable modifications, whereas 673

carbamidomethylations at cysteine residues were considered as fixed modification. Peptides 674

and proteins were accepted with a false discovery rate of 1%. Unique and razor peptides were 675

used for label-free quantification, and peptides with variable modifications were included in 676

the quantification. The minimal ratio count was set to two, and the ‘matched between runs’ 677

option was enabled. 678

Normalized intensities, as provided by MaxQuant, were analyzed using the Perseus 679

framework (version 1.5.0.15; MPI for Biochemistry, Planegg, Germany, Tyanova et al., 2016). 680

Only proteins containing at least two unique peptides and a minimum of three valid values in 681

at least one group were quantified. Proteins which were identified only by site or marked as a 682

contaminant (from the MaxQuant contaminant list) were excluded from the analysis. 683

Differential enrichment of proteins in the two groups (Col-0; bou-2) was assessed using 684

Student’s t-test. Significance analysis was applied on log2-transformed values using an S0 685

constant of 0 and a false discovery rate of 5%, as threshold cutoffs (Benjamini and Hochberg, 686

1995). 687

The MS proteomics data has been deposited to the ProteomeXchange Consortium via the 688

PRIDE partner repository with the data set identifier PXD014137. 689

Accession numbers 690

Sequence data from this article can be found in the GenBank/EMBL data libraries under 691

accession numbers AAF82782 (AlaAT1), AAF82781 (AlaAT2), AEC08469 (AspAT), 692

OAO95225 (BOU), AED92076 (FDH), AEE76603 (GABA-T), AED92515 (GluDH1), 693

AED91158 (GluDH2), AEE74011 (GluDH3), BAF44962 (sGFP), Q9ZP06 (mMDH1), Q9LKA3 694

(mMDH2), and OAO94076 (TOM5). 695

SUPPLEMENTAL DATA 696

Supplemental Figure S1. Schematic representation of the vector used to label mitochondria 697

with triple HA-tag. 698

Supplemental Figure S2. Phenotypic analysis of transgenic lines 699



23

Supplemental Figure S3. Original images of all immunoblots related to Figure 3. 700

Supplemental Figure S4. Activity of malate dehydrogenase (MDH) in mitochondria rapidly 701

isolated from 4-week-old Col-0 and bou-2 leaves expressing the UB10p-3HA-sGFP-TOM5 702

construct. 703

Supplemental Table S1. List of proteins identified and quantified in 10-day-old Col-0 and bou-704

2 plants expressing the 3HA-sGFP-TOM5 protein. 705

Supplemental Table S2. List of raw intensities and reliability of all quantified peptides. 706

707

ACKNOWLEDGEMENTS 708

This work was supported by the Deutsche Forschungsgemeinschaft (CRC 1208 and funding 709

under Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111“). 710

711

TABLES 712

Table 1: List of signal changes for proteins showing significant differences in protein 713

abundance between 10-day-old Col-0 UB10p-3HA-sGFP-TOM5 and bou-2 UB10p-714

3HA-sGFP-TOM5. Difference was calculated as change of log2 of normalized intensity. List 715

includes only proteins that show mitochondrial localization. List ranges from most 716

downregulated in bou-2 UB10p-3HA-sGFP-TOM5 (top) to most upregulated bou-2 UB10p-717

3HA-sGFP-TOM5 (bottom). Significance was calculated with Student’s t-test, *P < 0.05, ** P 718

< 0.01, *** P < 0.001. 719

AGI Gene symbol Gene description Difference Significance

AT5G46800 BOU Mitochondrial substrate carrier family

protein -8,66407

***

AT2G42310 AT2G42310 ESSS subunit of NADH:ubiquinone

oxidoreductase (complex I) protein -2,0953

*

AT5G41685 AT5G41685 Mitochondrial outer membrane

translocase complex, subunit Tom7 -1,90711

**

AT3G03100 AT3G03100 NADH:ubiquinone oxidoreductase,

17.2kDa subunit -1,33356

*



24

AT5G53650 AT5G53650 ABC transporter A family protein -1,25252 *

AT5G67590 FRO1 NADH-ubiquinone oxidoreductase-

like protein -1,0968

*

AT5G40810 AT5G40810 Cytochrome C1 family -1,06444 *

AT3G27280 PHB4 prohibitin 4 -0,94888 *

AT3G27380 SDH2-1 succinate dehydrogenase 2-1 -0,764682 *

AT4G37660 AT4G37660

Ribosomal protein L12/ ATP-

dependent Clp protease adaptor

protein ClpS family protein

-0,732468

*

AT2G42210 OEP16-3

Mitochondrial import inner membrane

translocase subunit

Tim17/Tim22/Tim23 family protein

-0,710023

*

AT3G55400 OVA1

methionyl-tRNA synthetase /

methionine-tRNA ligase / MetRS

(cpMetRS)

-0,705525

*

AT5G14780 FDH formate dehydrogenase -0,697987 **

AT2G38670 PECT1 phosphorylethanolamine

cytidylyltransferase 1 -0,606253

*

AT5G63400 ADK1 adenylate kinase 1 -0,603703 *

AT1G79230 MST1 mercaptopyruvate sulfurtransferase 1 -0,587758 *

AT4G30010 AT4G30010 ATP-dependent RNA helicase -0,545131 *

AT3G03420 AT3G03420 Ku70-binding family protein -0,531796 **

AT4G31810 AT4G31810 ATP-dependent caseinolytic (Clp)

protease/crotonase family protein -0,461768

*

AT1G19140 AT1G19140 ubiquinone biosynthesis COQ9-like

protein -0,455841

*

AT4G31460 AT4G31460 Ribosomal L28 family -0,430804 *



25

AT1G54220 AT1G54220 Dihydrolipoamide acetyltransferase,

long form protein -0,324755

*

AT3G59820 LETM1 LETM1-like protein 0,42657 *

AT5G27540 MIRO1 MIRO-related GTP-ase 1 0,447933 *

AT3G10160 DFC DHFS-FPGS homolog C 0,507662 ***

AT1G71260 ATWHY2 WHIRLY 2 0,51931 *

AT5G15980 AT5G15980 Pentatricopeptide repeat (PPR)

superfamily protein 0,8335

*

720

721



26

Table 2: List of changes in protein abundance of glycine decarboxylase proteins, serine 722

hydroxymethyltransferase (SHM) and the enzymes malate dehydrogenase (MDH), 723

formate dehydrogenase (FDH), aspartate aminotransferase (ASP), γ-aminobutyric acid 724

transaminase (POP2), alanine aminotransferase (AlaAT) and glutamate dehydrogenase 725

(GDH). Difference was calculated as change of log2 of normalized intensity. Significance was 726

calculated with Student’s t-test, P < 0.05. 727

AGI Gene symbol Gene description Difference Significant

AT4G33010 GLDP1 glycine decarboxylase P-protein 1 -0,373902 no

AT2G26080 GLDP2 glycine decarboxylase P-protein 2 -0,155379 no

AT2G35370 GDCH1 glycine decarboxylase H-protein 1 -0,760396 no

AT1G32470 GDCH3 glycine decarboxylase H-protein 3 0,173202 no

AT1G11860 GLDT Glycine cleavage T-protein family -0,164196 no

AT1G48030 GDCL1 mitochondrial lipoamide

dehydrogenase 1 -0,0284281 no

AT3G17240 GDCL2 mitochondrial lipoamide

dehydrogenase 2 -0,217162 no

AT4G37930 SHM1 serine hydroxymethyltransferase 1 0,109162 no

AT5G26780 SHM2 serine hydroxymethyltransferase 2 0,696879 no

AT5G14780 FDH formate dehydrogenase -0,697987 yes

AT5G18170 GDH1 glutamate dehydrogenase 1 0,91113 no

AT5G07440 GDH2 glutamate dehydrogenase 2 0,105005 no

AT3G22200 POP2 γ-aminobutyric acid transaminase 0,345683 no

AT1G17290 AlaAT1 alanine aminotransferase 1 0,640894 no

AT1G72330 AlaAT2 alanine aminotransferase 2 0,223008 no

AT2G30970 ASP1 aspartate aminotransferase 1 0,399954 no



27

AT1G53240 mMDH1 Lactate/malate dehydrogenase

family protein 0,564951 no

AT3G15020 mMDH2 Lactate/malate dehydrogenase

family protein 0,0343099 no

728

FIGURE LEGENDS 729

Figure 1: Confocal microscopy of epitope-tagged mitochondria in leaf and root tissues 730

of 10-day-old Arabidopsis Col-0 and bou-2 lines expressing the UB10p-3HA-sGFP-731

TOM5 construct. (A–D) Images of tagged Col-0 leaf (A) and root (B) tissues and tagged bou-732

2 leaf (C) and root (D) tissues expressing the 3HA-sGFP-TOM5 protein. Pictures were taken 733

of the epidermal layer of the first leaf pair (A, C) and the epidermal and cortex layer of the 734

main root (B, D). Green color represents GFP signal, whereas red color represents the signal 735

of mitochondrial marker MitoTracker™ Red CMXRos. Bright field is shown in grey and merged 736

images are shown in yellow. Scale bars = 10 µm. 737

Figure 2: Workflow showing the rapid isolation of epitope-tagged mitochondria via co-738

Immunopurification (co-IP) from Arabidopsis. Tagged lines harboring the UB10p-3HA-739

sGFP-TOM5 construct were harvested and homogenized in a Warren blender. The extract 740

was filtered and centrifuged to obtain a crude mitochondrial fraction. Epitope-tagged 741

mitochondria were purified via co-IP using 50–250 µl magnetic anti-HA beads. The purified 742

mitochondria were washed and either lysed for immunoblot analysis or extracted for 743

proteomics. As a control, non-tagged Col-0 was used. 744

Figure 3: Assessment of the contamination of enriched mitochondria via co-IP or 745

classical methods. Mitochondria isolations were performed on 10-day-old Col-0 and tagged 746

Col-0 lines via co-IP using 150 µl magnetic anti-HA beads or on 10-day-old Col-0 via classical 747

methods using differential centrifugation and gradient purification. Samples were taken after 748

tissue homogenization (Whole cell, Extract), enrichment of mitochondria via co-IP (Anti-HA-749

IP), differential centrifugation (DC) and density gradient purification (Purified). Protein amounts 750

were loaded as described in Materials and Methods. Names of organelle marker proteins are 751

shown on the left side of the blots, and their subcellular localization is indicated on the right. 752

Figure 4: Intactness of rapidly enriched mitochondria isolated from 10-day-old tagged 753

Col-0 lines via co-IP. Oxygen consumption rates (OCR) of purified mitochondria bound to 754

magnetic anti-HA beads were measured using Seahorse XFe96 Analyzer as described in 755

Materials and Methods (A). OCR were measured in the presence (black boxes) or absence 756



28

(blue boxes) of the respiratory chain inhibitor Antimycin A and before (b. i.) and after (a. i.) 757

injection of Antimycin A. Control (grey boxes) did not contain mitochondria. Data are shown in 758

Box and whiskers (Min to Max) of means of two (Mitochondria + Antimycin A, Control) and 759

three biological replicates (Mitochondria). Different letters indicate statistically significant 760

differences between means (P < 0.05; two-way ANOVA). Latency of malate dehydrogenase 761

(MDH) activity was measured as described in Materials and Methods (B). As control isolations 762

were performed on Col-0 lines. Data represent mean ± SD of three biological replicates (each 763

four technical replicates). Different letters indicate statistically significant differences between 764

means (P < 0.05; one-way ANOVA). Purified mitochondria retain the mitochondrial marker 765

MitoTracker™ Red CMXRos that is able to stain mitochondria with an active membrane 766

potential (C). Staining experiments were performed as described in Materials and Methods. 767

As control isolations were performed on Col-0 lines. Data represent mean ± SD of three 768

biological replicates (each four technical replicates). Asterisks indicate statistically significant 769

differences (***, P < 0.001; Student’s t-test). 770

Figure 5: Characterization of enzyme activities in mitochondria isolated via co-IP from 771

10-day-old tagged Col-0 and bou-2 lines. Plants were sampled following growth under 3,000 772

ppm CO2 (HC) and 5 days after shift to 380 ppm CO2 (5d LC). (A) Malate dehydrogenase 773

(MDH) activity. Average tagged Col-0 activity: 38.2 ± 1.5 µmol min-1 mg-1 (HC), 49.4 ± 1.5. 774

µmol min-1 mg-1 (5d LC). (B) Formate dehydrogenase (FDH) activity. Average tagged Col-0 775

activity: 17.8 ± 0.5 nmol min-1 mg-1 (HC), 33.6 ± 1.5 nmol min-1 mg-1 (5d LC). (C) Aspartate 776

aminotransferase (AspAT) activity. Average tagged Col-0 activity: 4.31 ± 0.08 µmol min-1 mg-777

1 (HC), 3.74 ± 0.01 µmol min-1 mg-1 (5d LC). (D) γ-aminobutyric acid transaminase (GABA-T) 778

activity. Average tagged Col-0 activity: 96.2 ± 4.3 nmol min-1 mg-1 (HC), 137.4 ± 1.0 nmol min-779

1 mg-1 (5d LC). (E) Alanine aminotransferase (AlaAT) activity. Average tagged Col-0 activity: 780

0.92 ±0.02 µmol min-1 mg-1 (HC), 0.75 ± 0.04 µmol min-1 mg-1 (5d LC). (F) Glutamate 781

dehydrogenase (GluDH) activity. Average tagged Col-0 activity: 0.83 ± 0.05 µmol min-1 mg-1 782

(HC), 2.03 ± 0.04 µmol min-1 mg-1 (5d LC). Activities were calculated from initial slopes. 783

Enzyme activities in tagged Col-0 grown under elevated CO2 conditions and five days after 784

shift to ambient CO2 conditions were each set to 100%. Data represent mean ± SD of three 785

biological replicates (each four technical replicates). Different letters indicate statistically 786

significant differences between means for each enzyme (P < 0.05; one-way ANOVA). 787



29

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Figure 1

Figure 1: Confocal microscopy of epitope-tagged mitochondria in leaf and root tissues of 10-day-old Arabidopsis Col-0 and bou-2 lines expressing the UB10p-3´HA-sGFP-TOM5construct. (A–D) Images of tagged Col-0 leaf (A) and root (B) tissues and tagged bou-2 leaf (C)and root (D) tissues expressing the 3´HA-sGFP-TOM5 protein. Pictures were taken of theepidermal layer of the first leaf pair (A, C) and the epidermal and cortex layer of the main root (B, D).Green color represents GFP signal, whereas red color represents the signal of mitochondrial markerMitoTracker™ Red CMXRos. Bright field is shown in grey and merged images are shown in yellow.Scale bars = 10 µm.

A

B

C

D

HA-GFP-TOM5 CMXRos Brightfield Merge



Figure 2

Figure 2: Workflow showing the rapid isolation of epitope-tagged mitochondria via co-Immunopurification (co-IP) from Arabidopsis. Tagged lines harboring the UB10p-3´HA-sGFP-TOM5construct were harvested and homogenized in a Warren blender. The extract was filtered and centrifuged toobtain a crude mitochondrial fraction. Epitope-tagged mitochondria were purified via co-IP using 50–250 µlmagnetic anti-HA beads. The purified mitochondria were washed and either lysed for immunoblot analysis orextracted for proteomics. As a control, non-tagged Col-0 was used.



Figure 3

Figure 3: Assessment of the contamination of enriched mitochondria via co-IP or classicalmethods. Mitochondria isolations were performed on 10-day-old Col-0 and tagged Col-0 lines via co-IPusing 150 µl magnetic anti-HA beads or on 10-day-old Col-0 via classical methods using differentialcentrifugation and gradient purification. Samples were taken after tissue homogenization (Whole cell,Extract), enrichment of mitochondria via co-IP (Anti-HA-IP), differential centrifugation (DC) and densitygradient purification (Purified). Protein amounts were loaded as described in Materials and Methods.Names of organelle marker proteins are shown on the left side of the blots, and their subcellularlocalization is indicated on the right.

Extrac

t

DC PurifiedWhole cell

Anti-HA IP

Col-0tagged Col-0 Classical isolation

+ +

+ +

AOX

IDH

VDAC1

RbcL

BiP

Cat

Histone H3

HSC70

HA

Inner mitochondrial membrane

Mitochondrial matrix

Outer mitochondrial membrane

Plastid

Endoplasmatic Reticulum

Peroxisomes

Nucleus

Cytosol

HA-GFP-TOM5



Figure 4

Figure 4: Intactness of rapidly enriched mitochondria isolated from 10-day-old tagged Col-0 linesvia co-IP. Oxygen consumption rates (OCR) of purified mitochondria bound to magnetic anti-HA beadswere measured using Seahorse XFe96 Analyzer as described in Materials and Methods (A). OCR weremeasured in the presence (black boxes) or absence (blue boxes) of the respiratory chain inhibitorAntimycin A and before (b. i.) and after (a. i.) injection of Antimycin A. Control (grey boxes) did not containmitochondria. Data are shown in Box and whiskers (Min to Max) of means of two (Mitochondria +Antimycin A, Control) and three biological replicates (Mitochondria). Different letters indicate statisticallysignificant differences between means (P < 0.05; two-way ANOVA). Latency of malate dehydrogenase(MDH) activity was measured as described in Materials and Methods (B). As control isolations wereperformed on Col-0 lines. Data represent mean ± SD of three biological replicates (each four technicalreplicates). Different letters indicate statistically significant differences between means (P < 0.05; one-wayANOVA). Purified mitochondria retain the mitochondrial marker MitoTracker™ Red CMXRos that is ableto stain mitochondria with an active membrane potential (C). Staining experiments were performed asdescribed in Materials and Methods. As control isolations were performed on Col-0 lines. Data representmean ± SD of three biological replicates (each four technical replicates). Asterisks indicate statisticallysignificant differences (***, P < 0.001; Student’s t-test).

A

B C***



Figure 5

Figure 5: Characterization of enzyme activities in mitochondria isolated via co-IP from 10-day-oldtagged Col-0 and bou-2 lines. Plants were sampled following growth under 3,000 ppm CO2 (HC) and 5days after shift to 380 ppm CO2 (5d LC). (A) Malate dehydrogenase (MDH) activity. Average tagged Col-0activity: 38.2 ± 1.5 µmol min-1 mg-1 (HC), 49.4 ± 1.5. µmol min-1 mg-1 (5d LC). (B) Formate dehydrogenase(FDH) activity. Average tagged Col-0 activity: 17.8 ± 0.5 nmol min-1 mg-1 (HC), 33.6 ± 1.5 nmol min-1 mg-1(5d LC). (C) Aspartate aminotransferase (AspAT) activity. Average tagged Col-0 activity: 4.31 ± 0.08 µmolmin-1 mg-1 (HC), 3.74 ± 0.01 µmol min-1 mg-1 (5d LC). (D) γ-aminobutyric acid transaminase (GABA-T)activity. Average tagged Col-0 activity: 96.2 ± 4.3 nmol min-1 mg-1 (HC), 137.4 ± 1.0 nmol min-1 mg-1 (5dLC). (E) Alanine aminotransferase (AlaAT) activity. Average tagged Col-0 activity: 0.92 ±0.02 µmol min-1mg-1 (HC), 0.75 ± 0.04 µmol min-1 mg-1 (5d LC). (F) Glutamate dehydrogenase (GluDH) activity. Averagetagged Col-0 activity: 0.83 ± 0.05 µmol min-1 mg-1 (HC), 2.03 ± 0.04 µmol min-1 mg-1 (5d LC). Activitieswere calculated from initial slopes. Enzyme activities in tagged Col-0 grown under elevated CO2 conditionsand five days after shift to ambient CO2 conditions were each set to 100%. Data represent mean ± SD ofthree biological replicates (each four technical replicates). Different letters indicate statistically significantdifferences between means for each enzyme (P < 0.05; one-way ANOVA).

A B C

D E F



Parsed CitationsBenjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R StatSoc Ser B 57: 289–300

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Chen WW, Freinkman E, Wang T, Birsoy K, Sabatini DM (2016) Absolute Quantification of Matrix Metabolites Reveals the Dynamics ofMitochondrial Metabolism. Cell 166: 1324-1337.e11


Clark SM, Di Leo R, Dhanoa PK, Van Cauwenberghe OR, Mullen RT, Shelp BJ (2009) Biochemical characterization, mitochondriallocalization, expression, and potential functions for an Arabidopsis gamma-aminobutyrate transaminase that utilizes both pyruvate andglyoxylate. J Exp Bot 60: 1743–57


Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J16: 735–43


De Col V, Fuchs P, Nietzel T, Elsässer M, Voon CP, Candeo A, Seeliger I, Fricker MD, Grefen C, Møller IM, et al (2017) ATP sensing inliving plant cells reveals tissue gradients and stress dynamics of energy physiology. Elife 6: 1–29


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https://www.ncbi.nlm.nih.gov/pubmed?term=Meyer EH%2C Welchen E%2C Carrie C %282019%29 Assembly of the Complexes of the Oxidative Phosphorylation System in Land Plant Mitochondria%2E Annu Rev Plant Biol 70%3A 23%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Millar AH%2C Eubel H%2C J�nsch L%2C Kruft V%2C Heazlewood JL%2C Braun H%2DP %282004%29 Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits%2E Plant Mol Biol 56%3A 77%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Millar AH%2C Sweetlove LJ%2C Gieg� P%2C Leaver CJ %282001%29 Analysis of the Arabidopsis mitochondrial proteome%2E Plant Physiol 127%3A 1711%26%23x&dopt=abstract

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https://scholar.google.com/scholar?as_q=Analysis of the Arabidopsis mitochondrial proteome.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Millar&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Miyashita Y%2C Dolferus R%2C Ismond KP%2C Good AG %282007%29 Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana%2E Plant J 49%3A 1108%26%23x&dopt=abstract

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https://scholar.google.com/scholar?as_q=Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Miyashita&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Monn� M%2C Daddabbo L%2C Gagneul D%2C Obata T%2C Hielscher B%2C Palmieri L%2C Miniero DV%2C Fernie AR%2C Weber APM%2C Palmieri F %282018%29 Uncoupling proteins 1 and 2 %28UCP1 and UCP2%29 from Arabidopsis thaliana are mitochondrial transporters of aspartate%2C glutamate%2C and dicarboxylates%2E J Biol Chem 293%3A 4213%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Murcha MW%2C Kmiec B%2C Kubiszewski%2DJakubiak S%2C Teixeira PF%2C Glaser E%2C Whelan J %282014%29 Protein import into plant mitochondria%3A Signals%2C machinery%2C processing%2C and regulation%2E J Exp Bot 65%3A 6301%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Murcha MW%2C Narsai R%2C Devenish J%2C Kubiszewski%2DJakubiak S%2C Whelan J %282015%29 MPIC%3A A mitochondrial protein import components database for plant and non%2Dplant species%2E Plant Cell Physiol 56%3A e&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Nickel C%2C Horneff R%2C Heermann R%2C Neumann B%2C Jung K%2C Soll J%2C Schwenkert S %282019%29 Phosphorylation of the outer membrane mitochondrial protein OM64 influences protein import into mitochondria%2E Mitochondrion 44%3A 93%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Oliver DJ%2C Neuburger M%2C Bourguignon J%2C Douce R %281990%29 Interaction between the Component Enzymes of the Glycine Decarboxylase Multienzyme Complex%2E Plant Physiol 94%3A 833%26%23x&dopt=abstract

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