Retrograde signals from mitochondria reprogramme
skoto-morphogenesis in Arabidopsis thaliana via
alternative oxidase 1a

Livia Merendino, Florence Courtois, Björn Grübler, Olivier Bastien, Vera Straetmanns, Fabien Chevalier, Silva Lerbs-Mache, Claire Lurin, Thomas Pfannschmidt

► To cite this version:

Livia Merendino, Florence Courtois, Björn Grübler, Olivier Bastien, Vera Straetmanns, et al. Retrograde signals from mitochondria reprogramme skoto-morphogenesis in Arabidopsis thaliana via alternative oxidase 1a. Philosophical Transactions of the Royal Society B: Biological Sciences, 2020, 375 (1801), pp.20190567. 10.1098/rstb.2019.0567. hal-02971816

HAL Id: hal-02971816

https://hal.science/hal-02971816

Submitted on 13 Nov 2020

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 **Title:** Retrograde signals from mitochondria reprogram skoto-morphogenesis in
2 *Arabidopsis thaliana* via Alternative Oxidase 1a

3

4 Authors:

5 Livia Merendino<sup>1,2,3*</sup>, Florence Courtois<sup>1</sup>, Björn Grübler<sup>1</sup>, Olivier Bastien<sup>1</sup>, Vera

6 Straetmanns<sup>1</sup>, Fabien Chevalier<sup>1</sup>, Silva Lerbs-Mache<sup>1</sup>, Claire Lurin<sup>2,3</sup>, Thomas

7 Pfannschmidt<sup>1,4*</sup>

8

9 <sup>1</sup> Univ. Grenoble-Alpes, CNRS, CEA, INRA, BIG-LPCV, 38000, Grenoble, France

10 <sup>2</sup> Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud,
11 Université Evry, Université Paris-Saclay, Bâtiment 630, Rue Noetzlin, 91405 Orsay, France

12 <sup>3</sup> Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, Bâtiment
13 630, Rue Noetzlin, 91405, Orsay, France

14 <sup>4</sup>Present address: Institute for Botany, Plant Physiology, Leibniz University Hannover,
15 Herrenhäuser Str. 2, 30419 Hannover, Germany

16

17 *To whom correspondence may be addressed:

18 Livia Merendino, E-mail: livia.merendino@u-psud.fr; ORCID: 0000-0003-1593-5442

19 Thomas Pfannschmidt, E-mail: t.pfannschmidt@botanik.uni-hannover.de; ORCID: 0000-0002-7532-3467

21

22

23

24
25

**Summary**

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

The early steps in germination and development of angiosperm seedlings often occur in the dark inducing a special developmental programme called skoto-morphogenesis. Under these conditions photosynthesis cannot work and all energetic requirements must be fulfilled by mitochondrial metabolization of storage energies. Here, we report the physiological impact of mitochondrial dysfunctions on the skoto-morphogenic programme by using the *Arabidopsis rpoTmp* mutant. This mutant is defective in the T7-phage-type organellar RNA polymerase shared by plastids and mitochondria. Lack of this enzyme causes a mitochondrial dysfunction resulting in a strongly reduced mitochondrial respiratory chain and a compensatory up-regulation of the alternative-oxidase (AOX)-dependent respiration. Surprisingly, the mutant exhibits a triple-response-like phenotype with a twisted apical hook and a shortened hypocotyl. Highly similar phenotypes were detected in other respiration mutants (*rug3* and *atphb3*) or in WT seedlings treated with the respiration inhibitor KCN. Further genetic and molecular data suggest that the observed skoto-morphogenic alterations are specifically dependent on the activity of the AOX1a enzyme. Microarray analyses indicated that a retrograde signal from mitochondria activates the ANAC017-dependent pathway that controls the activation of *AOX1A* transcription. In sum, our analysis identifies AOX as a functional link that couples the formation of a triple-response-like phenotype to mitochondrial dysfunction.

**Key words:** *Arabidopsis*; Mitochondrial respiration; Alternative oxidase; Retrograde signal; Organellar RPOTmp RNA polymerase; Skoto-morphogenesis.
## 47 Introduction

Mitochondria are essential cell organelles that convert organic matter into chemical energy in form of ATP and reducing power through the process of respiration [1]. To this end in seeds of oilseed plants as *Arabidopsis*, the triacylglycerol lipids present in the oil bodies are oxidatively degraded into citrate which is fed into the tricarboxylic acid (TCA) cycle within the mitochondrial matrix [2]. Here the carbon skeleton is completely oxidized and CO₂, ATP and reducing equivalents are released by the cycle. The reducing power then is used to drive an electron transport through the mitochondrial KCN-sensitive electron transport chain (ETC) within the inner mitochondrial membrane towards oxygen as end acceptor. ETC consists of three large multi-subunit protein complexes (complexes I, III and IV) that are connected by mobile electron carriers. The electron transport through this chain is coupled to a proton-translocation across the mitochondrial membranes, which is finally used by the ATP synthase to build ATP. The oxidized forms of the reducing equivalents produced by the ETC are then available again for the TCA cycle as electron sink. By this way TCA cycle and ETC are functionally coupled and any restriction in metabolic flux in one of them affects also the other [1].

Plant mitochondria display an extraordinary plasticity in their respiratory pathways as they possess additional oxidoreductases that can release excess levels of reducing power by bypassing parts of the ETC without concomitant ATP production [1,3]. At the ETC input site electrons can be donated to the alternative type II NADH dehydrogenases that bypass complex I. A second shunt is found at the end of the ETC where electrons can be transferred to oxygen by the alternative oxidase (AOX), which bypasses complexes III and IV. The role of both the alternative NADH dehydrogenases and AOX is still a matter of debate, but they are proposed to avoid generation of dangerous reactive oxygen species (ROS) and to stabilize the NAD+/NADH balance by preventing a metabolic feedback inhibition of the TCA cycle [1]. In addition, these enzymes are also suggested to serve as safety valves for photosynthesis under various stress conditions. Experimental evidences support the function of AOX as a releasing enzyme of excess reducing power transferred from the chloroplast under high light [3]. The additional enzymes also provide high flexibility to the ETC of plant mitochondria allowing genetic studies. Mutants in nuclear genes, that are necessary for the proper expression of mitochondrial-encoded proteins, are defined as surrogate mutants [4]. The surrogate mutants *rpoTmp* and *rug3* present a defective expression of ETC complex subunits but they are viable in that they maintain residual complexes. Even if they display delayed
growth phenotypes, they still generate rosettes that are comparable in size to wildtype and
produce seeds [5-7]. These mutants accumulate high levels of AOX protein. In *rpoTmp*
plants, the AOX pathway was shown to be activated [8].

Recent studies have shown that stress induced by block of mitochondrial translation or
respiration as well as by environmental constraints activates retrograde signaling pathways
from mitochondria towards the nucleus [9,10]. One of the target genes is *AOX1a*
(ALTERNATIVE OXIDASE 1a) that is now commonly used as a marker to investigate
mitochondrial retrograde signaling [7,11,12]. Also, recently identified regulators of *AOX1A*
expression (e.g. RAO1/CDKE1 and RAO2/ANAC017) seem to respond to mitochondrial as
well as to chloroplast retrograde signals [13,14]. Furthermore, chloroplast redox signals from
photosynthetic electron transport impact the expression of mitochondria-located genes [15].
These last studies investigating the signaling functions of the two energy-converting
organelles have been all performed on plants grown in the light where both chloroplasts and
mitochondria are active. Because of the multiple connections between these two organelles a
clear separation of mitochondrial and chloroplast retrograde signals, therefore, remains
difficult and partial overlaps and interactions occur in green tissues [14,16]. How a
mitochondrial dysfunction would affect plant growth and development when chloroplasts and
energy supply from photosynthesis are not available, i.e. during germination in the dark, is
however, not well investigated. Angiosperms such as *Arabidopsis* undergo a special
developmental program when they germinate and grow in the dark, the skoto-morphogenesis
[17]. When a germinating seed is buried by humus or soil the seedling re-allocates all storage
energy into shoot elongation while the cotyledons remain small, being directed down-wards
and forming an apical hook that protects the sensitive apical meristem. In this phase the
seedling relies completely on its storage energies and their metabolization by mitochondria.
As soon as the cotyledons breach the surface and perceive light, photo-morphogenesis is
initiated and chloroplasts are formed [18]. Mutants with mitochondrial dysfunction, therefore,
should be expected to show major defects in the performance of skoto-morphogenesis.

*Arabidopsis rpoTmp* mutants are defective in a phage-type single-subunit RNA polymerase
that is bi-localized to chloroplasts and mitochondria [19,20]. This RNA polymerase is
required for the proper expression of the organelle genomes, however, it was shown that its
impact in chloroplasts is restricted to the recognition of the PC promoter in front of the 16S
rRNA operon [21], while it displays a major deficiency in the transcription of several
mitochondrial genes including those for complex I and IV [5,22]. The mutant exhibits
113

strongly reduced complex I and complex IV accumulation, but displays only a mild phenotype in the light with a delayed growth and curly leaves in the fully developed rosette [5].

114

115

116

In this manuscript, we analyze the impact of restrictions of respiration on early seedling development in the dark using the respiration deficient *rpoTmp* mutant from *Arabidopsis*. We found that limitations of the ETC lead to drastic changes in the early developmental program of etiolated seedlings and that this reprogramming of skoto-morphogenesis is triggered by the AOX1a enzyme.

117

118

119

120
121 **Material and Methods**

122 *Plant material*

123 *Arabidopsis thaliana* wildtype (WT) (ecotype Columbia) and mutant lines (*rpoTmp-2* 124 SALK_132842 and *rpoTmp-3* SALK_086115 [21][5], *rug3* SALK_092071 [6], *atphb3* 125 SALK_020707 [23], *rpoTmp* SALK_132842/*anac017* (*anac017*, *rao2-1*) [7,24], *aox1A* 126 SALK_084897 [8] and *rpoTmp* SALK_132842/*aox1A* SALK_084897 [8]) were used in this 127 study. Seeds were surface-sterilized and spread on Murashige and Skoog (MS) agar plates 128 supplemented with sucrose (1%). Activated charcoal (in powder, SIGMA-ALDRICH, 0.08%) 129 was added to the plant culture medium in order to improve image quality of etiolated plants 130 by increasing background contrast. Seeds were stratified for 72 hours (at 4°C in darkness), 131 exposed to light (100 µE white light) for 6 hours and then grown at 23°C in darkness. Plants 132 were harvested after 3 days. For microarray, qRT-PCR and western-immuno-blot analyses the 133 etiolated plants were harvested in the dark under a green safe-light, immediately frozen in 134 liquid nitrogen, ground in a mortar and subjected to further techniques as described below. 135 The germination percentage was variable according to the seed lot and the drug treatment. For 136 chemical treatments MS agar plates were supplemented with 1-aminocyclopropane- 137 carboxylic acid (ACC, SIGMA-ALDRICH A3903, 20 µM), KCN (1 mM) or 138 salicylhydroxamic acid (SHAM, SIGMA-ALDRICH S7504, 1 mM in DMSO). In case of 139 KCN or SHAM treatments, seeds were first spread on black nitrocellulose membrane filters 140 (Whatman 10409770, 0.45 µm) on MS agar plates, stratified for 72 hours and light-exposed 141 for 6 hours, and then transferred (on filter) to KCN/SHAM-containing MS agar plates for 142 growth in the dark. DMSO-containing MS agar plates were used as mock-control for the 143 SHAM treatment. 144

145 *Measurements of phenotypic parameters*

146 Phenotypic appearance of plantlets was analyzed with a dissection microscope (Olympus 147 SZX12) using the ACT-1C for DXM1200C software. Digital images of individual plants 148 were taken and the angles formed in the apical hook between hypocotyl and cotyledons were 149 subsequently determined using the ImageJ program. In absence of any curvature (cotyledons 150 straight and in the same axis as the hypocotyl but oppositely oriented from the roots) the angle 151 was considered as 0°. In a hook where cotyledons were in the same axis as the hypocotyl, but 152 oriented towards the roots, the angle was considered as 180°. Any hook angle larger than 180° 153
153

was considered as twisted. The length of the hypocotyls was measured by determining the
shoot length between the origins of roots and cotyledons.

154

155

156

*Statistical analyses of phenotypic parameter measurements*

157

All statistical tests were performed using the computing environment R [25], with a
significance level of 0.05. Details about the statistical analyses are found in the supplementary
methods.

158

159

160

161

*Microarray analysis*

162

Total RNA purification and microarray analysis were performed as indicated in [26]. Total
RNA was isolated using RNeasy Plant Mini Kit (Qiagen) with on-column DNase I (Qiagen)
treatment. Affymetrix whole transcriptome microarray analysis was performed on three
biological replicates by the commercial service Kompetenz-Zentrum für Fluoreszente
Bioanalytik (KFB) (Regensburg, Germany). cDNAs were prepared using the Ambion®
Whole Transcriptome (WT) Expression Kit and fragmented and labeled using the Affymetrix
GeneChip® WT Terminal Labeling Kit. Expression analyses were performed using the
“GeneChip® Arabidopsis Gene 1.0 ST Array”. Since the “Ambion® WT Expression Kit”
uses a mixture of oligo-dT and random hexamer primers for the generation of the first strand
cDNA, the resulting hybridization signals reflect also the organellar transcript accumulation
besides the one from the nucleus.

163

164

165

166

167

168

169

170

171

172

173

174

*Analysis of microarray data*

175

Analysis of microarray data was performed as indicated in [26]. The cel files containing the
scanned chips received from KFB were analyzed with RobiNA [27] with the background
correction method RMA (robust multi-array average expression measure) [28], p-value
correction method Benjamini-Hochberg [29] and the analysis Strategy Limma for the quality
control [30]. For the main analysis the normalization method RMA with p-value correction
method after BH and a multiple testing strategy “nestedF” was chosen. In the analysis using
Excel (MS-Office), data with the description “Multiple Hits” were ignored and only results
with one-to-one correspondence to a given gene identity were regarded for further analysis.

176

177

178

179

180

181

182

183
184 RT-qPCR analysis of RNA levels

185 Ground plant material was resuspended in 3 volumes of solution A (10 mM Tris-HCl pH8.0;
186 100 mM NaCl; 1 mM EDTA; 1% SDS) and 2 volumes of phenol/chloroform/isoamyl alcohol
187 (25:24:1; v/v/v). After centrifugation, RNAs in the aqueous phase were again extracted twice
188 with phenol-chloroform and finally once with chloroform. After over-night precipitation in 2
189 M LiCl at 4°C, RNAs were then precipitated in ethanol, washed in ethanol 70% and
190 resuspended in water. 300 ng of DNAse (DNAse Max Kit, Quiagen) treated-RNAs were
191 retro-transcribed using random hexo-nucleotides and the Super-Script II enzyme (Invitrogen)
192 according to the manufacturer's protocol. The PCR reaction was performed in the Biorad
193 CFX384TM Real Time System PCR machine in presence of forward and reverse gene-
194 specific primers (0.5 µM, **Table S1**) using the SYBR® Premix Ex Taq™ (Tli RNaseH Plus)
195 from Takara Bio. Data were analyzed using the CFX Manager Software. The expression
196 levels of the transcript of interest were normalized with the levels of ACTIN 2-8 expression,
197 used as reference gene. The mean values of biological and/or technical replicates were plotted
198 together with the error bars corresponding to standard errors.

199

200 Protein immuno-western analysis

201 Total protein extracts were prepared by re-suspending 100 mg of ground plant material in 100
202 µl of protein lysis buffer (50 mM Tris pH 8.0; 2% SDS; 10 mM EDTA; protease inhibitors
203 (Roche (04693159001) cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail)) and
204 incubating at room temperature for 30 min. Extracts were cleared by centrifugation for 30 min
205 at 12000 × g at 4°C and dilutions of the supernatant were quantified with Bradford. Samples
206 were then denatured for 5 min at 95 °C in 4X reducing electrophoresis sample buffer.
207 Increasing amounts of wild-type and mutant protein extracts (10, 20, 40 µg) were fractionated
208 on a 12% gel by SDS-PAGE, electro-blotted on a nitrocellulose or PVDF membranes and
209 immuno-decorated with specific polyclonal rabbit antisera against mitochondrial protein
210 NAD9 [31]; mitochondrial AOX1/2 plant alternative oxidase 1 and 2 (Agrisera, AS04 054);
211 plastidial RBCL (Agrisera, AS03 037); vacuole V-ATPase (Agrisera, AS07 213). The blots
212 were developed with Clarity Western ECL substrate (Bio-Rad, Hercules, USA). Images of the
213 blots were obtained using a CCD imager (Chemidoc MP, Bio-Rad) and the Image Lab
214 program (Bio-Rad, Hercules, USA). Image analysis for AOX protein quantification was
215 performed using the Image Lab program and normalized with the levels of V-ATPase.
216

217 *In planta O₂ consumption measurements*

218 Etiolated plantlets were harvested from Petri dishes and immediately soaked in the dark with
219 1 mL of oxygenated phosphate buffer (10 mM sodium phosphate buffer pH 7.2; 10 mM KCl;
220 10 mM glucose) in the measurement cell of a Clark oxygen electrode (Hansatech). For
221 electron transport inhibition experiments, KCN (0.1 M stock, inhibitor of KCN-sensitive
222 chain at the level of complex IV) and SHAM (0.1 M stock in DMSO, inhibitor of the AOX-
223 dependent pathway) were added directly into the electrode chamber to a final concentration of
224 1 mM once the recorded signal reached a constant regime. The capacity (or maximum
225 activity) of the AOX-dependent pathway (corresponding to KCN-insensitive pathway) and of
226 the COX- dependent pathway (corresponding to KCN-sensitive pathway) was calculated as
227 ratio of O₂ consumption rate upon addition of KCN or SHAM and total O₂ consumption rate,
228 respectively. The contribution of extra mitochondrial O₂ consumption to mitochondrial
229 respiration was evaluated at the end of each measurement after addition of both KCN and
230 SHAM and systematically subtracted from O₂ consumption rates.
231 **Results**

232 *rpoTmp* mutants display a triple-response-like phenotype

233 To investigate the impact of mitochondrial dysfunction on skoto-morphogenesis we compared
234 3-day-old dark-grown (etiolated) WT with *rpoTmp* mutant plants grown under identical
235 conditions (Fig.1a). WT seedlings displayed a typical skoto-morphogenesis with elongated
236 hypocotyl, apical hook and yellow cotyledons. In contrast, seedlings of two allelic *rpoTmp*
237 mutant lines (*rpoTmp*-2 SALK_132842 and *rpoTmp*-3 SALK_086115) developed a
238 significant exaggeration of apical hook curvature (twist) and a marked shortening of the
239 hypocotyls. The phenotype of both alleles was identical indicating that indeed the genetic
240 defect in *rpoTmp* causes the massive change in the seedling developmental program. The
241 observed mutant phenotype was reminiscent of morphological changes reported for etiolated
242 seedlings exposed to high levels of ethylene, commonly known as triple-response [32,33]. To
243 investigate this in more detail we compared the *rpoTmp* phenotype to that of WT plants
244 grown in presence of 20 µM 1-aminocyclopropane-1-carboxylic acid (ACC), a biosynthetic
245 precursor of ethylene known to induce the triple-response [34] (Fig. 1a). We observed
246 comparable values for the increased apical hook curvature (Fig. 1b) and the hypocotyl
247 shortening (Fig. 1c) in the two plant samples. Only the widening of the hypocotyl diameter in
248 response to ACC treatment was not observable in *rpoTmp* (Fig. 1a) suggesting that the
249 genetic defect in the *RPOTmp* locus causes a strong but not perfect phenocopy of the ACC-
250 treatment. Therefore, we termed the observed phenotypic response a triple-response-like
251 phenotype.

252

253 The triple-response-like phenotype is dependent on an active AOX1a enzyme

254 In order to find a potential link between the genetic inactivation of the *RPOTmp* gene and the
255 morphological changes, we performed a transcriptomic profiling of etiolated mutant seedlings
256 in comparison to WT (Table S2) and compared these data to previous expression analyses
257 performed in light-grown plants. An overview of the gene expression profiles in etiolated
258 plants in relation to the corresponding cellular metabolic pathways using the MapMan tool
259 indicated that the general impact of the mutation in *RPOTmp* was relatively weak (Fig. S1).
260 Only a small number of genes displayed a strong expression change of more than log2 fold ≥2
261 (29 genes) or ≤-2 (60 genes, Table S2). These data suggest that the observed triple-response-
262 like phenotype is rather a specific effect than the result of pleiotropic disturbance in the
mutant. By analyzing the mitochondrial transcriptome, we found that from the twenty
mitochondrial genes that were presenting the highest degree of transcriptional variations in
previous analyses in light-grown *rpoTmp* adult plants [7], 17 were also differentially
expressed in etiolated *rpoTmp* seedlings and, here, in the same direction of expression change,
even though to a lower extent (Table S3). More particularly, the subset of mitochondrial
genes encoding ETC complex subunits that was previously described as RPOTmp-dependent
[5] was also down-regulated in etiolated *rpoTmp* seedlings (Fig. 2a). Analysis of the
accumulation of the mitochondrial-encoded NAD9 protein as marker for complex I
accumulation indicated that these gene expression defects were also reflected at the protein
level (Fig. 2b). While clearly present in WT, NAD9 was almost undetectable in *rpoTmp*
mutant plants in contrast to the control RBCL, the plastid-encoded large subunit of Ribulose-
1.5-bisphosphate-carboxylase/oxygenase. This indicated strong deficiency of complex I in
*rpoTmp* plants also in the dark, which was accompanied by a decrease in the capacity of the
KCN-sensitive pathway in *rpoTmp* seedlings (Fig. S2). By analyzing the transcriptomic
profile in more detail, we found that more than half of the 20 most up-regulated genes encode
proteins that are localized in the mitochondria (Fig. 2c). Functional sub-grouping of these
genes identified four groups: one class containing genes corresponding to markers of
mitochondrial stress and targets of the retrograde-mitochondrial pathway, as *AT12CYS* and its
co-expression partner *NDB4* [11] and three *DUF295 Organellar B* genes [35]; one tRNA
class including one nucleus- and three mitochondria-encoded *tRNAs*; one class corresponding
to general mitochondrial functions, including *TOM7-2* (coding for a member of the family of
the TOM7 translocases of outer membrane), *SPL2* phosphatase [36], ATP synthase or an
undefined gene; and a final group including genes coding for proline- and glycine-rich
proteins, often involved in stress response (Fig. 2c, d). Functional sub-grouping of the 20
most down-regulated genes also identified a group of genes coding for glycine rich proteins
and/or factors involved in response to stress. An additional group of genes was also defined
corresponding to the response to hormonal *stimuli*, and a final class of genes related to
reduction/oxidation activities (Fig. 2e, Table S4).

We have compared our transcriptomic data (threshold 1.5) concerning dark-grown *rpoTmp*
seedlings with previous analyses performed by Van Aken and colleagues with light-grown
adult plants [7]. We found 33 up DEGs (Differentially Expressed Genes) and 40 down DEGs
in common (Table S5). Of these genes, four are among the top 20 up DEGs in both studies
(highlighted in yellow and by an asterisk), eight are in the list of our top 20 up and seven are
among our top 20 down DEGs (highlighted in yellow). These data emphasize the strong similarity in the gene-expression profiles of *rpoTmp* plants that differ for developmental stages and growth conditions. Remarkably, among the common up DEGs, 20 genes encode mitochondrial localized proteins and at least nine genes are induced by mitochondrial dysfunction and transcriptionally regulated by mitochondrial retrograde signaling, as *AT12CYS2* [11], *NDB4* [11], five different *DUF 295 organellar genes* [35] and *UPOX* that is an hallmark of oxidative stress [37]. All these data pointed to the presence of a strong mitochondrial stress in *rpoTmp* plants and prompted us to explore the possibility that defects in mitochondrial respiration are at the origin of the observed skoto-morphogenetic alteration. In order to test this hypothesis by an independent experimental line we chose to grow etiolated WT plants on a medium containing 1 mM KCN, an inhibitor of complex IV in the ETC. Here, a statistically significant increment in apical hook bending could be detected (**Fig. 3a** and **Fig. S3**). This indicates that limitation of KCN-sensitive ETC results in the same phenotype as in the *rpoTmp* mutant suggesting that respiration restriction is indeed at the origin of the phenotypic deviations. To obtain independent proof for this assumption we observed the phenotype of dark-grown *rug3* and *atphb3 Arabidopsis* mutant seedlings (**Fig. 3b**). The *rug3* mutant is disturbed in complex I biogenesis [6] and *atphb3* mutant is defective in the mitochondrial membrane protein PROHIBITIN 3, that was suggest to be a structural support for membrane protein complexes, such as the electron transport chain [23]. Both mutants possess a fully functional RPOTmp enzyme, but like in *rpoTmp* mutants or KCN-treated WT we found a twisted apical hook. This supports our hypothesis of ETC limitation as cause of the reprogramming of skoto-morphogenesis and excludes also any side-effect from the defective RPOTmp enzyme in the plastid.

Up-regulation of the AOX pathway was already reported in light-grown *rpoTmp* plants [8]. In etiolated *rpoTmp* plants *AOX1a* expression was also strongly activated both at transcript and protein levels (**Fig. 4a, b**). Together with the *AOX1a* gene, also the levels of two other markers of the cellular response to oxidative stress, *AT12CYS* [11,38] and *UPOX* [37,38], were highly induced in *rpoTmp* plants (**Fig. S4a**). Transcriptional activation of *AOX1a* and partially of *AT12CYS* mRNAs was impaired by the inactivation of the mitochondrial retrograde master regulator ANAC017 [7] (**Fig. 4a, Fig. S4b**). The activation of the *AOX1a* gene expression corresponded to an increase in the AOX-dependent respiration capacity that is KCN-insensitive (**Fig. 4c, Fig S5**). To investigate whether or not the AOX-dependent pathway is involved in the triple-response-like phenotype we grew *rpoTmp* plantlets in
329

presence of 1 mM SHAM, a chemical inhibitor of the AOX-dependent pathway. Under this condition a statistically significant decrease of the twisting of the apical hook angle could be detected (Fig. 3a, Fig. S3 right panel).

330

331

332

To further prove independently the involvement of the AOX-pathway, we aimed to characterize the *rpoTmp/aox1a* double mutant plants in the dark in which the AOX1a isoform is lacking. The AOX enzyme is known to be expressed in various isoforms that potentially could replace each other. In western-blot analyses of *rpoTmp/aox1a* protein extracts using an antiserum detecting the five AOX isoforms no signal was detected (Fig. 4b, right panel) indicating that AOX1a is the main AOX isoform in the *rpoTmp* genetic background in the dark. Analysis of *rpoTmp/aox1a* mutant seedlings, thus, should provide conclusive evidence about the involvement of the AOX1a enzyme in the phenotypic appearance of *rpoTmp* mutants. A strong decrease in the AOX-capacity was observed in *rpoTmp/aox1a* double mutants when compared to *rpoTmp* plants (Fig. 4c, Fig. S5). Importantly, we observed a suppression of the twisting of the apical hook in *rpoTmp/aox1a* mutants (Fig. 4d, Fig. S6). However, the hypocotyl elongation was not restored suggesting that it is regulated by a different pathway.

333

334

335

336

337

338

339

340

341

342

343

344

345

346

## The triple-response-like phenotype requires a retrograde signal from mitochondria

347

While the genetic defect in *rpoTmp* mutant lines causes a dysfunction in mitochondria, the up-regulated AOX1a enzyme is encoded in the nucleus. The up-regulation for involvement in the observed developmental change, therefore, requires a mito-nuclear retrograde signal. Recently, the ER-bound transcription factor ANAC017 was shown to be responsible for the retrograde activation of the *AOX1a* gene [7,24]. We, thus, proposed that the double mutant *rpoTmp/anac017*, which keeps the basal *AOX1a* expression but lacks the capacity to upregulate it to its full extent, should also show a reduction of the triple-response-like phenotype. Indeed, we found intermediate levels of AOX proteins and AOX capacity in the double mutant *rpoTmp/anac017* plants when compared to *rpoTmp* and *rpoTmp/aox1a* mutant plants (Fig. 4b, c, S5). As expected, we detected a partial suppression of the twisting of the apical hook in the *rpoTmp/anac017* double mutant when compared to *rpoTmp* plants (Fig. 4d, e, S6) indicating that the ANAC017-dependent pathway is required for the induction of the triple-response-like phenotype.

348

349

350

351

352

353

354

355

356

357

358

359
In sum, the results with the *rpoTmp/aox1a* and *rpoTmp/anac017* double mutants provide compelling evidence that the hook exaggeration in *rpoTmp* requires AOX1a. A strong decrease in the AOX capacity was also observed in *aox1a* single mutants (**Fig. 4c, S5**), but these plants displayed a normal skoto-morphogenesis without any aberrations (**Fig. 4d**). We concluded that a lack of AOX1a becomes critical for skoto-morphogenesis only under conditions when ETC is limited.
366

**Discussion**

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

In the light, *Arabidopsis rpoTmp* mutants display defects in ETC but only mild phenotypes with slightly retarded growth and few macroscopic changes such as curled leaves [5,21]. In the dark we expected stronger impact on the phenotype as no energy compensation from photosynthesis can occur. Here we report that the expression of the mitochondrial sub-set of genes that were previously identified as RPOTmp-dependent [5] was highly impacted in *rpoTmp* plants also in the dark (**Fig. 2a, b; Table S3**). In addition, the strong activation of stress marker genes indicates the presence of a severe mitochondrial stress in etiolated mutant plants (**Fig. 2c, d, 4a** and **Fig. S4a**). Comparison of our transcriptomic data obtained from dark-grown seedlings with previous analyses done on light-grown adult plants [7] identified high similarities at the level of the response to mitochondrial dysfunction (**Table S5**). These data underline the robustness of the plant stress reaction to the genetic defect in the *RPOTMP* gene locus that is largely independent of the developmental stage and the light regime. However, to our surprise we did not observe any retardation in development of the dark-grown seedlings (as observed in the light), but instead we found a strong modification of the skoto-morphogenic programme (**Fig. 1**).

382

383

384

385

386

387

388

389

390

391

We could confirm that limitation of the ETC leads to shortened hypocotyls and twisted apical hooks during the development of the seedlings by both chemical means (in KCN-treated plants, **Fig. 3a** and **Fig. S3 left panel**) and independent genetic tools (in *rug3* and *atphb3* mutant plants, **Fig. 3b**). This phenotype is highly reminiscent of the triple response of seedlings exposed to ethylene (or precursors of ethylene such as ACC) (**Fig. 1**). The third parameter, a widened hypocotyl, could not be significantly determined. Therefore, we termed the observed phenotypic response a triple-response-like phenotype. In the *rpoTmp/aox1a* double mutant the twisting of the hook was prevented, however, the hypocotyl shortening was maintained (**Fig. 4d**). These observations suggest that the three phenotypic traits may be controlled by different specific pathways and/or different hormone thresholds.

392

393

394

395

396

397

A recent study reported that limitation of mitochondrial translation causes proteotoxic stress and activates an unfolded protein response in mitochondria by MAP kinase cascades which leads to shortened hypocotyls [10]. Partial inhibition of mitochondrial translation, however, leads also to a general down-regulation of mitochondrial respiration and, concomitantly, respiration stress does occur. This stress was apparently not as strong as in our system since an exaggerated hook was not observed in that study. Interestingly, an up-regulation of *AOX1a*
398
399

could be observed also in that study, but its potential involvement in the signaling of the proteotoxic stress was not investigated.

400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423

Here, we show that limitation of the ETC leads to drastic changes in skoto-morphogenesis and that the increased capacity of the AOX-pathway is the critical parameter that triggers the hook exaggeration (Fig. 5). Both pharmaceutical interference of AOX enzyme activity (by SHAM) or genetic impairment of the AOX1a isoform resulted in a significant suppression of the twisting phenotype in *rpoTmp* plants (Fig. 3a, S3 right panel, 4d, S6). Our western-analyses indicate also, that AOX1a appears to be the sole isoform being expressed in the dark (Fig. 4b). Recently, it was reported that the AOX1a enzyme expression is induced at the transcriptional level by the action of the ANAC017-dependent retrograde pathway in response to mitochondrial stress [7,24]. In our study the genetic impairment of the ANAC017-dependent transcriptional activation of the *AOX1a* gene (Fig. 4a) affects moderately both the increase of the accumulation (Fig. 4b) and the enzymatic capacity of AOX proteins (Fig. 4c) and the induction of the twisting phenotype (Figs.4d and e, S6) in *rpoTmp* seedlings. These data highlight the dependence of the triple-response-like phenotype on the AOX1a protein levels and the mitochondrial retrograde pathway. It is important to note that in the *rpotmp/anac017* double mutant seedlings, the basal *AOX1a* expression is not reduced (as in an *aox1a* knock out line). The plants just lack the ability to upregulate *AOX1a* expression to its full extent. As such the phenotypical effect is expected to be less pronounced than in *rpotmp/aox1a* mutants (Fig. 4d, S6). This observation suggests that transcriptional activation of *AOX1a* by the ANAC017-dependent retrograde pathway has not an exclusive role in the dark and likely additional translational or post-translational regulation of the AOX1a enzyme might be also implemented. Based on *in vitro* experiments, it was proposed that AOX activity might be regulated at the post-translational level, at the redox state and by the interaction with TCA cycle intermediates, leading to a very tight and rapid metabolic control of the mitochondrial respiration process [39,40].

424
425
426
427
428
429

We conclude that the limitation of respiration, therefore, induces the triple-like response by promoting the abundance and capacity of the AOX1a enzyme (Fig. 5). Our study strongly suggests that the AOX1a enzyme is not just a biomarker for stress [3,41] that accumulates as a side effect, but it represents a key component of the stress reaction network that triggers subsequent cellular responses to mitochondrial dysfunction. How precisely the AOX1a enzyme induces the reprogramming remains to be elucidated, but one can imagine that
430

especially under oxygen limitation high capacities of AOX1a activity could reduce alternative
substrates generating products that may serve as signaling molecules such as nitric oxide [42].

431

432

A physiological role for the triple response was obscure for a long time until recently a study
uncovered that the triple response can be triggered by the soil overlay of buried seeds [43].

433

434

The triple morphogenic change provides higher resistance to soil friction and allows the
seedling to breach the surface while protecting the apical meristem. A recent study
investigated the influence of hypoxic conditions on skoto-morphogenesis and uncovered that

435

437

a low oxygen tension applied after the apical hook maintenance phase prevented the opening
of the hook in subsequent light conditions [44]. The study identified that seedlings are able to
sense the oxygen tension *via* the N-end rule pathway-controlled stability of ERFVII

438

440

transcription factors. However, the hypoxic condition in that study was imposed to the
seedlings after the apical hook was formed and, thus, represents a phase later than that in our

441

442

An attractive hypothesis is that dense soil overlay may lead to hypoxic conditions that
in turn induce respiration stress (due to limitation of the final electron acceptor of the KCN-
sensitive ETC, the oxygen) and a subsequent triple response-like phenotype. How soil and O₂
sensing mechanisms relate to our proposed model will be subject of future research.

443

444

445

446

Our proposed model (Fig. 5) is based on respiration deficiency generated in the laboratory by
genetic or pharmaceutical blocking. However, it would fit also with other natural scenarios in
which the final electron acceptor for KCN-sensitive electron transport (i.e. oxygen) becomes
limiting for instance in response to water flooding, drought or acute ozone exposure [9]. In
addition, rapid responses to touch and wounding stress involve changes in the expression of
genes for mitochondria-localized proteins that are responsible for the availability of the
substrates required for mitochondrial energy metabolism [14]. It will be of great interest to
investigate whether the proposed model becomes active also in these conditions and which
developmental effects could be triggered.

447

448

449

450

451

452

453

454

455

456

**Acknowledgements:** This work was supported by grants from the Deutsche Forschungsgemeinschaft [PF323-5-2 to T.P.] and the DFG research group FOR 804; the Centre National de la Recherche Scientifique [PEPS to T.P.]; the French Ministry of Education and the Grenoble Alliance for Integrated Structural Cell Biology (LabEx GRAL, ANR-10-LABX-49-01). IPS2 benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). We thank Robert Blanvillain and Stéphane Ravanel from LPCV (Grenoble, France), Etienne Delannoy and Emmanuelle Issakidis-Bourguet from IPS2

457

458

459

460

461

462
463

(Orsay, France) for helpful discussions; Juliette Jouhet (LPCV, Grenoble, France), Géraldine

464

Bonnard (IBMP, Strasburg, France) for sharing NAD9 antisera. We thank Olivier van Aken

465

(Lund University, Sweden) for sharing *rpoTmp/anac017* and *atphb3* seeds, Renate Scheibe

466

(Universität Osnabrück, Germany) for *aox1a* seeds and Kristina Kühn (Universität Halle,

467

Germany) for *rpoTmp/aox1a* and *rug3* seeds. RNA sample processing and Affymetrix

468

microarray hybridization were carried out at the genomics core facility: Center of Excellence

469

for Fluorescent Bioanalytics (KFB, University of Regensburg, Germany).

470

**Author contribution.** LM and TP designed research, LM, FC, BG, VS, FCh and SB

471

performed experiments, OB performed statistical analyses, CL contributed analytical tools.

472

LM, FC, SLM, TP analyzed data, LM and TP wrote the manuscript with the help of all co-

473

authors. All authors read and approved the manuscript.

474

**Competing interests.** The authors declare no conflict of interest.
475 References

476 1. Millar AH, Whelan J, Soole KL, Day DA. 2011 Organization and Regulation of Mitochondrial Respiration in Plants. *Annu. Rev. Plant Biol.* **62**, 79–104. (doi:10.1146/annurev-arplant-042110-103857)

479 2. Pracharoenwattana I, Cornah JE, Smith SM. 2005 Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination. *Plant Cell* **17**, 2037–2048. (doi:10.1105/tpc.105.031856)

482 3. Vanlerberghe G. 2013 Alternative Oxidase: A Mitochondrial Respiratory Pathway to Maintain Metabolic and Signaling Homeostasis during Abiotic and Biotic Stress in Plants. *Int. J. Mol. Sci.* **14**, 6805–6847. (doi:10.3390/ijms14046805)

485 4. Colas des Francs-Small C, Small I. 2014 Surrogate mutants for studying mitochondrially encoded functions. *Biochimie* **100**, 234–242. (doi:10.1016/j.biochi.2013.08.019)

488 5. Kühn K *et al*. 2009 Phage-type RNA polymerase RPOTmp performs gene-specific transcription in mitochondria of Arabidopsis thaliana. *Plant Cell* **21**, 2762–79. (doi:10.1105/tpc.109.068536)

491 6. Kühn K *et al*. 2011 The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana. *Plant J.* **67**, 1067–80. (doi:10.1111/j.1365-313X.2011.04658.x)

494 7. Van Aken O, Ford E, Lister R, Huang S, Millar AH. 2016 Retrograde signalling caused by heritable mitochondrial dysfunction is partially mediated by ANAC017 and improves plant performance. *Plant J.* **88**, 542–558. (doi:10.1111/tpj.13276)

497 8. Kühn K *et al*. 2015 Decreasing Electron Flux through the Cytochrome and/or Alternative Respiratory Pathways Triggers Common and Distinct Cellular Responses Dependent on Growth Conditions. *Plant Physiol.* **167**, 228–250. (doi:10.1104/pp.114.249946)

501 9. Wagner S, Van Aken O, Elsässer M, Schwarzländer M. 2018 Mitochondrial Energy Signaling and Its Role in the Low-Oxygen Stress Response of Plants. *Plant Physiol.* **176**, 1156–1170. (doi:10.1104/pp.17.01387)

504 10. Wang X, Auwerx J. 2017 Systems Phytohormone Responses to Mitochondrial Proteotoxic Stress. *Mol. Cell* **68**, 540–551.e5. (doi:10.1016/j.molcel.2017.10.006)

506 11. Wang Y *et al*. 2016 Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and
508

Mitochondrial Function. *Mol. Plant* **9**, 696–710. (doi:10.1016/j.molp.2016.01.009)

509

12.

Schwarzländer M, König A-C, Sweetlove LJ, Finkemeier I. 2012 The impact of impaired mitochondrial function on retrograde signalling: a meta-analysis of transcriptomic responses. *J. Exp. Bot.* **63**, 1735–1750. (doi:10.1093/jxb/err374)

510

511

13.

Blanco NE, Guinea-Díaz M, Whelan J, Strand Å. 2014 Interaction between plastid and mitochondrial retrograde signalling pathways during changes to plastid redox status. *Philos. Trans. R. Soc. Lond. B. Biol. Sci.* **369**, 20130231. (doi:10.1098/rstb.2013.0231)

512

513

514

14.

Van Aken O, De Clercq I, Ivanova A, Law SR, Van Breusegem F, Millar AH, Whelan J. 2016 Mitochondrial and Chloroplast Stress Responses Are Modulated in Distinct Touch and Chemical Inhibition Phases. *Plant Physiol.* **171**, 2150–65. (doi:10.1104/pp.16.00273)

515

516

517

518

519

15.

Dietzel L et al. 2015 Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts. *Mol. Plant* **8**, 1237–52. (doi:10.1016/j.molp.2015.03.004)

520

521

522

16.

Van Aken O, Pogson BJ. 2017 Convergence of mitochondrial and chloroplastic ANAC017/PAP-dependent retrograde signalling pathways and suppression of programmed cell death. *Cell Death Differ.* **24**, 955–960. (doi:10.1038/cdd.2017.68)

523

524

525

17.

Solymosi K, Schoefs B. 2010 Etioplast and etio-chloroplast formation under natural conditions: the dark side of chlorophyll biosynthesis in angiosperms. *Photosynth. Res.* **105**, 143–166. (doi:10.1007/s11120-010-9568-2)

526

527

528

18.

Arsovski AA, Galstyan A, Guseman JM, Nemhauser JL. 2012 Photomorphogenesis. *Arab. B.* **10**, e0147. (doi:10.1199/tab.0147)

529

530

19.

Hedtke B, Börner T, Weihe A. 1997 Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. *Science* **277**, 809–11.

531

532

20.

Hedtke B, Börner T, Weihe A. 2000 One RNA polymerase serving two genomes. *EMBO Rep.* **1**, 435–40. (doi:10.1093/embo-reports/kvd086)

533

534

21.

Courtois F, Merendino L, Demarsy E, Mache R, Lerbs-Mache S. 2007 Phage-type RNA polymerase RPOTmp transcribes the rrn operon from the PC promoter at early developmental stages in Arabidopsis. *Plant Physiol.* **145**, 712–21. (doi:10.1104/pp.107.103846)

535

536

537

538

22.

Tarasenko VI, Katyshev AI, Yakovleva T V, Garnik EY, Chernikova V V, Konstantinov YM, Koulintchenko M V. 2016 RPOTmp, an Arabidopsis RNA polymerase with dual targeting, plays an important role in mitochondria, but not in chloroplasts. *J. Exp. Bot.* **67**, 5657–5669. (doi:10.1093/jxb/erw327)

539

540

541
542

23.

Van Aken O *et al*. 2007 Mitochondrial type-I prohibitins of Arabidopsis thaliana are required for supporting proficient meristem development. *Plant J.* **52**, 850–864. (doi:10.1111/j.1365-313X.2007.03276.x)

543

544

545

24.

Ng S *et al*. 2013 A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis. *Plant Cell* **25**, 3450–3471. (doi:10.1105/tpc.113.113985)

546

547

548

25.

Team R. 2017 R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2016.

549

550

26.

Grübler B *et al*. 2017 Light and plastid signals regulate different sets of genes in the albino mutant pap7-1. *Plant Physiol.* **175**. (doi:10.1104/pp.17.00982)

551

552

27.

Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M, Usadel B. 2012 RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics. *Nucleic Acids Res.* **40**, W622-7. (doi:10.1093/nar/gks540)

553

554

555

28.

Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. 2003 Exploration, normalization, and summaries of high density oligonucleotide array probe level data. *Biostatistics* **4**, 249–264. (doi:10.1093/biostatistics/4.2.249)

556

557

558

29.

Benjamini Y, Hochberg Y. 1995 Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. *J. R. Stat. Soc. Ser. B.* **57**, 289–300. (doi:10.2307/2346101)

559

560

561

30.

Smyth GK. 2005 limma: Linear Models for Microarray Data. In *Bioinformatics and Computational Biology Solutions Using R and Bioconductor*, pp. 397–420. New York: Springer-Verlag. (doi:10.1007/0-387-29362-0_23)

562

563

564

31.

Lamattina L, Gonzalez D, Gualberto J, Grienenberger JM. 1993 Higher plant mitochondria encode an homologue of the nuclear-encoded 30-kDa subunit of bovine mitochondrial complex I. *Eur. J. Biochem.* **217**, 831–8.

565

566

567

32.

Guzmán P, Ecker JR. 1990 Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. *Plant Cell* **2**, 513–23. (doi:10.1105/tpc.2.6.513)

568

569

33.

Neljubow D. 1901 Uber die horizontale Nutation der Stengel von Pisum Sativum und einiger anderen Planzen. *Beih Bot Zentralb* **10**, 128–139.

570

571

34.

Larsen PB, Chang C. 2001 The Arabidopsis eer1 mutant has enhanced ethylene responses in the hypocotyl and stem. *Plant Physiol.* **125**, 1061–73.

572

573

35.

Lama S, Broda M, Abbas Z, Vaneechoutte D, Belt K, Säll T, Vandepoele K, Van Aken O. 2019 Neofunctionalization of Mitochondrial Proteins and Incorporation into Signaling Networks in Plants. *Mol. Biol. Evol.* **36**, 974–989.

574

575
576 (doi:10.1093/molbev/msz031)

577 36. Uhrig RG, Labandera A-M, Tang L-Y, Sieben NA, Goudreault M, Yeung E, Gingras
578 A-C, Samuel MA, Moorhead GBG. 2017 Activation of Mitochondrial Protein
579 Phosphatase SLP2 by MIA40 Regulates Seed Germination. *Plant Physiol.* **173**, 956–
580 969. (doi:10.1104/pp.16.01641)

581 37. Ho LHM, Giraud E, Uggalla V, Lister R, Clifton R, Glen A, Thirkettle-Watts D, Aken
582 O Van, Whelan J. 2008 Identification of Regulatory Pathways Controlling Gene
583 Expression of Stress-Responsive Mitochondrial Proteins in Arabidopsis. *Plant Physiol.*
584 **147**, 1858. (doi:10.1104/PP.108.121384)

585 38. Van Aken O, Whelan J. 2012 Comparison of Transcriptional Changes to Chloroplast
586 and Mitochondrial Perturbations Reveals Common and Specific Responses in
587 Arabidopsis. *Front. Plant Sci.* **3**, 281. (doi:10.3389/fpls.2012.00281)

588 39. Selinski J, Scheibe R, Day DA, Whelan J. 2018 Alternative Oxidase Is Positive for
589 Plant Performance. *Trends Plant Sci.* (doi:10.1016/j.tplants.2018.03.012)

590 40. Del-Saz NF, Ribas-Carbo M, McDonald AE, Lambers H, Fernie AR, Florez-Sarasa I.
591 2018 An In Vivo Perspective of the Role(s) of the Alternative Oxidase Pathway.
592 *Trends Plant Sci.* **23**, 206–219. (doi:10.1016/j.tplants.2017.11.006)

593 41. Clifton R, Lister R, Parker KL, Sappl PG, Elhafez D, Millar AH, Day DA, Whelan J.
594 2005 Stress-induced co-expression of alternative respiratory chain components in
595 Arabidopsis thaliana. *Plant Mol. Biol.* **58**, 193–212. (doi:10.1007/s11103-005-5514-7)

596 42. Vishwakarma A, Kumari A, Mur LAJ, Gupta KJ. 2018 A discrete role for alternative
597 oxidase under hypoxia to increase nitric oxide and drive energy production. *Free
598 Radic. Biol. Med.* (doi:10.1016/j.freeradbiomed.2018.03.045)

599 43. Zhong S, Shi H, Xue C, Wei N, Guo H, Deng XW. 2014 Ethylene-orchestrated
600 circuitry coordinates a seedling's response to soil cover and etiolated growth. *Proc.*
601 *Natl. Acad. Sci. U. S. A.* **111**, 3913–20. (doi:10.1073/pnas.1402491111)

602 44. Abbas M et al. 2015 Oxygen Sensing Coordinates Photomorphogenesis to Facilitate
603 Seedling Survival. *Curr. Biol.* **25**, 1483–1488. (doi:10.1016/j.cub.2015.03.060)

604

605
606 Supporting Information

607 Fig. S1: Mapman representation of the relative gene expression profile and related
608 metabolic processes in *rpoTmp* dark-grown seedlings

609 Fig. S2: Capacity of the KCN-sensitive pathway in etiolated seedlings

610 Fig. S3: Images of etiolated inhibitor-treated plants

611 Fig. S4: Expression changes of oxidative stress marker genes in etiolated mutant plants
612 compared to WT

613 Fig. S5: Total O₂ consumption rate in etiolated mutant plants

614 Fig. S6: Images of etiolated *rpoTmp*, *rpoTmp/anac017* and *rpoTmp/aox1a* mutant plants.

615 Table S1: Primers used in qPCR for expression analyses

616 Table S2: Microarray-based relative expression profiling of etiolated *rpoTmp* seedlings

617 Table S3: Microarray-based relative mitochondrial expression profiling of etiolated
618 *rpoTmp* seedlings and comparison with heatmap of mitochondrial-encoded DEGs of
619 light-grown adult plants [7]

620 Table S4: List of the 20 most down-regulated genes in etiolated *rpoTmp* seedlings versus
621 WT

622 Table S5: List of the common up and down DEGs (threshold 1.5) between *rpoTmp*
623 etiolated seedlings (this work) and light-grown adult plants [7]. The genes that are
624 among the top 20 DEGs in *rpoTmp* etiolated seedlings are underlined in yellow; the
625 genes that are among the top 20 DEGs in *rpoTmp* light-grown plants are marked with an
626 asterisk

627
628 Supplemental information to Methods.

629
630 **Figure Legends**

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
663 *rpoTmp* plants. Details as in **a**). Mitochondrial (mt) localization and protein function are also indicated on the right side of the table. “Mt stress” stands for “response to mitochondrial stress”. **d**) Functional sub-grouping of the 20 most up-regulated genes listed in **c**). The four groups identified were color-coded as in **c**). For details see text. **e**) Functional sub-grouping of the 20 most down-regulated genes (**Table S4**). The four groups identified were color-coded as in **c**). The terms “hormones” and “stress” for protein functions stand for “response to hormonal stimuli” and “response to stress”, respectively.

670 **Fig. 3 Plants that are defective in respiration present a triple-response-like phenotype. a)**
671 Median values of apical hook angle measurements were box-plotted for WT plants that were
672 grown in presence of 1 mM KCN or H₂O as mock-control and mutant *rpoTmp* plants that were
673 grown in presence of 1 mM SHAM or DMSO as mock-control. N (measured in four
674 independent analyses) corresponds to 178 for H₂O-treated WT plants and to 128 for KCN-
675 treated WT plants. N (measured in two independent analyses) corresponds to 148 for DMSO-
676 treated *rpoTmp* plants and to 85 for SHAM-treated *rpoTmp* plants. Details of box-plot
677 representation are explained in **Fig. 1b. b)** Images of etiolated mutant *rug3* and *atphb3* plants
678 were taken under a dissection microscope. Scale bar corresponds to 1 mm.

679 **Fig. 4 The triple-response-like phenotype in rpoTmp mutant plants requires AOX1a. a)**
680 Linear fold change (FC) induction of *AOX1a* mRNAs in mutant *rpoTmp* and
681 *rpoTmp/anac017* plants *versus* WT determined by RT-qPCR. The *AOX1a* expression levels
682 were normalized with the mean of *ACTIN2-8* expression, used as reference gene. The mean
683 values of two biological replicates are plotted. Error bars correspond to standard errors. **b)**
684 Protein extracts from etiolated WT, *rpoTmp*, *rpoTmp/anac017* plants (left panel) and WT,
685 *rpoTmp/aox1A*, *rpoTmp* plants (right panel) were fractionated and immunoblotted with
686 specific antisera against the mitochondrial AOX isoforms and the vacuolar protein V-ATPase
687 as loading control. Linear fold change variation of AOX protein levels in mutant *rpoTmp*
688 plants *versus* WT using V-ATPase for normalization is described in the table (bottom panel).
689 **c)** Capacity of the AOX-dependent pathway in etiolated mutant plants. AOX capacity was
690 calculated upon addition of 1 mM KCN into the measurement cell (final concentration). The
691 figure presents the ratio in percentage of the KCN-insensitive O₂ consumption rate to the total
692 consumption rate. Median values (thick horizontal lines) of N independent measurements (full
693 circles) were scatter-plotted for WT (N=19), *rpoTmp* (N=12), *rpoTmp/aox1a* (N=5), *aox1a*
694 (N=4) and *rpoTmp/anac017* plants (N=4). **d)** Images of etiolated plants: WT, *aox1a*, *rpoTmp*,
695 *rpoTmp/aox1a*, *rpoTmp/anac017* were taken under a dissection microscope. Scale bar
696

corresponds to 1 mm. e) Median values of apical hook angle measurements are box-plotted for WT, mutant *rpoTmp* and *rpoTmp/anac017* plants. Values were measured in 2 independent analyses. N =53 for WT plants; N =57 for *rpoTmp* plants; N= 57 for *rpoTmp/anac017* plants. Details of box-plot representation are as given in **Fig. 1b**.

697

698

699

700

701

702

703

704

705

706

707

708

709

710

711

712

713

714

715

**Fig. 5 Scheme of the proposed functional link between the respiratory chain capacities (KCN-sensitive and AOX-dependent) and the skoto-morphogenic programme.** In mitochondria the KCN-sensitive transport of electrons (ETC) from NADH is used for $O_2$ reduction and ATP production. Only a limited fraction of electrons is diverted to the AOX enzyme for reduction of $O_2$ without concomitant ATP production. In etiolated *rpoTmp*, *rug3*, *atphb3* or KCN-treated WT plants, the KCN-sensitive electron flux is strongly decreased inducing a strong dysfunctional stress (red indication). This stress activates the canonical (ANAC017-dependent) mitochondrial retrograde pathway that activates *AOX1a* expression. It may be supported by yet unknown factors (represented by a yellow oval with question mark). Additional AOX regulation by dysfunctional stress at the level of protein accumulation and modification might be possible (thick back arrow). As ultimate effect the capacity of the AOX-dependent chain is up-regulated and more electrons can be diverted by this pathway releasing the stress (green indication). The dysfunctional stress in combination with the highly increased AOX capacity, however, sends a signal *via* an unknown transmitter (represented by a grey circle with question mark) that triggers the formation of the triple-like-response (or parts of it).