Mitochondrial perturbation negatively affects auxin signaling

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Mitochondrial Perturbation Negatively Affects Auxin Signaling

Pavel Ivanov Kercheva,b,2

, Inge De Clercq a,b,2

, Jordi Deneckera,b,3

,Per Mühlenbocka,b,3,4

, Robert

Kumpfa,b

,

Long Nguyenc, Dominique Audenaert

a,b,c, Wim Dejonghe

a,b & Frank Van

Breusegema,b,1

a Department of Plant Systems Biology, VIB, Ghent University, B–9052 Gent, Belgium

b Department of Plant Biotechnology and Bioinformatics, Ghent University, B–9052 Gent,

Belgium

c VIB Compound Screening Facility, B–9052 Gent, Belgium

1 To whom correspondence should be addressed. E-mail [email protected]

ugent.be, tel. 32 9 331 39 20, fax 32 9 331 38 09.

2,3 These authors contributed equally to this work.

4 Current address: Department of Plant Protection Biology, Swedish University of Agricultural

Sciences, SE-230 53 Alnarp, Sweden

Running title: Impact of mitochondria on auxin signaling

Short Summary

Mitochondria play a crucial role in stress signaling upon perturbation of there function. Here,

we show the negative regulatory role of auxin in the process of mitochondria-to-nucleus

signaling of stress responses.

Molecular Plant Advance Access published June 5, 2014 at R

ijksuniversiteit Gent on June 13, 2014

http://mplant.oxfordjournals.org/

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ABSTRACT

Mitochondria are crucial players in the signaling and metabolic homeostasis of the plant

cell. The molecular components that orchestrate the underlying processes, however, are

largely unknown. Using a chemical biology approach, we exploited the responsiveness of

Arabidopsis UDP-glucosyltransferase-encoding UGT74E2 towards mitochondrial

perturbation in order to look for novel mechanisms regulating mitochondria-to-nucleus

communication. The most potent inducers of UGT74E2 shared a (2-furyl)acrylate (FAA)

substructure that negatively affected mitochondrial function and was identified before

as an auxin transcriptional inhibitor. Based on these premises, we demonstrated that

perturbed mitochondria negatively affect the auxin signaling machinery. Moreover,

chemical perturbation of polar auxin transport and auxin biosynthesis was sufficient to

induce mitochondrial retrograde markers and their transcript abundance was

constitutively elevated in the absence of the auxin transcriptional activators ARF7 and

ARF19.

Keywords: mitochondrial perturbation; auxin signaling; retrograde communication

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INTRODUCTION

Mitochondria play a crucial role in maintaining cellular homeostasis by integrating a plethora

of signaling and metabolic pathways that operate between different cellular compartments

(Dutilleul et al., 2003; Noctor et al., 2007; Sweetlove et al., 2007). Apart from the production

of energy-rich adenosine triphosphate (ATP) coupled to the mitochondrial electron transport

chain (mETC), numerous mitochondrial biosynthetic reactions deliver metabolic

intermediates that are transported and utilized throughout the cell to sustain plant metabolism

(Day, 2004; Millar et al., 2011). The flux via the photorespiratory pathway, for example,

depends on mitochondrial glycine decarboxylase and serine hydroxymethyltransferase

activities and thus couples mitochondrial function to carbon assimilation (Foyer et al., 2009).

Moreover, the terminal step in the synthesis of ascorbic acid, the most abundant cellular redox

buffer, is coupled to the mETC (Bartoli et al., 2000). This diverse array of mitochondrial

functions underlies their high functional and morphological plasticity which is required to

execute specific developmental and stress programs.

Despite the fact that plant mitochondria contain their own genome, over 90% of the

mitochondrial proteins are encoded in the nucleus. Therefore, the synthesis and import of

nuclear-encoded mitochondrial proteins have to be tightly coordinated with the metabolic and

signaling requirements of the cell and further subjected to feedback regulatory mechanisms

reflecting the functional state of mitochondria (Woodson and Chory, 2008). The

communication between mitochondria and the nuclear transcription machinery, often referred

to as mitochondrial retrograde regulation (MRR), ensures adequate gene expression at the

mitochondrial and nuclear levels to maintain organellar as well as whole-cellular function. In

particular, a function has been attributed to MRR during plants responses to stresses that alter

or perturb the functioning of mitochondria (Rhoads and Subbaiah, 2007). The precise

molecular mechanism by which plant mitochondria signal to the nucleus are far from

elucidated but reactive oxygen species (ROS), calcium, and changes of energy and redox

status are likely to be involved in initiating and/or transmitting mitochondrial signals to the

nucleus (Rhoads and Subbaiah, 2007; Rhoads et al., 2006; Schwarzlander and Finkemeier,

2013; Subbaiah et al., 1998; Vanlerberghe et al., 2002).

Chemical and genetic perturbation of mitochondrial function has been extensively

exploited as a model system to study MRR (Dojcinovic et al., 2005; Schwarzlander et al.,

2012; Zarkovic et al., 2005) and led to the identification of a set of genes commonly referred




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to as Mitochondrial Dysfunction Stimulon (MDS) that are consistently and reproducibly

induced under such conditions (De Clercq et al., 2013; Van Aken et al., 2007; Van Aken and

Whelan, 2012). Perhaps the best characterized of these transcripts is ALTERNATVE OXIDASE

1a (AOX1a) encoding a cyanide-insensitive terminal oxidase that functions alongside

cytochrome c oxidase (Moller, 2001). Together with the alternative NADH dehydrogenases,

AOX forms a complete respiratory chain that does not generate a proton gradient across the

inner mitochondrial membrane and subsequently does not contribute to ATP production.

Similar to AOX1a, transcripts encoding alternative NAD dehydrogenases (NDB4) are also

part of the MDS. Redirection of electron flow via the alternative respiratory pathway

minimizes ROS accumulation by preventing the overreduction of the mETC (Cvetkovska and

Vanlerberghe, 2012; Maxwell et al., 1999). This can be crucial when the cytochrome pathway

is impaired or overreduced by adverse environmental conditions (Hernández et al., 1993;

Parsons et al., 1999; Prasad et al., 1994). The functions of other MDS genes extend beyond

mitochondrial homeostasis and comprise, amongst others, N-acetyltransferases, cytochrome

P450 enzymes, transmembrane transporters, etc. UGT74E2, for example, encodes a UDP-

glycosyltransferase and is one of the most strongly induced MDS genes. In vitro enzymatic

assays with recombinant UGT74E2 identified the auxin indole-3-butyric acid (IBA) as its

favored substrate. In vivo perturbation of auxin homeostasis through overexpression of

UGTE74E2 was associated with distinct morphological changes (increased shoot branching

and compact rosette) and enhanced survival under drought and salt stress (Tognetti et al.,

2010).

Various signaling components required for the information flow from mitochondria to

the nucleus have been identified in yeast and animal systems. Apart from the evolutionary

conserved RTG and NF-κB pathways that activate among others glycolysis upon impaired

respiration, other retrograde pathways that trigger various responses to specific mitochondrial

defects have been reported (Butow and Avadhani, 2004; Jazwinski and Kriete, 2012; Liu and

Butow, 2006; Srinivasan et al., 2010). In contrast, protein components playing a role in plant

MRR are largely unknown. The only identified players so far are cyclin-dependent kinase E1

(CDKE1), and the transcription factors ABSCISIC ACID INSENSITIVE 4 (ABI4),

WRKY40, and NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION

ACTIVATION FACTOR/CUP-SHAPED COTYLEDON 13 and 17 (ANAC013 and

ANAC017) (De Clercq et al., 2013; Giraud et al., 2009; Ng et al., 2013a; Ng et al., 2013b;

Van Aken et al., 2013). ANAC013 and ANAC017 target a conserved cis-regulatory element




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commonly found in the promoter regions of the MDS genes which is necessary and sufficient

for gene expression upon mitochondrial perturbation. ANAC013 and ANAC017 contain

transmembrane domains required for their anchoring to the endoplasmic reticulum (ER) and

ANAC017 localizes at the junctions of ER and F-actin. Upon proteolytic cleavage, likely

mediated by rhomboid proteases, ANAC017 migrates into the nucleus to activate gene

expression. Intimate contacts between mitochondria and ER have been documented in yeast

and animal systems and are required for interorganellar Ca2+

exchange. The link between

mitochondria and ER in the activation of the MRR has recently emerged in Arabidopsis.

Under salt stress, the induction of AOX1a is dependent on the Ca2+

-mediated communication

between ER and mitochondria (Vanderauwera et al., 2012). The involvement of the ER during

stress response is dependent on the unfolded protein response (UPR), which is evolutionary

conserved and maintains cellular function upon accumulation of misfolded proteins (Urade,

2007). UPR activation, for example, negatively impacts the auxin signaling machinery and its

severity is reduced in mutants affected in ER-localized auxin transporters (Chen et al., 2014).

The signaling pathways of plant MRR have been extensively studied using AOX1a as

a model system (Dojcinovic et al., 2005; Zarkovic et al., 2005; Giraud et al., 2009). However,

mitochondrial perturbation induces several other genes, among which one of the most

strongly induced is UGT74E2. Using the distinct advantages offered by small molecule

approaches, we performed a chemical screen looking for activators of UGT74E2. The two

most potent hit compounds shared a common 3-(2-furyl) acrylate substructure and

surprisingly inhibited auxin signaling, implying a mechanistic link between mitochondrial

function and auxin signaling pathways.




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RESULTS

Chemical Screen to Identify Compounds Activating Mitochondrial Retrograde Signaling

Pathways

We have previously shown that a set of nuclear-encoded genes which we referred to as

Mitochondrial Dysfunction Stimulon (MDS), are strongly induced upon mitochondrial

perturbation caused by chemical or genetic factors (De Clercq et al., 2013). The MDS is co-

regulated by NAC transcription factors (ANAC013 and ANAC017) targeting the conserved

Mitochondrial Dysfunction Motif (MDM) cis-regulatory element present in their promoter

regions (De Clercq et al., 2013; Ng et al., 2013b). To further explore the molecular

mechanism by which perturbed mitochondria signal to the nucleus, we designed a high-

throughput chemical screen based on the luciferase (LUC) reporter gene driven by the

UGT74E2 promoter (pUGT74E2:LUC). UGT74E2, being part of the MDS, is strongly

responsive to mitochondrial perturbations and contains the MDM cis-regulatory element in its

promoter region, making it a good marker to identify novel players in mitochondrial

retrograde signaling. To counterselect for chemicals that trigger a general stress response and

thus unspecifically induce UGT74E2 expression (Tognetti et al., 2010), we used the

chlorophyll fluorescence parameter photosystem II (PSII) maximum efficiency (Fv'/Fm') that is

a stable and sensitive stress marker (Baker, 2008; Mishra et al., 2011). Chemicals that

negatively affected photosynthetic performance, reflected by a Fv'/Fm' drop, were not retained

as potential hits (Supplemental Figure 1). Following a primary screen and two validation

rounds, we isolated seventeen hit compounds that induced the luciferase signal in

pUGT74E2:LUC lines above the control threshold without impairing PSII maximum

efficiency (Figure 1A, Supplemental Figure 1).

The two most potent hit compounds shared a characteristic (2-furyl)acrylate moiety

fused via an amide linkage to the rest of the molecule (Figure 1B). Both chemicals acted in a

dose-dependent manner and their ability to enhance UGT74E2 transcript abundances was

confirmed in a wild-type (Col-0) background by quantitative real-time (qRT)-PCR analysis

(Supplemental Figure 2). We then tested whether 3-(2-furyl) acrylic acid (FAA), which is

present as a substructure in both hit compounds, is sufficient to activate pUGT74E2. FAA

(20 µM) also strongly induced the luciferase signal in pUGT74E2:LUC seedlings without a

negative impact on the the Fv'/Fm' ratio (Figure 1C, Figure 1D).

FAA Treatment Activates the Mitochondrial Dysfunction Stimulon




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We then tested whether FAA activates other genes belonging to the MDS apart from

UGT74E2 by analyzing their transcript abundance in chemically treated seedlings using qRT-

PCR analysis. Treatment of Arabidopsis seedlings with 20 µM FAA induced the majority of

the tested MDS genes; the most strongly up-regulated transcripts were UGT74E2, UPOX,

AOX1a and sHsp23.5 (Figure 2A), suggesting that FAA activates mitochondrial retrograde

signaling pathways. To further corroborate these findings, we mined the publicly available

microarray data for perturbation experiments that had used structurally similar chemicals. We

identified an experiment in which global gene expression changes were analyzed upon

treatment of seven-day-old Arabidopsis seedlings with 10 µM furyl acrylate ester of a

thiadiazole heterocycle (Armstrong et al., 2004). The FAA part of the molecule was shown to

render its bioactivity (Sungur et al., 2007). Comparing the expression of the MDS genes after

treatment with the furyl acrylate ester and after treatment with chemicals perturbing

mitochondrial functions (rotenone, Antimycin A, and oligomycin) revealed a similar

transcriptional pattern (Figure 2B). Taken together, these results suggest that FAA-dependent

transcriptional activation is not restricted to the induction of UGT74E2, but rather mimics the

responses provoked by disturbance of mitochondrial function.

We further tested this hypothesis by using the fluorescent dye MitoTracker Red CM-H2XRos

which stains actively respiring mitochondria. Mitochondrial staining in root tip cells was

severely decreased upon pretreatment with 50 µM Antimycin A (AA) (Figure 2C). AA inhibits

the mitochondrial respiratory chain by binding to the quinone reduction site of Complex III.

Similarly, pretreatment with 20 µM FAA strongly decreased the fluorescent signal, pointing

toward a negative impact of FAA on mitochondrial function (Figure 2C).

FAA Activates the Mitochondrial Dysfunction Motif cis-Regulatory Element

The presence of the Mitochondrial Dysfunction Motif (MDM) cis-regulatory element was

shown to be necessary and sufficient for full induction of the MDS genes upon various

mitochondrial perturbations. The MDM consensus (CTTGNNNNNCA[AC]G) is present

twice in the AOX1a promoter and once in the UGT74E2 promoter (De Clercq et al., 2013). To

test whether FAA mediates promoter activation through this sequence, we used luciferase

reporter lines harboring either a hexamer of the MDM sequence from the UGT74E2 promoter

(6xMDM[UGT74E2]) or one of the MDM sequences from the AOX1a promoter

(6xMDM1[AOX1a]). The luminescent signal detected upon exposure of seedlings carrying either

of the promoter constructs to FAA was strongly induced in comparison to mock-treated




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controls (Figure 3). The treatment had no effect on the signal recorded from lines carrying a

mutated MDM sequence (6xMDM1mut[AOX1a]), implying that induction of the MDS

promoters by FAA is likely mediated through the conserved MDM cis-regulatory element co-

regulated by ANAC013 and ANAC017.

Mitochondrial Perturbation Negatively Impacts Auxin Signaling

FAA was previously characterized as the active moiety of a chemical that inhibits auxin-

dependent transcriptional activation (Armstrong et al., 2004; Sungur et al., 2007). Consistent

with these results, under our experimental conditions, FAA attenuated indole-3-acetic acid

(IAA)-dependent induction of the DR5:GUS reporter in Arabidopsis root tips (Ulmasov et al.,

1997) (Figure 4A). The fact that FAA affects mitochondrial function prompted us to

investigate whether the negative impact on the auxin signaling machinery by FAA could be a

direct consequence of mitochondrial perturbation. Therefore, we used AA to block the mETC

and perturb mitochondria. Similar to FAA, AA was able to abolish the induction of the

DR5:GUS reporter in the presence of auxin (Figure 4A). We further explored how

mitochondrial perturbation affects auxin signaling using the VENUS fluorescent protein fused

to the degron motif of Aux/IAA28 (DII-VENUS), which is degraded in response to auxin

stimuli (Brunoud et al., 2012). An inverse relation between auxin abundance and reporter

fluorescent intensity was demonstrated following IAA treatment, which rapidly reduced the

DII-VENUS signal intensity (Figure 4B and C). The presence of AA, however, counteracted

the auxin-triggered degradation of DII-VENUS and stabilized the fluorescent signal

(Figure 4B and C). This effect was not observed in the presence of FAA. Complementary

support for a direct effect of mitochondrial impairment on auxin signaling was derived from

the observation that within a compendium of microarray datasets from mitochondrial

perturbation experiments, auxin marker genes were consistently repressed (Supplemental

Figure 3).

Perturbation of Auxin Homeostasis Mimics the Effect of Mitochondrial Perturbation

The fact that mitochondrial perturbation negatively impacted auxin signaling prompted us to

test whether perturbation of auxin homeostasis might have an impact on the MDS genes. The

polar auxin transport inhibitors (NPA, TIBA, Gravacin, and Quercetin) and the auxin

biosynthesis inhibitor L-Kynurenine (He et al., 2011) were all able to strongly activate the

promoters of UGTE74E2 and AOX1a (Figure 5A). We further tested whether these chemicals

can induce the artificial MDM reporter constructs in order to evaluate whether the activation




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of the full promoters might be mediated through the MDM cis-regulatory element. A

significant induction of the 6xMDM[UGT74E2] promoter was recorded only upon treatment

with TIBA (Figure 5B) like this activation was modest in comparison to the effect of FAA.

The 6xMDM1[AOX1a] reporter was similarly induced by TIBA, but also by Gravacin and

Quercetin (Figure 5B). Again the induction was less pronounced compared to FAA

treatments.

To test whether genetic perturbation of auxin signaling induces MDS genes, we

quantified their transcript levels by qRT-PCR in a range of auxin-related mutants. No

consistent effects were observed in mutants impaired in components required for auxin

perception (tir1), Aux/IAA transcriptional repression (axr3), polar auxin transport (pin2 and

pin3 pin7), regulation of polar auxin transport (pid wag1 wag2), and intercellular trafficking

(gnom) (Supplemental Figure 4). Interestingly, however, the absence of auxin-regulated

transcriptional activators AUXIN RESPONSE FACTOR 7 and 19 (ARF7 and ARF19) in the

double mutant arf7 arf19 led to strong constitutive expression of the majority of MDS genes

(Figure 6).

Activation of Auxin Signaling Modulates the Effects of Mitochondrial Perturbation

To further investigate the interplay between mitochondrial function and auxin homeostasis,

we pretreated Arabidopsis seedlings carrying pUGT74E2 and pAOX1a luciferase constructs

with 5 µM IAA before disrupting mitochondrial function with 50 µM AA. Regardless of the

presence of exogenous auxin, AA activated both promoters to fully induced levels

(Supplemental Figure 5). However, when seedlings harboring the artificial MDM reporter

constructs were treated in a similar way, the induction of both 6xMDM[UGT74E2] and

6xMDM1[AOX1a] was alleviated by auxin (Supplemental Figure 5), suggesting that the

signaling cascades activated upon mitochondrial perturbation could be fine-tuned by auxin.

Several high-flux metabolic pathways operate in mitochondria and some of them are

intimately linked with the respiratory activity that partly regulates mitochondria-to-nucleus

communication. To assess how auxin could affect mitochondrial function and subsequently

modify AA-triggered stress responses, we profiled polar metabolites (by GC-MS) in

Arabidopsis seedlings treated with AA only, or pretreated with auxin before AA treatment.

The abundance of the majority of the measured metabolites induced by AA was not affected

by IAA pretreatment (Supplemental Figure 5). Nevertheless, a small number of metabolites

were differentially regulated. The AA-induced increase of serine and glycine content was




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significantly alleviated in the presence of auxin (Supplemental Figure 5). Similarly, elevated

glycine and serine levels were also detected in mitochondrial prohibitin 3 mutants (Van Aken

et al., 2007) (Supplemental Figure 6). Taken together, these results suggest that serine and

glycine accumulation might be a general consequence of mitochondrial perturbation, which

can be fine-tuned by auxin.

DISCUSSION

We have used a small molecule approach to identify novel plant mitochondrial retrograde

signaling components. In contrast to the majority of previous studies which used AOX1a as a

marker for MRR (Giraud et al., 2009; Ng et al., 2013a; Ng et al., 2013b), we exploited the

responsiveness of UGT74E2 towards mitochondrial perturbation and selected for compounds

that activate the UGT74E2 promoter without negatively impacting the chlorophyll fluorescent

parameter Fv’/Fm’ which is widely used as an indicator of stress response (Baker, 2008;

Mishra et al., 2011). UGT74E2 is induced by a variety of abiotic and biotic stress conditions

(Tognetti et al., 2010) and by monitoring Fv’/Fm’ values we aimed to discard cytotoxic

chemicals.

FAA has been previously shown to impair auxin-triggered Aux/IAA protein degradation and

subsequent auxin-dependent gene expression without interfering with the interaction between

IAA7/AXR2 and the SCFTIR1

complex, leaving the cellular targets and the mode of action of

FAA enigmatic (Sungur et al., 2007). Here, we further revealed that FAA induces the MDS

genes among which is the well-characterized MRR marker AOX1a; implying that FAA

treatment might directly or indirectly affect mitochondrial function. To unequivocally test

whether mitochondrial perturbation directly negatively affects auxin signaling, we used AA, a

specific inhibitor of mitochondrial Complex III, and demonstrated that auxin signaling is

attenuated upon AA treatment through a stabilization of Aux/IAA repressors (Figure 4). Thus,

even though we cannot exclude the possibility that FAA is not targeting mitochondria directly,

its ultimate impact on mitochondria is most probably linked to the observed attenuation of

auxin signaling. Intriguingly, under our experimental conditions, FAA failed to stabilize the

DII-VENUS reporter. This is at first sight in conflict with the previously reported FAA-

triggered stabilization of the IAA17/AXR3-GUS fusion protein in the presence of exogenous

auxin (Sungur et al., 2007). The heat shock-inducible production of the reporter protein in the

HS:AXR3NT-GUS lines (Sungur et al., 2007), the different degron motifs used in both




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constructs and the higher sensitivity of DII-VENUS could explain the different responses of

both reporters.

Perturbation of mitochondrial function (e.g. by AA) has previously been associated with

enhanced mitochondrial ROS levels (Maxwell et al., 1999). The interaction between oxidative

stress signaling and auxin has surfaced in the last years, but the molecular knowledge is

limited and mainly based on circumstantial evidence. The perturbed redox homeostasis in the

triple glutathione-deficient mutant (cad2 ntra ntrb) leads to developmental defects

reminiscent of the phenotype of several mutants affected in auxin transport or biosynthesis

(Bashandy et al., 2010). Moreover, apoplastic ROS negatively affect the abundance of several

transcripts encoding auxin receptors, Aux/IAA transcriptional repressors, and auxin efflux and

influx carriers without noticeable changes in IAA levels (Blomster et al., 2011). The widely

used auxin transport inhibitor TIBA mimics the negative effect of apoplastic ROS on auxin-

responsive gene expression (Blomster et al., 2011). Our analysis displayed that conditions

perturbing mitochondrial function provoke similar auxin-related transcriptional changes

(Supplemental Figure 3). Similar repression of auxin signaling was observed in meristematic

tissues under mild osmotic stress (Skirycz et al., 2010). Meristems are crucial in determining

plant growth with auxin orchestrating many of the underlying processes and bear

mitochondria with altered shapes (Skirycz et al., 2010). Moreover, the global transcriptional

pattern of the proliferating leaves under mild osmotic stress conditions displayed a

characteristic ethylene signature, pointing towards an auxin-ethylene crosstalk. An intriguing

aspect in this context is the tissue-specific regulation of UGT74E2 and other MDS genes.

During adverse environmental conditions, they are specifically induced in proliferating

tissues, which potentially implies a mechanistic link to the maintenance of mitochondrial

function and cell proliferation. In this respect, the fact that UGT74E2 glycosylates IBA and its

overexpression perturbs auxin homeostasis implies that induction of UGT74E2 might be

instrumental to rearrange auxin pools in the meristematic tissues during stress response.

Interestingly, prohibitin mutants which also have abnormally shaped mitochondria

specifically induce the MDS genes, show lower meristematic activity and display

developmental defects (Van Aken et al., 2007). A common theme between various adverse

conditions is a general growth retardation (Potters et al., 2007; Pasternak et al., 2005) and

UGT74E2 and the other MDS (e.g. ABCB4 is known as an auxin transporter, Terasaka et al.,

2005)) genes might be involved in this response. Recently, it was also found that auxins

counteract photorespiration-dependent cell death, indicating that auxin plays a role in




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https://www.researchgate.net/publication/51721443_Apoplastic_Reactive_Oxygen_Species_Transiently_Decrease_Auxin_Signaling_and_Cause_Stress-Induced_Morphogenic_Response_in_Arabidopsis?el=1_x_8&enrichId=rgreq-b274a4e2-0d7b-4b9b-bef2-716de286e2b7&enrichSource=Y292ZXJQYWdlOzI2MjkzMDk0NztBUzoxMDc1Mjk0OTc0ODUzMTJAMTQwMjY0ODQzODY1OQ==

https://www.researchgate.net/publication/41465990_Interplay_between_the_NADP-Linked_Thioredoxin_and_Glutathione_Systems_in_Arabidopsis_Auxin_Signaling?el=1_x_8&enrichId=rgreq-b274a4e2-0d7b-4b9b-bef2-716de286e2b7&enrichSource=Y292ZXJQYWdlOzI2MjkzMDk0NztBUzoxMDc1Mjk0OTc0ODUzMTJAMTQwMjY0ODQzODY1OQ==

12

adapting tissues to ROS challenge (Kerchev et al., 2014). It is tempting to speculate that this

adaptation occurs through the fine tuning of mitochondrial function through regulation of the

MDS genes. The constitutive induction of the MDS genes in the arf7arf19 knock-out mutant is

highly intriguing and deserves further attention in the future. ARF7 and ARF19 function

together not only in regulating auxin signaling, but also participate in ethylene responses and

the coordination of lipid remodeling during phosphate starvation (Li et al., 2006; Narise et al.,

2010). The attenuated accumulation of nonphosphorous glycolipids under limited phosphate

conditions in arf7arf19 hints towards a role in stress response which is further supported by

our observations.

The role of auxin homeostasis during stress responses has recently emerged in the light

of the ROS-auxin crosstalk (Tognetti et al., 2012). Auxin depends on a complex signaling and

transport machinery distributing it differentially within plant tissues (Vanneste and Friml,

2009). The way plants perceive stress responses is equally modulated by tissue-specific

mechanisms, that are likely to be co-orchestrated by mitochondria. The fact that

mitochondrial perturbation negatively impacts auxin signaling will for sure have implications

for our understanding how growth is rearranged during adverse environmental conditions.

METHODS

Plant Material and Growth Conditions

Promoter:LUC (pAOX1a:LUC, pUGT74E2:LUC, 6xMDM1[AOX1a]:LUC and

6xMDM1mut[AOX1a]:LUC) Arabidopsis lines were described in De Clercq et al. (2013).

Unless otherwise stated, seeds were surface-sterilized with chlorine gas, stratified at 4 ºC for

3-4 days, germinated on half-strength Murashige and Skoog (½MS ) medium containing 1%

(w/v) sucrose solidified with 0.8% (w/v) agar and grown for 14 days until stage 1.04 (Boyes

et al., 2001). Plants were grown under long-day conditions (16h/8h light/dark photoperiod, 21

ºC, 150 µE m-2

s-1

) and treated as described for the individual experiments.

Generation of Transgenic Arabidopsis Plants

The artificial promoter construct (6xMDM[UGT74E2]) was synthesized as an oligonucleotide

(Invitrogen), annealed by heating followed by gradual cooling, and subsequently, cloned into

pDONRP4-P1r. The 6xMDM[UGT74E2] consists of a six-tandem repeat of the MDM cis-

regulatory element from the UGT74E2 promoter with 5-bp flanking sequences at each end

(ACAACTTTGTATAGAAAAGTTGcacatCTTGGTCGCCACGgaacacacatCTTGGTCGCCA




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https://www.researchgate.net/publication/50890727_Stress_homeostasis_-_the_redox_and_auxin_perspective_Plant_Cell_Environ?el=1_x_8&enrichId=rgreq-b274a4e2-0d7b-4b9b-bef2-716de286e2b7&enrichSource=Y292ZXJQYWdlOzI2MjkzMDk0NztBUzoxMDc1Mjk0OTc0ODUzMTJAMTQwMjY0ODQzODY1OQ==

13

CGgaacacacatCTTGGTCGCCACGgaacacacatCTTGGTCGCCACGgaacacacatCTTGGTCG

CCACGgaacacacatCTTGGTCGCCACGgaacaCAAGTTTGTACAAAAAAGCAGT). The

fusion of this construct upstream of the minimal Cauliflower mosaic virus 35S promoter and

the LUC reporter gene, and its subsequent transformation in Arabidopsis Col-0 was performed

as described in De Clercq et al. (2013). The pUGT74E2:LUC line used in this screen, was

described in Tognetti et al., 2010.

Chemical Screen

Surface-sterilized pUGT74E2:LUC seeds were distributed in 96-well white CulturePlate-96

(Perkin-Elmer) (5-10 seeds/well) containing 150 µL ½MS medium (0.5% w/v sucrose) per

well. Following a cold treatment (3-4 days at 4ºC), parafilm®-sealed plates were transferred

to a growth chamber with controlled environmental conditions (16h/8h light/dark

photoperiod, 100 μmol m−2

s−1 light intensity, 21ºC, 50% relative humidity). Nine days post

germination 12,000 chemicals from the DIVERSet screening library (ChemBridge

Corporation, USA) were added to individual wells to a final concentration of 50 µM (1%

(v/v) DMSO). A treatment with 1% (v/v) DMSO and 10 mM H2O2 (1% (v/v) DMSO) were

used as a negative and positive control, respectively. Twenty four hours following chemical

addition, the PSII maximum efficiency (Fv'/Fm') was recorded. Subsequently, 100 µl luciferin

(Promega ONE-Glo™ Luciferase Assay System) was added to individual wells and after 8-

min dark-incubation, luminescence was acquired with a LUMIstar Galaxy luminometer

(BMG labtechnologies, Offenburg, Germany).

Chlorophyll fluorescence imaging

Chlorophyll fluorescence measurements were performed using an Imaging-PAM M-Series

chlorophyll fluorometer (Heinz Walz, Germany) on light-adapted plants. In the absence of

actinic illumination the minimal fluorescence level of light-adapted plants (F'o) was

determined. For assessment of the maximum fluorescence yield of light-adapted plants (F'm) a

saturation pulse of blue light (450 nm) with an intensity of ca. 2800 µmol m-2

s-1

was applied

for 1 second. Both fluorescence parameters were used to calculate the PSII maximum

efficiency (F'v/F'm=(F'm-F'o)/F'm) (Baker, 2008).

Chemical Treatments

3-(2-furyl)acrylic acid (FAA), indole-3-acetic acid (IAA), 2,3,5-triiodobenzoic acid (TIBA),

1-N-naphthylphthalamic acid (NPA), L-kynurenine, antimycin A (AA), and quercetin were




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14

purchased from Sigma-Aldrich. Gravacin (3-[5-(3,4-dichlorophenyl)-2-furyl]acrylic acid) was

obtained from ChemBridge Corporation (USA). Chemicals were applied from 50 mM stock

solutions in DMSO to final concentrations described in the text. Mock treatments of 0.1%

[v/v] DMSO were used as a control.

GC-MS Profiling

For metabolite profiling, 15 Arabidopsis Col-0 seeds were germinated in 24-well plates

containing 2 ml ½MS medium (0.5 % w/v sucrose) and grown for 14 days. Whole seedlings

(~60 mg fresh weight) were homogenized with a retsch mill and processed as described

before (Desbrosses et al., 2005; Kaplan et al., 2004). Subsequently, dried aliquots from the

extracted samples were incubated with 40 µl methylhydroxylamine hydrochloride (20 mg/ml

in pyridine) for 1.5h at 30°C. The samples were further incubated with 70 µl N-methyl-N-

(trimethylsilyl)trifluoroacetamide (MSTFA) and 10 µl alkane retention mixture for 30 min at

37°C. HP6890 gas chromatograph coupled to HP5973 quadrupole mass detector was used to

analyze the samples. Following a splitless injection (1 µl) at 230°C, constant helium flow (1

ml/min) was used to introduce the vaporized sample to a 30 m capillary column (FactorFour

VF-5ms column, Varian). The oven temperature was maintained constant at 70°C for 5 min,

and then ramped to 325°C at a rate of 5°C/min. The detector was operating in a scanning

mode (60 to 600 atomic mass units; 7.8 to 68.8 min).

Peak alignment and integration were performed using the functions implemented in

the xcms package with the following parameters: fwhm=3, max=300, snthresh=2, step=0.1,

steps=2, mzdiff=0.5. The peaks of interest were annotated with the AMDIS software using a

mass spectra library (Q_MSRI_ID) from the Golm Metabolome Database (Schauer et al.,

2005).

Luciferase Analysis

Luciferase measurements were performed as described before (De Clercq et al., 2013).

Quantitative RT-PCR

Total RNA was prepared with TRIzol reagent (Invitrogen) and total RNA concentrations were

determined with an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE).

First-strand cDNA was prepared from 2 µg of total RNA with the cDNA synthesis kit (Bio-

rad) according to the manufacturer’s instructions. Half a microliter of a 1:8 diluted first-strand




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15

cDNA was used as a template in the subsequent PCR, which was performed on the iCycler iQ

(BioRad, Hercules, CA) or on the LightCycler 480 (Roche Diagnostics) in 384-well plates

with the SYBR Green I Master kit (Roche Diagnostics), according to the manufacturer’s

instructions. All individual reactions were done in triplicate. Primers were designed with the

Universal ProbeLibrary Assay Design center ProbeFinder software (Roche; http://www.roche-

applied-science.com/). Primers used to amplify the MDS genes are shown in Supplemental

Table 1. The Δ cycle treshold was applied for relative expression analysis using three

reference genes (ACTIN-RELATED PROTEIN7 [ARP7], UBIQUITIN-CONJUGATING

ENZYME 21 [UBC21] (At5g25760), and At2g28390) for normalization.

Histological Analysis

To analyze DR5:GUS activity 5-day-old DR5:GUS seedlings were transferred from vertical

½MS (0.8 % agar) plates to liquid ½MS medium supplemented with 5 µM IAA, 20 µM FAA

or 50 µM AA or combinations of 5 µM IAA plus either 20 µM FAA or 50 µM AA in a 6-well

plate format. Following a 4 h incubation, GUS assays were performed as described previously

(Beeckman and Engler, 1994). Images were acquired at a 20-fold magnification (Zeiss,

AxioImager).

Five-day-old Arabidopsis seedlings grown on vertical ½MS (0.8 % agar) plates were

used to analyze the effect of FAA and AA on the DII VENUS reporter. For FAA and AA

treatment, seedlings were incubated for 5 min with 20 µM FAA or 50 µM AA before being

subjected to the following treatments. The seedlings were further placed in glass bottomed

dishes and covered with media containing 50 nM IAA, 20 µM FAA or 50 µM AA or

combinations of 50 nM IAA plus either 20 µM or 50 µM AA. The time series started 5 min

after the seedlings had been placed in contact with the media and captured over 35 min (every

5min) with a Zeiss 710 confocal microscope (20x objective). ZEN2009 software was used to

process the images.

For mitochondrial staining 6-day-old Arabidopsis Col0 seedlings grown vertically

were transferred to liquid ½MS media containing 0.1% (v/v) DMSO, 50 µM AA (0.1% [v/v]

DMSO) and 20 µM FAA (0.1% [v/v] DMSO). Following 1h incubation, seedlings were

transferred to equivalent solutions containing Mito Tracker Red CMH2Xros (M-7513,

Molecular Probes) in a final concentration of 250 nM and further incubated for an additional

hour. Fluorescence from five to ten seedlings was detected using a Zeiss confocal microscope.




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Meta-analysis of Transcriptome Data

For meta-analyses of public microarray data, normalized expression values were obtained

from Genevestigator (Zimmermann et al., 2008): AA treatment, AT-00561, GSE41136, Ng et

al. (2013a); FAA – ester treatment, AT-00167, GSE1491, Armstrong et al. (2004); rotenone

and Oligomycin treatment, AT-00522, GSE3709, Clifton et al. (2005). The exported data were

clustered based on Pearson correlation using the TMEV4 software.

SUPPLEMENTARY DATA

Supplementary Data are available at Molecular Plant Online.

FUNDING

This work was supported by Ghent University (Multidisciplinary Research Partnership

“Biotechnology for a Sustainable Economy”, Grant 01MRB510W), the Agency for Innovation

by Science and Technology (IWT; Phoenix project 070347), VIB and Marie Curie

(OMICS@VIB PCOFUND-GA-2010-267139), and the Interuniversity Attraction Poles

Programme (IUAP P7/29 “MARS”), initiated by the Belgian Science Policy Office. This

work was also supported by a Marie Currie fellowship to P.M., by FWO (Fonds

Wetenschappelijk Onderzoek – Vlaanderen) through a Pegasus postdoctoral grant funded to

P.K and by IWT through a predoctoral fellowship to I.D.C.

ACKNOWLEDGEMENTS

We thank Kristof Verleye for fruitful discussions and technical assistance. We also thank

Steffen Vanneste and Michael Metzlaff for discussions and suggestions, Wei Xuan for seed

material, and Annick Bleys for help in preparing the manuscript. No conflict of interest

declared.

FIGURE LEGENDS




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Figure 1. Chemical Screen to Identify Compounds Activating Mitochondrial Retrograde

Signalling Pathways.

(A) A schematic representation of the chemical screen.

(B) Chemical formulas of the two most potent hit compounds identified in the screen

(Compound 2 and Compound 3) and the shared substructure (3-(2-Furyl)acrylic acid (FAA)).

(C) Induction of pUGT74E2 by Compound 2, Compound 3, and FAA. 10 mM H202 was used

as a positive control. The 1,5-kb upstream sequences of UGT74E2 was fused upstream of the

luciferase gene and stably transformed in Arabidopsis plants. Luciferase activity following

chemical (20 µM) and mock (DMSO) treatment is shown (±SD; n=8 biological replicates).

Different letters represent statistically significant differences according to one-way ANOVA

with Tukey’s post hoc test (p<0.05). RLU, relative luminescence units.

(D) Photosystem II maximum efficiency (Fv'/Fm') of seedlings treated with chemicals (20

µM), 10 mM H202 or DMSO for 24 h (left panel). Fv'/Fm' levels are depicted by color codes;

blue represents high values and yellow-green corresponds to low values. Quantification of

Fv'/Fm' values in chemically treated seedlings (right panel). Bars represent means of 8

replicates ± SD. Different letters represent statistically significant differences according to

one-way ANOVA with Tukey’s post hoc test (p<0.05).

Figure 2. FAA Negatively Impacts Mitochondria.

(A) Induction of MDS genes following FAA treatment. Transcript levels of fourteen of the

MDS genes were analyzed by qRT-PCR in 10-day-old wild-type Arabidopsis plants grown as

liquid cultures and treated with 20 µM FAA or DMSO for 24 h. Bars represent average fold

changes relative to the mock treatment from four biological replicates (± SE). Asterisks

indicate significant differences to the mock (Student’s t test; * p<0.05, ** p<0.01, and ***

p<0.001).




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(B) Comparison of publicly available expression profiles of the MDS genes between FAA

ester treatment and treatments with mitochondrial blockers. Color codes represent linear fold

changes relative to control extracted from Genevestigator and hierarchically clustered using

Pearson correlation as a distance measure in the TMEV4 software. Antimycin A: GSE41136

(Ng et al., 2013b); oligomycin and rotenone: GSE3709 (Clifton et al., 2005); FAA ester:

GSE1491 (Armstrong et al., 2004).

(C) Mitochondrial staining of Arabidopsis root tips with MitoTracker Red CM-H2XRos

fluorescent dye (left panel). Six-old-day Arabidopsis Col0 seedlings were pretreated with

20 µM FAA, 50 µM AA and DMSO in liquids ½MS media for 1 h before transferring to

identical solutions containing additional 250 nM Mito Tracker Red CMH2XRos for an

additional 1 h. Bright field images (right panel). Scale bars equal 5 µM.

Figure 3. Induction of the MDM Cis-Regulatory Elements by FAA. Regulatory activity of the

synthetic sequence containing six consecutive repeats of the MDM sequence from the

UGT74E2 promoter (6xMDM[UGT74E2]) and from the AOX1a promoter

(6xMDM1[AOX1a]) in transgenic Arabidopsis plants treated with 20 µM FAA or DMSO for

24 h. The construct mutated in the MDM sequence (6xMDM1mut[AOX1a]) was included as a

negative control. Bars represent averages ±SE of 8 biological replicates. RLU, relative

luminescence units.

Figure 4. Inhibition of Auxin Signaling by Mitochondrial Dysfunction.

(A) Effect of AA and FAA on the auxin-responsive reporter line DR5:GUS. Five-day-old

DR5:GUS seedlings were treated for 4 h with 5 µM IAA alone or with combinations of 5µM

IAA and either 50 µM AA or 20 µM FAA. DMSO was used as a mock treatment.

(B) Quantification of the DII-Venus fluorescent signal intensity in the median section of the

root tip. Data points represent averages based on quantification of three independent roots




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http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1491


19

relative to the first time point (5 min = 1) ± SD. The roots were pre-incubated for 5 min with

either 50 µM AA or 20 µM FAA before being placed in contact with agar-solidified ½MS

media containing combinations of auxin (50 nM) and either 50 µM AA or 20 µM FAA and

imaged for 35 min. For IAA and mock (DMSO) treatments, roots were placed in contact with

the media without a prior incubation.

(C) DII-Venus fluorescence signal (range indicator: blue to green) in propodium iodid (red)

stained Arabidopsis root tips at 5 min and 25 min after exposure to exogenous IAA (50 nM);

IAA + FAA (50 nM and 20 µM), IAA + AA (50 nM and 50 µM), and DMSO as described

above.

Figure 5. Effect of Polar Auxin Transport Inhibitors on Mitochondrial Retrograde Markers.

(A) The UGT74E2 and AOX1a promoters are activated by polar auxin transport inhibitors and

an inhibitor of auxin biosynthesis. The 1,5-kb upstream sequences of UGT74E2 and AOX1a

were fused upstream of the luciferase gene and stably transformed in Arabidopsis plants.

Mean luciferase activities after 24 h treatment of UGT74E2:LUC and AOX1a:LUC lines with

20 µM NPA, 20 µM TIBA, 20 µM gravacin, 500 µM quercetin, 20 µM L-kynurerine, and

DMSO are shown ±SE (n = 8 biological replicates). RLU, relative luminescence units.

(B) The MDM cis-regulatory element is sufficient for promoter activation by polar auxin

transport inhibitors and an inhibitor of auxin biosynthesis. Regulatory activity of the synthetic

sequence containing six consecutive repeats of the MDM sequence from the UGT74E2

promoter (6xMDM[UGT74E2]) and from the AOX1a promoter (6xMDM1[AOX1a]) in

transgenic Arabidopsis plants treated with NPA, TIBA, gravacin, quercetin, L-kynerine and

DMSO in concentrations described above. The construct mutated in the MDM sequence

(6xMDM1mut[AOX1a]) was included as a negative control. Bars represent average luciferase




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20

activity ± SE (n = 8 biological replicates). Asterisks indicate significant differences to the

mock treatment (Student’s t test; *p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 6. Constitutive Activation of MDS Gene Expression in the arf7arf19 Mutant

Background. Transcript abundances of fourteen of the MDS genes were quantified by qRT-

PCR analysis in two-week-old arf7arf19 plants relative to the wild type. Bars represent

average linear fold changes from four biological replicates (± SE). Asterisks indicate

significant differences to the wild type (Student’s t test; * p<0.05, ** p<0.01, and ***

p<0.001).




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26

SUPPLEMENTARY DATA

Supplemental Figure 1. High-throughput screen for chemicals inducing pUGT74E2 without

causing a general stress response. A) Normalized luminescence data for 12,000 compounds

from a DIVERSet library screened in the primary screen. Luminescence values are indicated

in percent activity, calculated by normalizing the relative luminescence units (RLU) induced

by each compound to the mean of the signals of the negative controls. The arbitrary chosen

700% activity threshold is indicated by the dashed line. Encircled data points show

compounds that have a percent activity equal or above 700 but decrease the F’v/F’m value

below a threshold of 0.677. B). Photosystem II maximum efficiency (F’v/F’m) values of the

12,000 compounds screened in the primary screen. A threshold value of 0.677 was set

(indicated by the dashed line) to retain compounds for further analysis. This threshold

corresponds to the mean of all F’v/F’m values from all negative controls (DMSO treated)

included in primary screen minus three times the standard deviation of these negative controls

(μc - 3σc, with μc = mean F’v/F’m of all negative controls; σc = standard deviation of the

F’v/F’m values of all negative controls) . Encircled data points indicate compounds inducing

the luciferase signal above the 700% activity threshold but decreasing F’v/F’m below the 0.677

threshold.

Supplemental Figure 2. Dose-responsiveness and UGT74E2 transcript abundancies induced

by compounds 2 and 3. A). Dose-response of compound 2. Induction of pUGT74E2:LUC by

different concetrations of compound 2. Data points represent means of 8 biological replicates

± SD. RLU, relative luminescence units. B) Dose-response of compound 3. Induction of

pUGT74E2:LUC by different concentrations of compound 3. Data points represent means of

8 biological replicates ± SD. RLU, relative luminescence units. C) Transcript abundance of

UGT74E2 quantified by qRT-PCR in 10-day-old seedlings treated with either 20 µM




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27

compound 2 or 20 µM compound 3 for 24 h. Bars represent average fold changes relative to

the mock treatment from four biological replicates ± SE.

Supplemental Figure 3. Expression profile of auxin response marker genes during

mitochondrial perturbation conditions. Auxin response marker genes were obtained from

Paponov et al. (2008). Expression data were obtained from publicly available microarray data

sets: Antimycin A, GSE41136 (Ng et al., 2013b); oligomycin and rotenone, GSE3709 (Clifton

et al., 2005); FAA-ester, GSE1491(Armstrong et al., 2004) . Color codes represent linear fold


Pearson correlation as a distance measure in the TMEV4 software.

Supplemental Figure 4. Expression profiles of MDS genes in different auxin mutant

backgrounds. MDS transcript abundances in transport inhibitor response 1 (tir1), auxin

resistant 3 (axr3), pin-formed 2 (pin2), pin-formed 3 and 7 (pin3 pin7), gnom and pid wag1

wag2 triple mutant backgrounds grown in vitro under long day conditions. Expression was

analyzed by qRT-PCR and bars represent average fold changes relative to wild-type (Col-0)

plants from three biological replicates ± SE.

Supplemental Figure 5. Activation of Auxin Signaling Modulates the Effects of

Mitochondrial Dysfunction. A) Auxin alleviates antimycin-mediated promoter activation.

Average fold changes of luciferase activity relative to the mock following treatments with

50 µM AA for 24 h alone or after pretreatment with 5 µM IAA for 24 h and subsequent

exposure to a combination of 50 µM AA and 5 µM IAA for additional 24 h. Asterisks indicate

significant differences according to Student’s t test; *p < 0.05. Arabidopsis lines carrying the

synthetic promoter sequences consisting of a six-tandem repeat of the MDM from the




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28

UGT74E2 promoter (6xMDM[UGT74E2]) and the MDM from the AOX1a promoter

(6xMDM1[AOX1a]), and the 1.5-kb upstream sequences of the UGT74E2 and the AOX1a

genes were used in the experiments. B) Auxin alleviates antimycin-mediated accumulation of

glycine and serine. Wild type Arabidopsis plants grown in liquid MS media were treated as

described above and subjected to metabolite profiling by GC-MS. Bars represent means of six

biological replicates ± SD. Different letters indicate significant differences according to one-

way ANOVA with Tukey’s post hoc test (p<0.05). C) GC-MS metabolite profiling of

Arabidopsis plants grown in liquid MS media and treated with 50 µM AA for 24 h alone or

pretreated with 5 µM IAA for 24 h and subsequently exposed to a combination of 50 µM AA

and 5 µM IAA for additional 24 h. Control plants were treated similarly with DMSO. Colors

bars represent log transformed (glog) and Pareto scaled values cluster hierarchically clustered

using Pearson correlation as a distance measure. Color codes represent log transformed (glog)

and Pareto scaled values hierarchically clustered using Pearson correlation as a distance

measure.

Supplemental Figure 6. GC-MS metabolite profiling of prohibitin3 mutant plants in

comparison to the wild type (Col-0). Metabolites were extracted from shoots of two-week-old

plants grown in vitro on horizontal plants under long-day conditions. Per treatment, results

from three biological replicates are shown. Colors codes represent log transformed (glog) and

Pareto scaled values hierarchically clustered using Pearson correlation as a distance measure.

Supplemental Table 1. Primers used for quantitative RT-PCR analysis.




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a)

Chemical

treatment

Control Fv'/Fm' Decreased Fv'/Fm'

9-day-old pUGT74E2:LUC seedlings

Induced

luminescence

Fv'/F

m'

0

0.2

0.4

0.6

0.8 a a a a

b

high

low

Fv'/F

m'

DM

SO

20

µM

Com

p 2

20

µM

Com

p 3

20

µM

FA

A

10

mM

H2 O

2

Compound 2

3-(2-Furyl)acrylic acid (FAA)

Compound 3

b)

Hit compound c)

0

5000

10000

15000

20000

25000

b b

c

a

d

DMSO 20 µM

Comp 2

20 µM

Comp 3

20 µM

FAA

10 mM

H2O2 d)

RL

U

Figure 1. Chemical Screen to Identify Compounds Activating Mitochondrial Retrograde Signalling

Pathways.

(A) A schematic representation of the chemical screen.

(B) Chemical formulas of the two most potent hit compounds identified in the screen (Compound 2

and Compound 3) and the shared substructure (3-(2-Furyl)acrylic acid (FAA)).

(C) Induction of pUGT74E2 by Compound 2, Compound 3, and FAA. 10 mM H202 was used as a

positive control. The 1,5-kb upstream sequences of UGT74E2 was fused upstream of the luciferase

gene and stably transformed in Arabidopsis plants. Luciferase activity following chemical (20 µM)

and mock (DMSO) treatment is shown (±SD; n=8 biological replicates). Different letters represent

statistically significant differences according to one-way ANOVA with Tukey’s post hoc test

(p<0.05). RLU, relative luminescence units.

(D) Photosystem II maximum efficiency (Fv'/Fm') of seedlings treated with chemicals (20 µM), 10 mM

H202 or DMSO for 24 h (left panel). Fv'/Fm' levels are depicted by color codes; blue represents high

values and yellow-green corresponds to low values. Quantification of Fv'/Fm' values in chemically

treated seedlings (right panel). Bars represent means of 8 replicates ± SD. Different letters

represent statistically significant differences according to one-way ANOVA with Tukey’s post hoc

test (p<0.05).




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DM

SO

20 µ

M F

AA

50 µ

M A

A

oligomycin (4h)

antimycin A

rotenone (3h) oligomycin (1h)

rotenone (12h)

furyl acrylate ester

3.0 -3.0

At2

g0

31

30

At3

g2

72

80

A

t2g2

16

40

AB

CB

4

HR

E2

A

t2g3

20

20

A

t2g4

17

30

A

t3g2

52

50

U

GT

74

E2

A

t2g0

37

60

A

t3g5

09

30

A

NA

C0

13

A

t2g0

40

50

C

RF

6

At5

g1

47

30

sH

sp2

3.5

A

OX

1a

At4

g3

73

70

A

t5g0

95

70

A

t1g0

50

60

A

t5g4

34

50

A

t2g0

40

70

N

DB

4

At5

g5

52

00

c) C *

***

***

*

***

***

**

*

***

b)

a)

Figure 2. FAA Negatively Impacts Mitochondria.

(A) Induction of MDS genes following FAA treatment. Transcript levels of fourteen of the MDS

genes were analyzed by qRT-PCR in 10-day-old wild-type Arabidopsis plants grown as liquid

cultures and treated with 20 µM FAA or DMSO for 24 h. Bars represent average fold changes

relative to the mock treatment from four biological replicates (± SE). Asterisks indicate

significant differences to the mock (Student’s t test; * p<0.05, ** p<0.01, and *** p<0.001).

(B) Comparison of publicly available expression profiles of the MDS genes between FAA

ester treatment and treatments with mitochondrial blockers. Color codes represent linear fold


Pearson correlation as a distance measure in the TMEV4 software. Antimycin A: GSE41136

(Ng et al., 2013b); oligomycin and rotenone: GSE3709 (Clifton et al., 2005); FAA ester:

GSE1491 (Armstrong et al., 2004).

(C) Mitochondrial staining of Arabidopsis root tips with MitoTracker Red CM-H2XRos

fluorescent dye (left panel). Six-old-day Arabidopsis Col0 seedlings were pretreated with

20 µM FAA, 50 µM AA and DMSO in liquids ½MS media for 1 h before transferring to identical

solutions containing additional 250 nM Mito Tracker Red CMH2XRos for an additional 1 h.

Bright field images (right panel). Scale bars equal 5 µM.




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Figure 3. Induction of the MDM Cis-Regulatory Elements by FAA. Regulatory activity of the

synthetic sequence containing six consecutive repeats of the MDM sequence from the

UGT74E2 promoter (6xMDM[UGT74E2]) and from the AOX1a promoter (6xMDM1[AOX1a])

in transgenic Arabidopsis plants treated with 20 µM FAA or DMSO for 24 h. The construct

mutated in the MDM sequence (6xMDM1mut[AOX1a]) was included as a negative control.

Bars represent averages ±SE of 8 biological replicates. RLU, relative luminescence units.




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IAA IAA + FAA IAA + AA DMSO

a)

5 m

in

25

min

IAA IAA + FAA IAA + AA DMSO

IAA IAA + FAA IAA +AA DMSO

c)

0

0.5

1

1.5

5 10 15 20 25 30 35

No

rmal

ized

sig

nal

inte

sity

b)

min IAA + AA DMSO

IAA + FAA IAA

Figure 4. Inhibition of Auxin Signaling by Mitochondrial Dysfunction.

(A) Effect of AA and FAA on the auxin-responsive reporter line DR5:GUS. Five-day-old DR5:GUS

seedlings were treated for 4 h with 5 µM IAA alone or with combinations of 5µM IAA and either 50

µM AA or 20 µM FAA. DMSO was used as a mock treatment.

(B) Quantification of the DII-Venus fluorescent signal intensity in the median section of the root tip.

Data points represent averages based on quantification of three independent roots relative to the

first time point (5 min = 1) ± SD. The roots were pre-incubated for 5 min with either 50 µM AA or

20 µM FAA before being placed in contact with agar-solidified ½MS media containing

combinations of auxin (50 nM) and either 50 µM AA or 20 µM FAA and imaged for 35 min. For

IAA and mock (DMSO) treatments, roots were placed in contact with the media without a prior

incubation.

(C) DII-Venus fluorescence signal (range indicator: blue to green) in propodium iodid (red)

stained Arabidopsis root tips at 5 min and 25 min after exposure to exogenous IAA (50 nM); IAA +

FAA (50 nM and 20 µM), IAA + AA (50 nM and 50 µM), and DMSO as described above.




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a)

**

**

***

*

b) a)

Figure 5. Effect of Polar Auxin Transport Inhibitors on Mitochondrial Retrograde Markers.

(A) The UGT74E2 and AOX1a promoters are activated by polar auxin transport inhibitors and

an inhibitor of auxin biosynthesis. The 1,5-kb upstream sequences of UGT74E2 and AOX1a

were fused upstream of the luciferase gene and stably transformed in Arabidopsis plants.

Mean luciferase activities after 24 h treatment of UGT74E2:LUC and AOX1a:LUC lines with

20 µM NPA, 20 µM TIBA, 20 µM gravacin, 500 µM quercetin, 20 µM L-kynurerine, and DMSO

are shown ±SE (n = 8 biological replicates). RLU, relative luminescence units.

(B) The MDM cis-regulatory element is sufficient for promoter activation by polar auxin

transport inhibitors and an inhibitor of auxin biosynthesis. Regulatory activity of the synthetic

sequence containing six consecutive repeats of the MDM sequence from the UGT74E2

promoter (6xMDM[UGT74E2]) and from the AOX1a promoter (6xMDM1[AOX1a]) in transgenic

Arabidopsis plants treated with NPA, TIBA, gravacin, quercetin, L-kynerine and DMSO in

concentrations described above. The construct mutated in the MDM sequence

(6xMDM1mut[AOX1a]) was included as a negative control. Bars represent average luciferase

activity ± SE (n = 8 biological replicates). Asterisks indicate significant differences to the mock

treatment (Student’s t test; *p < 0.05, **p < 0.01, and ***p < 0.001).




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**

*

*

***

**

***

*

**

*

*

*

*

Figure 6. Constitutive Activation of MDS Gene Expression in the arf7arf19 Mutant

Background. Transcript abundances of fourteen of the MDS genes were quantified by qRT-

PCR analysis in two-week-old arf7arf19 plants relative to the wild type. Bars represent

average linear fold changes from four biological replicates (± SE). Asterisks indicate

significant differences to the wild type (Student’s t test; * p<0.05, ** p<0.01, and *** p<0.001).




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a) b)

Supplemental Figure 1. High-throughput screen for chemicals inducing pUGT74E2 without

causing a general stress response. A) Normalized luminescence data for 12,000 compounds

from a DIVERSet library screened in the primary screen. Luminescence values are indicated in

percent activity, calculated by normalizing the relative luminescence units (RLU) induced by each

compound to the mean of the signals of the negative controls. The arbitrary chosen 700% activity

threshold is indicated by the dashed line. Encircled data points show compounds that have a

percent activity equal or above 700 but decrease the F’v/F’m value below a threshold of 0.677. B).

Photosystem II maximum efficiency (F’v/F’m) values of the 12,000 compounds screened in the

primary screen. A threshold value of 0.677 was set (indicated by the dashed line) to retain

compounds for further analysis. This threshold corresponds to the mean of all F’v/F’m values from

all negative controls (DMSO treated) included in primary screen minus three times the standard

deviation of these negative controls (μc - 3σc, with μc = mean F’v/F’m of all negative controls; σc =

standard deviation of the F’v/F’m values of all negative controls) . Encircled data points indicate

compounds inducing the luciferase signal above the 700% activity threshold but decreasing

F’v/F’m below the 0.677 threshold.




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0

10000

20000

30000

40000

50000

60000

0 2.5 5 10 15 20 25

Compound 2 [µM]

RL

U

0

10000

20000

30000

40000

50000

60000

0 2.5 5 10 15 20 25

Compound 3 [µM]

RL

U

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

Fold

chan

ge

(rel

ativ

e to

mock

)

a)

b)

c)

Supplemental Figure 2. Dose-responsiveness and UGT74E2 transcript abundancies induced by

compounds 2 and 3. A). Dose-response of compound 2. Induction of pUGT74E2:LUC by different

concentrations of compound 2. Data points represent means of 8 biological replicates ± SD. RLU,

relative luminescence units. B) Dose-response of compound 3. Induction of pUGT74E2:LUC by

different concentrations of compound 3. Data points represent means of 8 biological replicates ±

SD. RLU, relative luminescence units. C) Transcript abundance of UGT74E2 quantified by qRT-

PCR in 10-day-old seedlings treated with either 20 µM compound 2 or 20 µM compound 3 for 24

h. Bars represent average fold changes relative to the mock treatment from four biological

replicates ± SE.




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AUX1

LAX2

LBD41

ARF19

GH3.6

PIN1

SAUR36

SAUR66

SAUR72

GH3.1

LAX3

IAA3

LBD16

LBD25

ARF4

IAA29

LBD33

IAA30

IAA13

LBD15

PIN3

IAA19

IAA5

PIN7

SAUR15

SAUR59

IAA4

IAA27

SAUR62

ARF16

SAUR37

AMI1

SAUR64

GH3.5

IAA2

IAA28

LBD17

IAA1

IAA11

SAUR67

LBD29

LBD4

CYP79B2

GH3.4

FH3.3

LBD40

LBD18

SAUR32

GH3.17

CYP79B3

SAUR31

SAUR9

rote

no

ne

(12

h)

oli

go

myci

n (

1h)

rote

no

ne

(3h)

anti

myci

n A

oli

go

myci

n 4

h)

fury

l ac

ryla

te e

ster

-4.0 1.0 4.0

Supplemental Figure 3. Expression profile of auxin response marker genes during mitochondrial

perturbation conditions. Auxin response marker genes were obtained from Paponov et al. (2008).

Expression data were obtained from publicly available microarray data sets: Antimycin A,

GSE41136 (Ng et al., 2013b); oligomycin and rotenone, GSE3709 (Clifton et al., 2005); FAA-

ester, GSE1491 (Armstrong et al., 2004). Color codes represent linear fold changes relative to

control extracted from Genevestigator and hierarchically clustered using Pearson correlation as a

distance measure in the TMEV4 software.




ownloaded from


0

0.5

1

1.5

2

2.5

3

3.5

tir1

0

0.5

1

1.5

2

2.5

3

3.5

4

axr3 Fo

ld c

han

ge (

rela

tive

to

wild

typ

e)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

pin3 pin7

0

0.5

1

1.5

2

2.5

3

3.5pid wag1 wag2

Fold

ch

ange

(re

lati

ve t

o w

ild t

ype)

-2

0

2

4

6

8

10

12

14

gnom

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Fold

ch

ange

(re

lati

ve t

o w

ild t

ype)

pin2

Supplemental Figure 4. Expression profiles of MDS genes in different auxin mutant

backgrounds. MDS transcript abundances in transport inhibitor response 1 (tir1), auxin resistant

3 (axr3), pin-formed 2 (pin2), pin-formed 3 and 7 (pin3 pin7), gnom and pid wag1 wag2 triple

mutant backgrounds grown in vitro under long day conditions. Expression was analyzed by qRT-

PCR and bars represent average fold changes relative to wild-type (Col-0) plants from three

biological replicates ± SE.




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0

1

2

3

-1

-2

-3

-4

Nicotinic acid

Pyroglutamic acid

Serine

ß-Alanine

Homoserine

Sucrose

Valine

Phosphate

Glucose-6-phosphate

Glucose

Alanine

Succinic acid

Glycine

Glutamine

Arginine

Lysine

Tyrosine

Glycerol

Lactic acid

Threonic acid

Phenylalanine

Malic acid

Fructose

Tryptophan

Fumaric acid

Threonic acid

Glyceric acid

Putrescine

Ornithine/Arginine

Aspartic acid

Myo-Inositol

Citric acid

Galactonic acid

Ribonic acid

2-Keto-L-gluconic acid

Gluconic acid

ɣ-aminobutyric acid

Isoleucine

Threonine

Asparagine

Proline

Glutamic acid

Mock IAA AA IAA+AA

b)

c)

* *

a)

a a a b

a

a

b

c

Supplemental Figure 5. Activation of Auxin Signaling Modulates the Effects of Mitochondrial

Dysfunction. A) Auxin alleviates antimycin-mediated promoter activation. Average fold changes of

luciferase activity relative to the mock following treatments with 50 µM AA for 24 h alone or after

pretreatment with 5 µM IAA for 24 h and subsequent exposure to a combination of 50 µM AA and

5 µM IAA for additional 24 h. Asterisks indicate significant differences according to Student’s t test;

*p < 0.05. Arabidopsis lines carrying the synthetic promoter sequences consisting of a six-tandem

repeat of the MDM from the UGT74E2 promoter (6xMDM[UGT74E2]) and the MDM from the AOX1a

promoter (6xMDM1[AOX1a]), and the 1.5-kb upstream sequences of the UGT74E2 and the AOX1a

genes were used in the experiments. B) Auxin alleviates antimycin-mediated accumulation of glycine

and serine. Wild type Arabidopsis plants grown in liquid MS media were treated as described above

and subjected to metabolite profiling by GC-MS. Bars represent means of six biological replicates ±

SD. Different letters indicate significant differences according to one-way ANOVA with Tukey’s post

hoc test (p<0.05). C) GC-MS metabolite profiling of Arabidopsis plants grown in liquid MS media and

treated with 50 µM AA for 24 h alone or pretreated with 5 µM IAA for 24 h and subsequently exposed

to a combination of 50 µM AA and 5 µM IAA for additional 24 h. Control plants were treated similarly

with DMSO. Colors bars represent log transformed (glog) and Pareto scaled values cluster

hierarchically clustered using Pearson correlation as a distance measure. Color codes represent log

transformed (glog) and Pareto scaled values hierarchically clustered using Pearson correlation as a

distance measure.




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Aspartic acid

Threonine

Maleic acid

Proline

Ornithine/Arginine

Threonic acid

Valine

Phenylalanine

Xylose

Isoleucine

Lactic acid

Phosphate

Glutamic acid

Ribonic acid

Putrescine

Serine

Myo-inositol

Glucose-6-phosphate

Fumaric acid

Threonic acid

Glyceric acid

Glyceric acid-3-phosphate

2-Keto-L-gluconic acid

Citric acid

Sucrose

Glucose

Succinic acid

Pyroglutamic acid

Fructose

Shikimic acid

ɣ-aminobutyric acid

Glycine

Alanine 2TMS

Alanine 3TMS

Malic acid

Wild type (Col0) prohibitin3

0

1

2

-1

-2

Supplemental Figure 6. GC-MS metabolite profiling of prohibitin3 mutant plants in comparison

to the wild type (Col-0). Metabolites were extracted from shoots of two-week-old plants grown in

vitro on horizontal plants under long-day conditions. Per treatment, results from three biological

replicates are shown. Colors codes represent log transformed (glog) and Pareto scaled values

hierarchically clustered using Pearson correlation as a distance measure.




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Supplemental Table 1. Primers used for quantitative RT-PCR analysis.

Primer Sequence

ANAC013_RT_FWD ACCAGACAGATAAACAATGGATCA

ANAC013_RT_REV CAGAAGGAACAGGGTTTAGGAA

ABCB4_RT_FWD CAAAGTCTCCAAAGTTGCTCTG

ABCB4_RT_REV CTCGCTGCTTGTCTCTCTCC

AOX1a_RT_FWD TGGTTGTTCGTGCTGACG

AOX1a_RT_REV CACGACCTTGGTAGTGAATATCAG

ARP7_RT_FWD ACTCTTCCTGATGGACAGGTG

ARP7_RT_REV CTCAACGATTCCATGCTCCT

at2g04050_RT_FWD CCACAATGGTGAGCTCCAG

at2g04050_RT_REV CACCCGCTAACCCAAACA

at2g04070_RT_FWD CTCCAGCTCTCCGGTGTC

at2g04070_RT_REV GTGAACCCACTAACCCAAACA

AT2G28390_FWD AACTCTATGCAGCATTTGATCCACT

AT2G28390_REV TGATTGCATATCTTTATCGCCATC

at2g41730_RT_FWD GTCACCAAGGCATCGTAAGG

at2g41730_RT_REV AAAGCTGGTGGTGAATCGAG

at5g09570_RT_FWD GAAACCGTTGTTTCTCAGGTTC

at5g09570_RT_REV CCAAAATGGTTGACGCAAT

CRF6_RT_FWD TGGCTTGGGACTTTTGTCA

CRF6_RT_REV GAGATGAATCGCGGCTCTA

CYP81D8_RT_L CGTCTTTCTCGGAACTTTTCA

CYP81D8_RT_R AACACCGTCTCCGTAGTAACG

HRE2_RT_FWD GAAGCGTAAACCCGTCTCAGT

HRE2_RT_REV AATCTCCGCTGCCCATTT

HSP23.5_RT_FWD TCAAACCGACATGTTTCTCG

HSP23.5_RT_REV AAGCTTCTCGTTGGAGTAAACG

ST_RT_FWD GGTCACCAATCCACACCTTC

ST_RT_REV CGAAATCTGGGGACTCGTAG

UBC21_RT_FWD TCCTCTTAACTGCGACTCAGG

UBC21_RT_REV GCGAGGCGTGTATACATTTG

UGT74E2_RT_FWD TAACTTCTTCCACACTTCTCATAATCT

UGT74E2_RT_REV ACAACAAAAACTAGAGTCAGTAACAAC

UPOX_RT_FWD TTCAAAAACACCATGGACAAGA

UPOX_RT_REV GCCTCAATTTGCTTCTCTGC




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Mitochondrial perturbation negatively affects auxin signaling

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