Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in Arabidopsis


Low-oxygen response is triggered by an ATP-
dependent shift in oleoyl-CoA in Arabidopsis
Romy R. Schmidta,1, Martin Fuldab, Melanie V. Paulc, Max Andersa, Frederic Pluma, Daniel A. Weitsa, Monika Kosmaczd,
Tony R. Larsone, Ian A. Grahame, Gerrit T. S. Beemsterf, Francesco Licausig,h, Peter Geigenbergerc, Jos H. Schippersa,
and Joost T. van Dongena,1

aInstitute of Biology I, Rheinisch-Westfälische Technische Hochschule Aachen University, 52074 Aachen, Germany; bAlbrecht von Haller Institute of Plant
Sciences, Goettingen University, 37077 Goettingen, Germany; cDepartment Biology I, Ludwig Maximilian University of Munich, 82152 Planegg-Martinsried,
Germany; dMax Planck Institute of Molecular Plant Physiology, 14476 Potsdam, Germany; eDepartment of Biology, University of York, Heslington, YO10
5DD York, United Kingdom; fIntegrated Molecular Plant Physiology Research Group, University of Antwerp, G.U.613, 2020 Antwerpen, Belgium; gPlantLab,
Institute of Life Sciences, Scuola Superiore Sant’Anna, 56017 Pisa, Italy; and hDipartimento di Biologia,Università di Pisa, 56126 Pisa, Italy

Edited by Julia Bailey-Serres, University of California, Riverside, CA, and approved November 5, 2018 (received for review June 10, 2018)

Plant response to environmental stimuli involves integration of
multiple signals. Upon low-oxygen stress, plants initiate a set of
adaptive responses to circumvent an energy crisis. Here, we reveal
how these stress responses are induced by combining (i) energy-
dependent changes in the composition of the acyl-CoA pool and
(ii) the cellular oxygen concentration. A hypoxia-induced decline
of cellular ATP levels reduces LONG-CHAIN ACYL-COA SYNTHETASE
activity, which leads to a shift in the composition of the acyl-CoA
pool. Subsequently, we show that different acyl-CoAs induce
unique molecular responses. Altogether, our data disclose a role
for acyl-CoAs acting in a cellular signaling pathway in plants. Upon
hypoxia, high oleoyl-CoA levels provide the initial trigger to release
the transcription factor RAP2.12 from its interaction partner ACYL-COA
BINDING PROTEIN at the plasma membrane. Subsequently, according
to the N-end rule for proteasomal degradation, oxygen concentration-
dependent stabilization of the subgroup VII ETHYLENE-RESPONSE
FACTOR transcription factor RAP2.12 determines the level of
hypoxia-specific gene expression. This research unveils a specific
mechanism activating low-oxygen stress responses only when a de-
crease in the oxygen concentration coincides with a drop in energy.

low-oxygen stress | integrative signaling | acyl-CoA | ERFVII | ACBP

Flooding contributes almost 60% to the worldwide cost anddamage to crops provoked by natural disasters (1). Due to
heavy precipitation and concomitant waterlogging or flooding
events in large areas of the world, climate change will cause
plants to be even more frequently exposed to oxygen-limiting
conditions (hypoxia) in the near future (2).
In plants, subgroup VII ETHYLENE-RESPONSE FACTOR

(ERFVII) transcription factors act as key regulators of hypoxic
gene expression (3–6). During nonstress conditions, the ERFVII
protein RELATED TO APETALA 2.12 (RAP2.12) is seques-
tered to the plasma membrane via direct interaction with ACYL-
CoA BINDING PROTEIN (ACBP) (3, 7–9). Upon hypoxia,
RAP2.12 is released from the plasma membrane and subsequently
accumulates in the nucleus (3, 7, 9). Further, the stability of
ERFVII proteins is tightly controlled in an oxygen-dependent
manner employing the Cys branch of the N-end rule (3, 4).
That is, ERFVII protein degradation is prevented under hypoxic
conditions when N end rule-assisted degradation is impaired due
to oxygen limitation (10). Although the homeostatic regulation of
adaptive responses to low-oxygen stress in plants is well in-
vestigated (3, 4, 11), the identity of the initial trigger to release
RAP2.12 from its membrane docking protein ACBP remains
unknown and the existence of multiple signal queues that are
integrated into low-oxygen specific responses is likely (12).
ACBPs represent an evolutionarily conserved protein family

found in Escherichia coli, yeast, animals, and plants (13, 14) and
participate in the regulation of unbound acyl-CoA levels by seques-
tration and transportation of acyl-CoAs (15, 16). The interaction
between members of a protein family capable of reversibly binding

acyl-CoAs with the ERFVII proteins RAP2.12 (3, 7) and RAP2.3 (8,
9) provided a first indication that acyl-CoAs can be involved in the
release of ERFVII transcription factor protein during hypoxia. We
elaborated this mode of action with experiments on RAP2.12 as a
representative member of ERFVII transcription factors.
Acyl-CoAs are intermediates in both lipid catabolism and

anabolism. In the catabolic pathway, fatty acids are activated in
the cytosol by ACYL-CoA SYNTHETASES before their trans-
port into mitochondria or peroxisomes where β-oxidation occurs.
In plants, lipid anabolism occurs through two pathways: de novo
fatty acid synthesis takes place in plastids and the generated fatty
acids can be incorporated into complex lipids within the plastid
by the so-called prokaryotic pathway. Alternatively, the fatty acid
may be exported from the plastid to the cytosol to become
substrate for the eukaryotic lipid biosynthesis pathway in the
endoplasmic reticulum (ER). Transport of fatty acids from the
plastid, through the cytosol into the ER is mainly mediated via
palmitoyl-CoA (C16:0-CoA) and oleoyl-CoA (C18:1-CoA) that
are produced from palmitic and oleic acid by the enzyme LONG-
CHAIN ACYL-CoA SYNTHETASES (LACS) at the outer
plastid membrane in root and shoot tissues (17–20). In addition

Significance

To control adaptive responses to the ever-changing environ-
ment that plants are continuously exposed to, plant cells must
integrate a multitude of information to make optimal deci-
sions. Here, we reveal how plants can link information about
the cellular energy status with the actual oxygen concentration
of the cell to trigger a response reaction to low-oxygen stress.
We reveal that oleoyl-CoA has a moonlighting function in an
energy (ATP)-dependent signal transduction pathway in plants,
and we provide a model that explains how diminishing oxygen
availability can initiate adaptive responses when it coincides
with a decreased energy status of the cell.

Author contributions: R.R.S., M.F., T.R.L., I.A.G., F.L., P.G., J.H.S., and J.T.v.D. designed
research; R.R.S. coordinated the experiments; R.R.S., M.F., M.V.P., M.A., F.P., D.A.W.,
M.K., T.R.L., G.T.S.B., F.L., and J.H.S. performed research; R.R.S., M.F., M.V.P., T.R.L.,
I.A.G., G.T.S.B., P.G., J.H.S., and J.T.v.D. analyzed data; and R.R.S., J.H.S., and J.T.v.D. wrote
the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDerivatives License 4.0 (CC BY-NC-ND).

Data deposition: The data reported in this paper have been deposited in the Gene Ex-
pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE97186).
1To whom correspondence may be addressed. Email: roschmidt@bio1.rwth-aachen.de or
dongen@bio1.rwth-aachen.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1809429115/-/DCSupplemental.

Published online December 3, 2018.

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to their involvement in lipid metabolism, acyl-CoAs are also
known to modulate the activity of numerous enzymes, ion
channels, and transcription factors in animals and microorgan-
isms (15). Examples of acyl-CoAs directly affecting transcription
factor activity by functioning as ligands have been reported for
humans (HNF-4α) (21) and E. coli (FadR) (22). For plants, di-
rect involvement of acyl-CoAs controlling transcription factor
activity was not demonstrated yet, although an indirect regula-
tory role has been suggested (23).
Hypoxia has detrimental effects on the plant’s cellular ho-

meostasis, in the first place, because oxidative phosphorylation in
mitochondria is reduced, which ultimately leads to a decrease of
the cellular energy charge. This results in ATP-consuming met-
abolic processes being attenuated, including fatty acid synthesis
and processing (24). Consequently, the export of newly synthe-
sized fatty acids from the plastid into the cytosol is affected, since
the activation to acyl-CoAs is ATP dependent (18, 25). There-
fore, an energy-related impact of hypoxia on acyl-CoA levels in
the cytosol is expected and investigated here.
In this study, we reveal a combinatory signaling network by

which the reduced energy level under low-oxygen stress is in-
tegrated into the ERFVII-dependent hypoxic signaling cascade.
We show that dynamic responses of C18:1-CoA and C16:0-CoA
levels to hypoxia constitute an early molecular trigger, leading to
dissociation of the ACBP:RAP2.12 complex thereby activating
the molecular low-oxygen response cascade. We describe an in-
tegrative signaling mechanism in which adaptive gene expression
upon low-oxygen stress results from the specific combination of
(i) low-energy triggered release of the transcription factor
RAP2.12 from ACBP as mediated by an acyl-CoA signal; and (ii)
low-oxygen dependent stabilization of the RAP2.12 protein
according to the N-end rule of protein degradation.

Results
C18:1-CoA Promotes Dissociation of the ACBP:RAP2.12 Complex in
Vitro and in Vivo. In Arabidopsis, the ERFVII transcription fac-
tor RAP2.12 is constitutively expressed but sequestered at the
plasma membrane by binding to ACBP1 during normoxic con-
ditions, while it accumulates in the nucleus under oxygen con-
centrations below 10% (vol/vol) (3, 7). The interaction domains
of ACBP1 and RAP2.12 were previously identified in Arabi-
dopsis (3, 26, 27) and appear to be well conserved among plant
species (Fig. 1 A and B), indicating that complex formation of
both proteins is a general feature in plants. Importantly, changing
the expression of ACBP1 affects tolerance to low oxygen (28) (Fig.
1 C and D) similar to what was shown previously for RAP2.12 (3).
During hypoxia GFP-tagged ACBP1 remains at the plasma
membrane (Fig. 1E), while GFP-tagged RAP2.12 was shown to
accumulate in the nucleus upon hypoxia (3, 7). This indicates that
RAP2.12 dissociates from ACBP1 before its relocation to the
nucleus. Consequently, the release of RAP2.12 from ACBP1 is
considered as a trigger that activates adaptive gene expression in
response to hypoxia.
To investigate if acyl-CoAs interfere with the interaction be-

tween ACBP1 and RAP2.12, we performed an in vitro affinity
assay (SI Appendix, Fig. S1) in the presence of either oleoyl-CoA
(C18:1-CoA), which is a preferred substrate for ACBP1, or
palmitoyl-CoA (C16:0-CoA) that is not a strongly interacting
agent (29, 30). In vitro exposure of an ACBP1:RAP2.12 protein
complex to C18:1-CoA, but not C16:0-CoA, significantly de-
creased the binding affinity between the two proteins as indicated
by the reduced ratio of Flag-tagged ACBP1 and CFP-tagged
RAP2.12 protein (Fig. 2 A and B). Apparently, interaction be-
tween ACBP1 and C18:1-CoA reduces the binding capacity of
ACBP1 for the transcription factor RAP2.12. To confirm that
C18:1-CoA–induced dissociation of RAP2.12 from ACBP1 also
occurs in vivo, we exposed detached leaves of plants expressing
35S:RAP2.12-GFP to various acyl-CoAs under aerobic conditions.

Application of C18:1-CoA, but not C18:0-CoA or C16:0-CoA,
significantly induced nuclear accumulation of RAP2.12-GFP, in-
dicating that RAP2.12 was released from ACBP1 in vivo after the
application of C18:1-CoA, but not of C16:0-CoA (Fig. 2 C and D).
Translocation of RAP2.12 to the nucleus was previously described
to occur during hypoxic conditions (3, 7). Therefore, we tested if
application of acyl-CoA to leaves might have induced hypoxia due
to increased beta-oxidation or mitochondrial respiration. How-
ever, no increase of the oxygen consumption rate by leaf tissue
after treatment with acyl-CoAs was observed, indicating that our
experimental treatment did not affect the oxygen concentration of
the tissue in this experiment (SI Appendix, Fig. S2). We concluded,
remobilization of the transcription factor RAP2.12 from the
plasma membrane into the nucleus can be triggered in vivo by
increasing the level of C18:1-CoA.

Acyl-CoAs Provoke Distinct Transcript Responses. To determine if
specific transcriptional responses are provoked by the applica-
tion of different acyl-CoAs, RNA-Seq transcriptome analysis was
performed on wild-type seedlings exposed to either 1 mM C18:1-
CoA, C18:0-CoA, or C16:0-CoA. Uptake of these externally
applied acyl-CoAs is mediated by ABCD transporters that first
cleave the CoA group from the acyl chain, allowing the resulting
fatty acid to cross lipid membranes. Once inside the cell, CoA is
immediately reattached, which traps the acyl-CoA in the cellular
compartment in which it has been imported (31). This analysis
revealed that each of these acyl-CoAs modulates distinct sets of
genes (Fig. 3A and Dataset S1). The high specificity of induced
changes in gene expression underlines the eligibility of acyl-CoAs
as signaling molecules in plants. To investigate the biological
function of the differentially regulated genes, a Gene Ontology
(GO) enrichment analysis (32) was carried out. While applica-
tion of C18:0-CoA or C16:0-CoA mainly affected the expression
of genes related to reproductive development and hormone
signaling, C18:1-CoA mainly modulated the expression of genes
associated with hypoxia and low-oxygen responses (Fig. 3B and
Dataset S2). This result was confirmed by qPCR-assisted ex-
pression profiling executed on wild-type plants incubated with
C18:1-CoA. Indeed, RAP2.12-regulated hypoxia-response genes
were induced by C18:1-CoA treatment, while C18:0-CoA and
C16:0-CoA had only minor effects on the expression of these
genes (Fig. 3C and SI Appendix, Table S1). This observation is
readily explained by our earlier observation that RAP2.12 reloc-
alizes to the nucleus upon C18:1-CoA application (Fig. 2 C and
D). Therefore, it is concluded that C18:1-CoA provides a specific
cellular signal that is substantially involved in the control of gene
expression by releasing RAP2.12 from ACBP1.

Increase of C18:1-CoA to C16:0-CoA Ratio Induces Hypoxic Gene
Expression in Vivo. In a physiological context, C18:1-CoA–mediated
dissociation of RAP2.12 from ACBP1 only makes sense when the
endogenous acyl-CoA pool responds to hypoxia. Indeed, HPLC-
assisted quantification of acyl-CoAs revealed a shift to elevated
C18:1-CoA (Fig. 3D) and C20:0-CoA (SI Appendix, Fig. S3) levels
with less C16:0-CoA (Fig. 3E) after 3 h of hypoxia, while no sig-
nificant changes of the total pool of acyl-CoAs included in our
analyses were observed (SI Appendix, Fig. S3). These dynamic re-
sponses of specific acyl-CoAs to changing environmental conditions
as exemplified here for hypoxia are in line with the suggestion that
acyl-CoAs in plants can play a role in stress signaling.
A similar shift of the acyl-CoA pool as observed during low-

oxygen conditions was observed in transgenic plants in which two
LACS genes, LACS4 and -9, were knocked out (33) (Fig. 3 F and G
and SI Appendix, Fig. S4). Under aerobic conditions, lacs4lacs9
double knockout plants have elevated C18:1-CoA and reduced
C16:0-CoA levels. To provide further proof that endogenous
changes of the C18:1-CoA or C16:0-CoA level can have an effect on
low-oxygen responses of plants, we tested the induction of hypoxic

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gene expression in lacs4lacs9 plants in air. Indeed, many RAP2.12-
dependent hypoxia-response genes were activated under aerobic
conditions in lacs4lacs9 (Fig. 3H and SI Appendix, Table S2). In
addition, also tolerance to hypoxia was altered. Performance of
lacs4lacs9 was impaired under both anoxia and submergence con-
ditions compared with wild-type plants (Fig. 4). Similar observations
were described for plants with constitutive activation of the hypoxia-
stress response pathway (34), although it should be mentioned that
the impact of constitutive activation of the ERFVII signaling

pathway on plant performance under stress conditions also appears
to depend on the experimental growth conditions and the recovery
treatment (4, 35). Altogether, our data give rise to a model de-
scribing that the control of endogenous acyl-CoA levels through
LACS activity is a prerequisite to properly induce hypoxia-tolerance
responses in plants.

LACS Activity Is Reduced by Depletion of ATP. To explain the mech-
anism by which LACS activity in plants is affected by hypoxic

Fig. 1. ACBP is an important element of the low-oxygen stress response pathway in plants. (A) Multiple sequence alignment of class II ACBP ankyrin domain
compared with ACBP1 and ACBP2 ankyrin domains in A. thaliana (27). (B) Multiple sequence alignment of ERFVII DNA-binding domain compared with
RAP2.12 and RAP2.2 in A. thaliana (26). Sequences of homologous proteins were obtained from Phytozome v12.0 and aligned with Clustal X. (C) Eleven-day-
old seedlings of wild type and acbp1 after 9 h of anoxia and 3-d recovery. (Scale bar: 1.0 cm.) (D) Survival scores for wild type and acbp1 after 9-h anoxia and
3-d recovery. Data are mean values ± SD; *P < 0.05, n = 4 (15 seedlings per replicate). (E) Effect of 2 h of hypoxia treatment (1% O2) on the localization of GFP-
tagged ACBP1 in epidermal cells. Representative pictures are shown. (Scale bar: 10 μm.)

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conditions, we analyzed LACS activity in dependence of its sub-
strate ATP. First, we monitored the cellular energy status in wild-
type plants grown under hypoxia. Already after 2 h, the ATP level
as well as the ATP-to-ADP ratio were significantly reduced
compared with the air-treated control (Fig. 5 A–C). To examine at
which ATP concentration LACS activity becomes substrate lim-
ited, an in vitro LACS activity assay was performed. This experi-
ment showed that the activity of LACS4 was ATP dependent at
ATP concentrations below 0.8 mM (Fig. 5D), which is close to the
estimated cytosolic ATP concentration under aerobic conditions
in planta (36–38). Since ATP concentrations in plant cells de-
crease during hypoxia (24), LACS activity is expected to diminish
concomitantly. However, it should be mentioned that variations of

the ATP concentration between tissues or even within a cell
(between organelles) might lead to different local ATP-limiting
conditions for LACS during hypoxic stress. Moreover, the
hypoxia-induced low-energy status will also influence other pro-
cesses that are not covered by the analyses that we describe here.
To verify that a drop in ATP can trigger hypoxia responses of

plants in air, we chemically inhibited mitochondrial respiration
using antimycin-A. Indeed, after 3 h of 50 μM antimycin-A
treatment of rosettes in air, several hypoxia-responsive genes
were significantly induced (Fig. 5E and SI Appendix, Table S3),
while ATP levels and the ATP-to-ADP ratio were reduced (Fig.
5 F–H). Since application of antimycin-A strongly reduced the
oxygen consumption rate of the tissue (Fig. 5I), it is unlikely that

Fig. 2. Application of C18:1-CoA induces RAP2.12 relocalization into the nucleus. (A) Representative Western blot showing in vitro ACBP1:RAP2.12 complex
stability after treatment with C18:1-CoA or C16:0-CoA. Pluronic F68 treatment served as control. (B) Quantification of ACBP1-to-RAP2.12-ratio as shown in A.
Data are mean values ± SD *P < 0.05, n = 8. (C) Percentage of epidermal cells with nuclear localization of RAP2.12-GFP after treatment with different acyl-
CoAs. Data are mean values ± SD; *P < 0.05. (D) Localization of RAP2.12-GFP in detached leaves incubated with 0.1% C18:1-CoA, C18:0-CoA or C16:0-CoA
dissolved in 0.01% pluronic F68 under normoxic conditions for 3 h. Treatment with pluronic F68 only served as negative control. DAPI staining was used to
identify nuclei. Arrows indicate nuclei with GFP signal. (Scale bar: 10 μm.)

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the induction of hypoxia-responsive genes is the consequence of
low-oxygen concentrations here. Also an antimycin-A–induced
ROS burst appeared unlikely to be responsible for the gene in-
duction, since the induced genes responded similarly when in
addition to antimycin-A also 1 mM of the hydrogen peroxide
scavenger dimethylthiourea (DMTU) (39, 40) was supplied (Fig.
5E). Although it cannot be concluded that reduced ATP levels
are exclusively responsible for triggering hypoxia responses in
plants without performing dose–response analyses of individual
and combined compounds, the data provide evidence that the

cellular energy status is involved in the regulation of hypoxic
gene expression.

Discussion
In this study, we demonstrate that acyl-CoAs provoke distinct
transcriptional responses in plants, suggesting that they are involved
in different signaling pathways. Specifically, binding of C18:1-CoA
to ACBP triggers dissociation of the ACBP:RAP2.12 complex upon
an hypoxia-induced energy crisis, resulting in mobilization of the
transcription factor RAP2.12 into the nucleus. Consequently,
RAP2.12-mediated gene expression is induced. Therewith we reveal

Fig. 3. Changing oleoyl-CoA levels induces low-oxygen responsive gene expression. (A) Number of significantly differentially expressed genes in leaves after
1.5-h treatment with different acyl-CoAs as determined by RNA-Seq (FDR-adjusted P value <0.05). (B) Number of GO classes in which differentially expressed
genes are significantly overrepresented under acyl-CoA treatments as shown in A. (C) qPCR analysis of differential expression of hypoxia-responsive genes
after acyl-CoA treatment in air (reference: F68 only). Data are presented as mean values *P < 0.05, n = 5. (D) C18:1-CoA levels increase upon hypoxia in wild
type. Data are mean values ± SD; *P < 0.05, n = 3. (E) C16:0-CoA levels decrease upon hypoxia in wild type. Data are mean values ± SD; *P < 0.05, n = 3. (F)
C18:1-CoA levels are increased in lacs4lacs9 double mutants grown in air. Data are mean values ± SD; *P < 0.05, n = 3. (G) C16:0-CoA levels are lowered in
lacs4lacs9 double mutants. Data are mean values ± SD; *P < 0.05, n = 3. (H) Expression data for hypoxia-responsive genes comparing wild type and lacs4-1 lacs9-2
in air or hypoxia (2 h 1% O2; mean values, *P < 0.05, n = 4).

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the trigger of the ERFVII-mediated signaling cascade to activate
cellular hypoxia-tolerance responses in plants (Fig. 6).
When the oxygen availability to cells diminishes, mitochondria

produce less ATP due to a reduced activity of oxidative phos-
phorylation (41). Indeed, our plants showed a rapid decrease of
their energy status upon hypoxia. Already within 2 h, the ATP-
to-ADP ratio dropped significantly and remained decreasing
throughout the rest of the experiment (Fig. 5C). Consequently,
the activity of ATP-mediated reactions within the cell is expected
to reduce too (24). Here, we show that ATP concentration-
dependent LACS activity reaches its maximum at an ATP con-
centration of 1 mM (Fig. 5D) which resembles the concentration
in a nonstressed plant cell (36–38). This means that any decrease
of the cellular energy status is translated into a reduction of
LACS activity. The enzyme LACS is among others located in the
plastidial outer envelope where it is provided with C16:0 and
C18:1 fatty acids from the stroma by thioesterases that are lo-
cated in the inner envelope (42, 43). Using CoA and ATP as
cosubstrates, LACS activates fatty acids and releases them as
C16:0-CoA and C18:1-CoA into the cytosol (18). When the level
of ATP drops and LACS activity decreases, the export rate of
fatty acids will be reduced. As the elongation and rapid desatu-
ration reactions in the plastidial stroma from C16:0 to C18:1
commence, the ratio C18:1 compared with C16:0 that is provided
to LACS from the stroma is likely to increase through time under
these conditions. As a consequence, the ratio of C18:1-CoA to

C16:0-CoA that is released by LACS into the cytosol will in-
crease and would readily explain why we observe an increased
level of C18:1-CoA compared with C16:0-CoA in plants that
were exposed to low oxygen (Fig. 3 D and E) as well as in lac-
s4lacs9 double knockout lines in air (Fig. 3 F and G).
Acyl-CoA fatty acid esters bind to ACBP proteins. Here, we

show that specifically the interaction between C18:1-CoA with
ACBP1 results in release of RAP2.12 from the ACBP1:
RAP2.12 complex while C16:0-CoA does not affect the in-
teraction between RAP2.12 and ACBP1 (Fig. 2). A shift of the
ratio between C18:1-CoA and C16:0-CoA as described above,
will therefore lead to the release of RAP2.12 from ACBP1. In-
deed, application of C18:1-CoA, but not C16:0-CoA, increased
the number of nuclei in which GFP-tagged RAP2.12 accumu-
lated (Fig. 2 C and D). Consequently, also up-regulation of
hypoxia-responsive marker genes was observed (Fig. 3 A–C).
Similar to this external application of acyl-CoAs, also an en-

dogenous shift of the C18:1-CoA to C16:0-CoA ratio as observed
in lacs4lacs9 mutant lines resulted in the up-regulation of
hypoxia-responsive genes already during aerobic conditions (Fig.
3H). Altogether, these data describe how the ultimate trigger for
release of RAP2.12 from ACBP1 is constituted by an energy
crisis-provoked response of the acyl-CoA pool under hypoxia
(Fig. 6).
Changes of the cellular energy status happen all of the time as

most biotic and abiotic stress conditions affect energy metabolism

Fig. 4. Decreased tolerance of lacs4 lacs9 knockout lines to anoxia and submergence. (A) Eleven-day-old seedlings of wild type and lacs4-1 lacs9-2 after 9 h of
anoxia and 3-d recovery. (Scale bar: 1.0 cm.) (B) Survival scores for wild type and lacs4-1 lacs9-2 after 9-h anoxia and 3-d recovery. Data are mean values ± SD;
*P < 0.05, n = 4 (15 seedlings per replicate). (C) Phenotype of wild type and lacs4 lacs9 mutant grown in air (control), or after 3- or 4-d submergence-induced
hypoxic treatment. (Scale bar: 2 cm.) Photographs were taken 4 d after the submergence treatment. (D) Absolute dry weight of wild-type and lacs4-1 lacs9-2
plants grown in air. Data represent mean ± SD (three replicate experiments with every 12 plants per genotype). Asterisk indicates significant differences after
one-way ANOVA (P < 0.05). (E) Absolute fresh weight of wild-type and lacs4-1 lacs9-2 plants grown in air. Data represent mean ± SD (three replicate ex-
periments with every 12 plants per genotype). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (F) Relative fresh weight of wild-type
and lacs4-1 lacs9-2 plants grown in air, or after 3 or 4 d of submergence followed by 4 d of recovery. Data represent mean ± SD (three replicate experiments
with every 12 plants per genotype). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (G) Percentage of plants that survived the 3 or
4 d of flooding-induced hypoxia, respectively (mean values ± SD, three replicate experiments with every 12 plants per genotype). *P < 0.05 according to one-
way ANOVA.

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in one way or another (44). It would be most detrimental for plant
fitness, when each fluctuation of the ATP level immediately led to
the activation of hypoxic gene expression (34). Therefore, the
lifetime of ERFVII proteins depends on the actual cellular oxygen
concentration via the Cys branch of the N-end rule for proteaso-
mal protein degradation (3, 4, 11). Only when an energy crisis
is provoked by low-oxygen conditions, the stabilization of
RAP2.12 enables the protein to accumulate in the nucleus in a
sufficient amount to activate hypoxic gene expression. However,
when RAP2.12 is released from ACBP1 due to an energy deficit
that is not related to low-oxygen stress, the protein will be rapidly

degraded due to proteasomal activity. Therefore, we propose that
the ACBP1:RAP2.12 complex forms the initial hub capable of
integrating signal inputs related to the cellular energy charge with
oxygen concentration-dependent determination of the lifetime of
RAP2.12 protein (Fig. 6). Subsequently, RAP2.12 protein that is
newly synthesized after the onset of hypoxia does still undergo N-
end rule-assisted stabilization but may not be linked directly to the
energy status of the cell.
Constitutive activation of the molecular stress response to low

oxygen in the lacs4lacs9 mutant background led to reduced tol-
erance to low oxygen as well as to flooding stress (Fig. 4). This

Fig. 5. Decreasing the cellular ATP level constitutes limiting conditions for LACS activity and induces the expression of low-oxygen responsive genes. (A) ATP
levels under hypoxia (mean ± SD, *P < 0.05, n = 5). (B) Concentration of ADP in wild-type seedlings grown under long-day conditions and exposed to hypoxia.
Data shown are given in nanomoles per gram fresh weight and represent the mean ± SD of independent replicates (n = 5). (C) ATP-to-ADP-ratio under
hypoxia (mean ± SD, *P < 0.05, n = 5). (D) In vitro LACS activity depends on ATP concentration (mean ± SD, *P < 0.05, n = 5). The gray area marks the ATP-
concentration range usually determined in plant cells. (E) Differential expression of hypoxia-responsive genes after 3 h of 1 mM DMTU and/or 50 μM
antimycin-A (AA) treatment under aerobic conditions (reference: mock-treated control). Data are presented as mean ± SD, *P < 0.05, n = 5. (F) ATP levels after
3 h of 50 μM AA treatment (mean ± SD, *P < 0.05, n = 5). (G) Concentration of ADP in wild-type seedlings exposed to 3 h of 50 μM AA treatment. Data
represent mean ± SD (n = 5). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (H) ATP-to-ADP-ratio after 3 h of 50 μM AA treatment
(mean ± SD, *P < 0.05, n = 5). (I) Oxygen consumption rate in wild-type leaves upon 3 h of 50 μM AA treatment. Data represent mean ± SD (n = 7). Asterisk
indicates significant difference after Student’s t test (P < 0.05).

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observation is congruent with earlier observations that consti-
tutive activation of the hypoxia-stress response in plants via
overexpression of a stable version of RAP2.12 protein reduced
tolerance to hypoxia (34), although other studies indicate that
the latter phenotype is likely conditional to growth conditions
and recovery treatment too (4, 34). This underlines the impor-
tance of a timely control of stress responses that are optimally
adjusted to the actual environmental conditions. The integration
of (i) energy-dependent changes in C18:1-CoA levels as cellular
trigger signal with (ii) the homeostatic control of the lifetime of
RAP2.12 in an oxygen concentration-dependent manner pro-
vides a highly specific control mechanism to initiate hypoxic re-
sponses. The mechanism guarantees that a full low-oxygen
response is activated only when hypoxia is detrimental for the
plant’s energy status.
The ATP dependence of oleoyl-CoA synthesis by LACS in

combination with the impact of oleoyl-CoA on the interaction
between RAP2.12 and ACBP exposes mitochondrial activity as
an early trigger for hypoxia signaling. Consistently, manipulating
mitochondrial ATP synthesis using inhibitors of specific re-
spiratory complexes like antimycin-A induced RAP2.12-controlled
hypoxic gene expression even under aerobic conditions (45) (Fig.
5E). It is striking that the induction of hypoxic gene expression in
air by acyl-CoAs (Fig. 3 C and H) or antimycin-A (Fig. 5E) was
lower compared with the induction of these genes by hypoxic
conditions (Fig. 3H). This observation stresses the impact of ad-
ditional oxygen-dependent regulatory mechanisms, such as the N-
end rule-mediated reduction of RAP2.12 lifetime in air. In this
context, it is worth mentioning that the control of low-oxygen
stress responses is not only linked to the oxygen and energy sta-
tus of the cell, but it is also known to be influenced by other
cellular factors such as nitric oxide (46, 47), hydrogen peroxide
(48), calcium (49, 50), and potassium (51). In the near future, it
will be intriguing to expand the mechanistic explanation of how
the energy and oxygen status of a cell is integrated upon low-
oxygen stress with these additional signaling components (12).
ACBPs are found in all kingdoms of life, while ERFVIIs are

highly conserved among higher plants. Moreover, the interaction
domains of both protein families are highly conserved in plants
(Fig. 1). Therefore, the ACBP:ERFVII signaling hub as pre-
sented here may represent a universal mechanism in plants to
initiate hypoxia-induced stress responses via the integration of
multiple cellular signals. Moreover, the specific interaction of
ACBPs with various acyl-CoAs on the one hand (Fig. 2) and the
distinct cellular responses provoked by individual acyl-CoAs on
the other hand (Fig. 3 A and B) suggest that many additional

possibilities may exist of how acyl-CoAs can modulate cellular
signaling pathways in plants.

Materials and Methods
Plant Materials. Arabidopsis thaliana ecotype Col-0 was used as wild type for
all analyses. The 35S:RAP2.12-GFP line and lacs4-1 lacs9-2 and lacs4-2 lacs9-7
double knockout lines were described previously (3, 33). The acbp1 knockout
line (SAIL_683_C03) was obtained from the Nottingham Arabidopsis Stock
Center (SI Appendix, Fig. S5).

Growth Conditions and Analysis of Oxygen Deprivation Response. For testing
anoxia tolerance of seedlings, seeds were sown on half-strength MS medium
containing 0.5% (wt/vol) sucrose, stratified for 48 h at 4 °C, and germinated
at 21 °C day/18 °C night with a photoperiod of 16 h light (150 μmol·m−2·s−1)
and 8 h dark. At day 11, seedlings were exposed to full anoxia, by placing the
culture plates in an environment containing 100% nitrogen, for 9 h in the dark
to avoid photosynthetic oxygen production. After 3 d of recovery, the survival
score was determined as previously described (4). For submergence assays,
seeds were sown in moist soil, stratified at 4 °C in the dark for 48 h, and
germinated at 21 °C day/18 °C night with a photoperiod of 16 h light and 8 h
darkness. The 4-wk-old plants were submerged in water in 40-cm high plastic
containers and kept in the dark to avoid photosynthetic oxygen production.
Leaves stayed 10 cm under the water surface. After 3 or 4 d, water was re-
moved from the boxes and plants were placed back under photoperiodic
conditions (16 h/8 h, light/dark). Submergence tolerance was assayed after 4 d
of recovery.

For acyl-CoA pool measurements, plants were grown on horizontal agar
plates containing 0.8% agar in 1/2 MS medium (pH 5.7) with 15 mM sucrose
for 2 wk under long-day conditions (16 h in 150 μmol photons·m−2·s−1 at 21 °C
and 8 h in 0 μmol photons·m−2·s−1 at 19 °C). After 2.5 h in light, they were
subjected to hypoxia in the dark by exposition to a stream of air containing
1% (vol/vol) oxygen, supplemented with nitrogen and 350 ppm carbon di-
oxide. Plants were harvested by freezing in liquid nitrogen after 2 h, 3 h, and
4 h of hypoxia. For normoxic control, untreated plants were harvested si-
multaneously with the start of hypoxic treatment.

For expression analysis after acyl-CoA treatment, wild-type seeds were
sown in 24-well plates containing half-strength liquid MS medium, stratified
for 48 h at 4 °C, and germinated at 21 °C day/18 °C night with a photoperiod
of 16 h light (150 μmol·m−2·s−1) and 8 h dark. At day 14, seedlings were
treated with different acyl-CoAs at a final concentration of 0.1% in 0.01%
pluronic F68. Pluronic is a nonfatty acid-based detergent which means that
the detergent properties of pluronic in solubilizing fatty acyls are not con-
founded by fatty acyls derived from the detergent itself (52).

Cloning of Constructs. Coding sequences (CDSs) were amplified from a cDNA
template using Phusion High Fidelity DNA polymerase (Thermo Fisher Sci-
entific). The ACBP1 CDS was cloned into pENTR-D and recombined into
pK7FWG2 (51) to fuse it with GFP. For in vitro expression, the CDS of
ACBP1 was fused N terminally with a Flag tag by PCR and cloned into pF3A
WG BYDV (Promega), while the CDS of RAP2.12 was fused C terminally to

Fig. 6. Triggering low-oxygen responses in plants integrates the cellular energy and oxygen status via modulation of oleoyl-CoA levels. Oxygen limitation
reduces cellular ATP levels, which results in increased C18:1-CoA levels. Dissociation of ERFVII protein (as shown here for RAP2.12) bound to ACBP1 at the
plasma membrane is promoted by C18:1-CoA. Free ERFVII protein is stable under low-oxygen conditions and relocalizes into the nucleus to activate hypoxic
responses.

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https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1809429115/-/DCSupplemental
https://www.pnas.org/cgi/doi/10.1073/pnas.1809429115


CFP by PCR and cloned into pF3A WG BYDV (Promega). A complete list of all
primers used is provided in SI Appendix, Table S4.

Plant Transformation. Transgenic ACBP1-GFP plants were generated by trans-
forming wild-type plants with the vector pK7FWG2 (53) containing the ACBP1
CDS fused in frame to GFP at its C terminus. T0 seeds were screened for
kanamycin resistance and the presence of GFP signals by confocal microscopy.

qRT-PCR. RNA extraction, digestion of genomic DNA, cDNA synthesis, and
qRT-PCR analysis were performed as described previously (54). For all experi-
ments, four to five independent biological replicates were used, as indicated in
the figure and table legends. For normalization, UBIQUITIN10 expression was
used according to ref. 3. Primers for hypoxia core genes, LACS genes, and
UBI10 are given in SI Appendix, Table S4.

RNA-Seq Analysis. Illumina HiSeq sequencing was performed according to
standardized protocols as described in detail in SI Appendix. Transcriptome
analysis was performed by means of CLC Genomics Workbench v.6 using the
A. thaliana reference sequence (Tair10). Expression values were normalized
using quantile normalization and pairwise statistical analyses comparing the
treatments performed using false discovery rate (FDR)-corrected P values
based on Baggerly’s test (55).

Confocal Imaging. For protein localization studies, GFP signals were imaged
and analyzed with a Leica DM6000 TCS SP8 confocal microscope (Leica
Microsystems). Nuclear staining was performed by using DAPI (molecular
probes) according to the manufacturer’s instructions. For quantification of
nuclear translocation of RAP2.12 after acyl-CoA treatment, 20 DAPI-stained
nuclei per plant (five plants in total) were analyzed per treatment. Leaves of
5-wk-old soil-grown 35S:RAP2.12-GFP plants were incubated for 3 h with
different acyl-CoAs at a final concentration of 0.1% in 0.01% pluronic F68 in
a 24-well plate under continuous shaking in the light. The experiments were
repeated three times.

In Vitro Binding Assay. The method to determine whether acyl-CoAs affect the
interaction between ACBP1 and RAP2.12 is explained in detail in SI Appendix,
Supplementary Information Text and Fig. S1. In brief, both proteins were
synthesized using wheat germ extract (54). The full CDS of RAP2.12 was
fused C terminally with the CDS of CFP, and the CDS of ACBP1 was N-
terminally fused with a FLAG tag. RAP2.12-CFP protein was bound to GFP–
Trap-A beads (Chromotek) and incubated with ACBP1 protein overnight.
Subsequently, beads were resuspended in wash buffer containing different

acyl-CoAs at a final concentration of 0.1% in 0.01% pluronic F68, or as a
mock control of only 0.01% F68. After one night of incubation, the buffer
was replaced and the composition of the protein complex retained to the
beads was analyzed on a Western blot.

Analysis of Acyl-CoA Esters. Acyl-CoAs were extracted, derivatized, and an-
alyzed using HPLC as described earlier (56). Detailed information about the
method is provided in SI Appendix.

LACS4 in Vitro Enzyme Assay. The in vitro LACS enzyme assay was carried out
as described previously (17) using protein that was heterologously expressed
in Escherichia coli (57). Details of the method are explained in SI Appendix.

ATP and ADP Quantification. ATP and ADP were extracted with 16% tri-
chloroacetic acid (33) and analyzed after derivatization by HPLC as described
previously (58). Details of the procedure are described in SI Appendix.

Analysis of Oxygen Consumption Rates. For the determination of oxygen
consumption rates, 4-wk-old plants grown on soil under short-day conditions
(8 h in 160 μmol photons m−2·s−1 at 20 °C and 16 h in 0 μmol photons m−2·s−1

at 16 °C) were used. Starting 3 h before measurement, the plants were
sprayed every hour either with 50 mM Mes buffer (pH 6.5) containing 50 μM
antimycin-A (59) or with 50 mM Mes buffer for control. This treatment was
done in the last hour of dark phase and 2 h into the light phase, and then
measurement of oxygen consumption rates at normoxic conditions was
performed as described before (34) using the respective spraying solution. In
a similar setup, the effect of different acyl-CoAs on the oxygen consumption
rate was tested by incubating leaf disks in acyl-CoAs at a final concentration
of 0.1% in 0.01% pluronic F68.

Statistical Analysis. Statistical evaluation of significant variation between
treatments or genotypes was done by performing Student’s t test or one-way
ANOVA where appropriate.

Data Availability. The RNA-Seq gene expression data are available in NCBI’s
Gene Expression Omnibus (GEO) through GEO Series accession no. GSE97186.

ACKNOWLEDGMENTS. We thank Sandro Parlanti and Frauke Augstein for
valuable support. This work was supported by grants (to J.T.v.D.) (DO 1298/2-2)
and (to P.G.) (GE 878/7-2) from the German Science Foundation (DFG). M.F.
was supported by the DFG (Grant DFG FU 430/5-1).

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