HYPOTHESIS AND THEORY ARTICLE
published: 19 June 2012
Retrograde signaling in plants: from simple to
Plant Molecular Biology (Botany), Department Biology I, Ludwig Maximilians University, Munich, Germany
Tatjana Kleine, Ludwig-Maximilians-
Universität München, Germany
Shan Lu, Nanjing University, China
Karl-Josef Dietz, Universität Bielefeld,
Dario Leister, Plant Molecular Biology
(Botany), Department Biology I,
Ludwig Maximilians University
Munich, Großhaderner Str. 2,
Germany. e-mail: email@example.com
The concept of retrograde signaling posits that signals originating from chloroplasts or
mitochondria modulate the expression of nuclear genes. A popular scenario assumes that
signaling factors are generated in, and exported from the organelles, then traverse the
cytosol, and act in the nucleus. In this scenario, which is probably over-simplistic, it is tacitly
assumed that the signal is transferred by passive diffusion and consequently that changes
in nuclear gene expression (NGE) directly reflect changes in the total cellular abundance of
putative retrograde signaling factors. Here, this notion is critically discussed, in particular
in light of an alternative scenario in which a signaling factor is actively exported from the
organelle. In this scenario, NGE can be altered without altering the total concentration of
the signaling molecule in the cell as a whole. Moreover, the active transport scenario would
include an additional level of complexity, because the rate of the export of the signaling
molecule has to be controlled by another signal, which might be considered as the real
retrograde signal. Additional alternative scenarios for retrograde signaling pathways are
presented, in which the signaling molecules generated in the organelle and the factors
that trigger NGE are not necessarily identical. Finally, the diverse consequences of signal
integration within the organelle or at the level of NGE are discussed. Overall, regulation
of NGE at the nuclear level by independent retrograde signals appears to allow for more
complex regulation of NGE than signal integration within the organelle.
Keywords: retrograde signaling, plastid signaling, nuclear gene expression, signal integration
WHAT IS A RETROGRADE SIGNAL?
and involve massive changes in the transcript profiles of nuclear
genes. The concept of retrograde signaling therefore posits that
signals originating in chloroplasts and/or mitochondria modulate
signal is generated in the organelle(s), exported, and traverses the
cytosol to act in the nucleus. Several such classical retrograde sig-
nals have been tentatively identified. However, messenger roles
have also been proposed for factors which display some, but
not all, of the characteristics attributed to classical retrograde
THE SPECTRUM OF PUTATIVE RETROGRADE
SIGNALS – A CURRENT INVENTORY
been identified during the last three decades, and their number
continues to increase. However, unambiguous experimental veri-
fication of a compound as a signaling molecule is an extremely
difficult task, and in no case is the status of a candidate as a
signaling molecule yet secure. Therefore, instead of presenting
a detailed and exhaustive description of individual candidate sig-
2005; Beck, 2005; Nott etal., 2006; Pesaresi etal., 2007; Pogson
etal., 2008; Kleine etal., 2009a; Chan etal., 2010; Pfannschmidt,
2010; Inaba etal., 2011), I wish to concentrate here on some basic
aspects of retrograde signaling mechanisms and their impact on
the character of the response.
ious sources, including the tetrapyrrole pathway, organellar gene
expression (OGE), reactive oxygen species (ROS), or the redox
state of the organelle (Table 1). Conversely, one can ask which of
the principal types of biomolecules produced in organelles have
the potential to act as a retrograde signal, i.e., encode sufficient
information on the state of the organelle to trigger an appro-
priate response in the nucleus. Nucleic acids can efficiently store
and transfer information, and their relocation from organelle to
nucleus over evolutionary time is well established (reviewed in
Kleine etal., 2009b). Hence, more than three decades ago, it was
hypothesized that organelle-derived RNAs might regulate cyto-
support for this idea has not yet been forthcoming. ROS are by-
products of several organellar processes and their accumulation is
associated with changes in NGE (Apel and Hirt, 2004). However,
either because they are probably too short-lived to reach the
nucleus [in the case of singlet oxygen (1O2)] or too unspecific
(H2O2) to act as information carriers (reviewed in Møller and
Sweetlove, 2010). Although specific transporters for the export of
chloroplast proteins, in particular transcription factors, have yet
to be identified,evidence is accumulating that chloroplast-located
transcription factors can be conveyed to the nucleus (Sun etal.,
2011; Isemer etal.,2012). Nevertheless,metabolites still appear to
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Table 1 | Overview of different classes of putative retrograde signaling
Putative retrograde ExamplesReference
RNA Bradbeer etal. (1979)
Protein: transcription Whirly 1Isemer etal. (2012)
PTM Sun etal. (2011)
ABI4 Koussevitzky etal. (2007)
Protein: degradationReviewed in
productsMøller and Sweetlove (2010)
species Apel and Hirt (2004)
Reviewed in Apel and Hirt (2004)
Metabolite Strand etal. (2003)
HemeWoodson etal. (2011)
ABA reviewed in Baier and Dietz (2005)
PAP Estavillo etal. (2011)
β-Cyclocitral Ramel etal. (2012)
Unknown Organellar gene Reviewed in
expression Gray etal. (2003)
Thylakoid redoxReviewed in
state Pfannschmidt (2003);
Pfannschmidt etal. (2003)
be the most likely candidates for retrograde signaling molecules.
This is because (i) the profile of metabolites exported from an
organelle to the cytosol does indeed reflect the metabolic state
of the organelle and (ii) numerous transporters exist that facil-
itate the controlled exchange of metabolites between organelles
and the cytosol. Therefore, changes in the metabolic profile of
the cytosol triggered by the organelle might well be used by
the cell to adjust NGE, either by using a metabolite directly as
a signaling molecule or by converting an appropriate metabo-
lite into an active signaling molecule during its sojourn in the
WHAT SORTS OF NUCLEAR GENES ARE REGULATED BY
ing or the identification of putative signaling mutants are LHCB1,
2CPA, and APX2 (Oelmüller and Mohr, 1986; Karpinski etal.,
protein, a chloroplast peroxidase and a cytosolic ascorbate perox-
idase, respectively. Transcriptomics-based studies in Arabidopsis
thaliana have identified large sets of nuclear genes as possible
targets of retrograde signaling by employing either putative sig-
naling mutants and/or conditions thought to trigger retrograde
signaling. Thus, nuclear genes coding for chloroplast proteins
have been associated with GUN signaling (Strand etal., 2003)
and a subsequent study showed that the ACGT motif, the core
of both the light-responsive G box (CACGTG) and the abscisic
acid (ABA) response element (ABRE), was over-represented in
Analysis of the time-dependent impact of redox signals showed
rapid and dynamic changes in nuclear transcript accumulation,
resulting in differential and specific expression patterns for genes
associated with photosynthesis and metabolism (Bräutigam etal.,
2009). Another study analyzed the overlap between the differ-
ential gene expression profiles of several mutants affected in
represent either nuclear target genes of photosynthesis-relevant
retrograde signaling or genes that are differentially expressed to
compensate for the effects of suppression of retrograde signaling
(Pesaresi etal., 2009). Interestingly, genes involved in many dif-
ferent processes, including stress responses, post-transcriptional,
as well as metabolism, were represented in this set; however,
few of them appeared to be directly involved in the photo-
synthetic process (Pesaresi etal., 2009). ROS-responsive genes
were also identified and it was possible to discriminate between
H2O2- and1O2-responsive genes (op den Camp etal., 2003).
Interestingly, nuclear genes coding for chloroplast proteins gen-
erally seem to be co-regulated to a certain degree irrespective of
their biochemical function (Richly etal., 2003; Biehl etal., 2005;
Leister etal., 2011), prompting the postulate that a transcrip-
tional master switch might exist (Richly etal., 2003). Nuclear
photosynthesis-associated genes, including genes coding for pho-
tosystem subunits, display a distinct regulation pattern, which is
independent of the master switch, highlighting the unique role of
photosynthesis as representing both stimulus and target of ret-
rograde regulation (Biehl etal., 2005). The large-scale analysis
of the transcript profiles of chloroplast- and mitochondrion-
relevant genes in A. thaliana confirmed that the activity of gene
sets involved in organellar energy production (OEP) or OGE
in each of the organelles and in the nucleus is highly coordi-
nated (Leister etal., 2011). Moreover, the same analysis indicated
that dynamic inter- and intracompartmental transcriptional net-
works for OEP and OGE genes adjust the activity of organelles
in response to the cellular energy state and environmental stresses
(Leister etal., 2011).
Taken together, these studies suggest that large numbers of
unclear whether they are directly targeted by retrograde signal-
ing or are regulated indirectly as part of broader adjustments that
compensate for the original stimulus (in most cases mutations).
The use of transient stimuli and kinetic analysis of transcriptional
responses,as practiced by Bräutigam etal. (2009),seems the most
appropriate way to address this question.
THE SIMPLEST SCENARIO: THE CLASSICAL
As outlined above,classical signaling molecules are assumed to be
generated in the organelle and make their way to the nucleus,
where they modify NGE by either inducing or repressing the
expression of specific genes (Figure 1A). Therefore, when gene
expression patterns in retrograde signaling mutants are compared
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FIGURE 1 | Characteristics and transport of classical retrograde signals.
(A) Schematic overview of the mode of operation of the simplest scenario for
retrograde signaling, the case of a classical retrograde signal. (B) Overview of
how changes in the relative abundance of the retrograde signal (RS) in the
nucleus might affect NGE. Relative levels of the RS increase from state 1
(0%), state 2 (50%) to state 3 (100%), resulting in either induction (gene a) or
repression (gene b) of the expression of a nuclear reporter gene (indicated in
grayscale).The repressive effect of the RS is symbolized in the top panel by
negative values (“−”), the inducing effect by positive values (“+”). In a
signaling mutant rs, no expression changes occur and therefore differential
expression (rs1/WT) in terms of down-regulation (gene a) or up-regulation
(gene b) is observed (shown in color scale). (C) Passive diffusion versus active
transport of RS. If the signaling molecule is disseminated by diffusion (upper
panels, “Diffusion”), the total concentration of the signaling molecule in all
cellular components increases during the transition from state 1 to state 3,
leading to changes in NGE. In this case, a linear correlation between the total
concentration of the signaling molecule and NGE is expected. If the signaling
molecule is actively transported into the nucleus (lower panel, “Active
transport”), NGE can be altered by specifically changing the abundance of the
signaling molecule in the nucleus – without altering its total concentration in
the cell as a whole. In consequence, analyses of total cell extracts would fail
to identify any correlation between the overall abundance of the signaling
molecule and NGE.The tetrapyrrole Mg-protoporphyrin IX, for which total
cellular levels fail to correlate with changes in NGE, might represent such a
signal. Note that the “active transport scenario” would require regulation of
the activity of the transport by another signal. In fact, in such a scenario the
signal that up-regulates export might be considered as the real signal, and the
transported compound as second messenger.
with the profile in wild-type (WT) plants, differential expres-
sion of the regulated genes will be observed (Figure 1B). Two
principal modes of transfer of retrograde signaling molecules
present themselves: transfer by passive diffusion or active trans-
port involving specific transporters (Figure 1C). In principle,
the latter mode of transfer would include already an addi-
tional level of complexity, because the rate of the export of
the signaling molecule has to be controlled by another sig-
nal, which might be considered as the real retrograde signal
(Figure 1C). Here, three putative classical retrograde signaling
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Leister Concepts of retrograde signaling
molecules are discussed in the context of these alternative modes
of relocation: (i) the phosphonucleotide 3?-phosphoadenosine
5?-phosphate (PAP) and (ii) β-cyclocitral (β-CC), an oxida-
tion product of β-carotene, both of which might be relocated
by diffusion; and (iii) the tetrapyrrole Mg-protoporphyrin IX
PAP AND β-CYCLOCITRAL – TWO NOVEL CANDIDATE
3?-Phosphoadenosine 5?-phosphate accumulates in A. thaliana in
response to drought and high light (HL) stress (Estavillo etal.,
2011). The enzyme SAL1, which is found in both mitochon-
dria and chloroplasts, regulates PAP levels by dephosphorylating
PAP to AMP; therefore, in sal1 mutants PAP accumulates 20-fold
(Estavillo etal., 2011). Moreover, sal1 mutants display consti-
tutive upregulation of many HL-regulated genes (Wilson etal.,
2009). Except in the case of chloroplasts, where PAP could be
detected by HPLC, attempts to identify PAP in subcellular frac-
tions have failed for technical reasons (Estavillo etal., 2011).
However, targeting of SAL1 to either the nucleus or chloroplasts
in sal1 mutants reduces total PAP levels and decreases expres-
sion of the nuclear marker gene APX2, indirectly demonstrating
that PAP must be able to move between cellular compartments
(Table 2). Because total cellular levels of PAP and nuclear marker
gene expression correlate well (Estavillo etal., 2011), a plausible
mechanism for the effects of PAP on NGE is the “free diffusion”
scenario (Figure 1C). In this scenario, the total concentration of
the signaling molecule in the organelle must reach a certain level
to enable diffusion into the nucleus to supply sufficient amounts
toitsputativesiteof action. Consequently,PAPseemstorepresent
a classical signaling molecule which is synthesized in organelles
of a given type, leaves that organelle and reaches the nucleus
by passive diffusion. Moreover, the physiological relevance of
PAP as a possible retrograde signal is strongly supported by the
fact that PAP levels increase in response to at least two abiotic
Oxidation products of β-carotene, like β-CC, have been sug-
levels (Ramel etal., 2012; see below). β-CC is volatile; therefore,
it should be capable of traversing the cytosol to regulate gene
expression in the nucleus. β-CC is generated under physiologi-
cal conditions, following exposure to excess light which promotes
generation of1O2, which in turn oxidizes β-carotene. Moreover,
treatment of plants with exogenous β-CC induces changes in the
expression of many1O2-responsive genes. Therefore, it is tempt-
ing to speculate that β-CC represents a classical retrograde signal
which is disseminated by diffusion. Because this HL-induced sig-
naling pathway may involve at least two signaling molecules,1O2
in the next section.
IS Mg-PROTO IX A CLASSICAL RETROGRADE SIGNALING
MOLECULE OR PART OF A MORE COMPLEX PATHWAY?
When it became clear that most gun mutants were affected
in chloroplast tetrapyrrole biosynthesis (Mochizuki etal., 2001),
the obvious next step was to test whether an intermediate in
tetrapyrrole biosynthesis might be responsible for changes in
NGE. Therefore,the possibility that the concentrations of various
tetrapyrroles might be correlated with the expression of nuclear
marker genes in A. thaliana was analyzed in several studies with
Moulin etal., 2008). Strand etal. (2003) claimed to have found a
proto IX and NGE, although an earlier study by the same group
had come to a different conclusion (Mochizuki etal., 2001). Later
investigations employing more precise and reproducible tests, as
well as additional genotypes, confirmed that no correlation exists
in the total concentration of Mg-proto IX in plant leaves and
alterations in the expression profiles of certain nuclear marker
genes does not suffice to rule out the possibility that Mg-proto
IX acts as a classical plastid signal. The finding simply argues
against diffusion as a transport mechanism for the signal. It
does not exclude the possibility that the signaling molecule is
actively transported from its source to the nucleus. If the signaling
Table 2 | Characteristics of putative retrograde signaling molecules.
LocalizationPhysiological stimulus Reference
cpUnclear Unclear Excess light Apel and Hirt (2004), Fischer etal. (2007)
cp + mt + per
Yes LikelyMultiple Apel and Hirt (2004), Mubarakshina etal. (2010)
Unclear UnclearUnclear Strand etal. (2003)
Heme cpYes UnclearUnclear Thomas and Weinstein (1990), Woodson etal. (2011)
PAPcp + mt
Likely Indirect evidence Drought and excess lightEstavillo etal. (2011)
β-CyclocitralLikely LikelyExcess light Ramel etal. (2012)
ABA PrecursorYesCandidate receptorExcess light Wasilewska etal. (2008)
present in cpidentified
cp, chloroplast; mt, mitochondria; per, peroxisomes.
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there could still increase (with corresponding effects on NGE),
while the total cellular concentration of the signaling molecule
remained unchanged (Figure 1C). Whether or not this scenario
holds for Mg-proto IX remains to be clarified; as a phototoxic
molecule, it seems rather unlikely to act as a signaling molecule
(Mochizuki etal., 2010). Heme is a somewhat better candi-
date retrograde signaling molecule deriving from the tetrapyrrole
biosynthesis pathway (Woodson etal., 2011), but levels of total
heme and LHCB1 mRNA also fail to correlate (Voigt etal., 2010;
Woodson etal., 2011).
Nevertheless, a correlation between perturbations in chloro-
plast tetrapyrrole biosynthesis and NGE changes undoubtedly
exists,and future research has to clarify what retrograde signaling
mechanisms mediate this relationship.
MORE COMPLEX SCENARIOS OF RETROGRADE
the molecule that is generated in the organelle itself relays infor-
mation to the nucleus, might be true for the phosphonucleotide
PAP during drought and HL stress and for β-CC after HL stress
(Table2),but need not be valid for other retrograde signals. Thus,
Figure2A).Alternatively,the original metabolite triggering retro-
grade signaling might not even leave the organelle (Figure 2B).
For instance, singlet oxygen (1O2) is generated in chloroplasts
and regulates the expression of a set of nuclear genes, although
it is thought to be largely restricted to plastids with little, if any,
leakage to the cytosol (Apel and Hirt, 2004; Fischer etal., 2007).
In consequence, an unidentified second messenger should exist
which relays the information from the plastid to the nucleus, and
the two chloroplast proteins Executer 1 and 2 (reviewed in Kim
etal.,2008),as well as ABA (Kim etal.,2009),seem to be involved
gested to act as a “downstream” messenger of1O2(Ramel etal.,
2012), possibly representing a classical retrograde signal which
traverses the cytosol to regulate gene expression in the nucleus
So far, proteins that specifically transport signaling molecules
from the organelle to the nucleus have not been identified. In
an extreme case, the signaling molecules could be delivered
directly to its target if organelle and nucleus were physically con-
nected. In fact,stromules might represent such connection points
between plastids and the nucleus (Figure 2C). Stromules are
stroma-filled tubules that extend from the surface of plastids, are
extremely variable in length and are highly dynamic structures
(reviewed in Hanson and Sattarzadeh, 2008). Interestingly, stro-
mules are induced by stress treatments, including drought and
salt stress, and application of ABA (Gray etal., 2012) – condi-
tions which are also thought to be associated with retrograde
INTEGRATION OF MULTIPLE SIGNALS
The existence of multiple candidate signaling molecules, includ-
signal β-CC), Mg-Proto IX and PAP, suggests that it is very likely
that more than one retrograde signaling pathway exists. More-
over, the generation of signaling molecules in the organelle will
itself be affected by multiple factors. Two model cases that take
account of this complexity are discussed here. In scenario 1, each
of two retrograde signals (RS1 and RS2) can act independently to
activate or repress NGE. In scenario 2, only one retrograde signal
(RS) operates, but its formation is controlled by two organellar
factors (O1 and O2) that can act synergistically or in opposite
senses (Figure 3A). To simulate the effects on NGE of the two
retrograde signals in scenario 1 and the two organellar factors
in scenario 2, three discrete doses for each retrograde signal or
organellar factor were considered: 0, 50, and 100%. If both ret-
rograde signals or organellar factors are assumed to act in the
same sense, i.e., both stimulate (or repress) gene expression or
generation of the retrograde signal, the two scenarios result in
identical effects on NGE in WT plants (Figures 3B,C). Further-
more, where mutants for the two retrograde signaling pathways
or organellar factors are available, the differential expression pat-
terns determined in the two scenarios will be identical. Therefore,
FIGURE 2 | More complex scenarios for retrograde signaling. (A)The
signaling molecule generated in and exported from the organelle (filled
circles), and the signaling molecule that enters the nucleus (open circles)
might not be identical. ABA, which is synthesized in the cytosol from a
chloroplast precursor, is a possible example. (B)The signaling molecule
generated in the organelle might not even leave the organelle.1O2serves
here as an example, as it could, in principle, generate volatile oxidation
products of carotenoids that serve as “downstream” messengers.
(C) Direct delivery of the signaling molecule to the nucleus via
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FIGURE 3 | Signal integration. (A) Schematic overview of the integration of
two retrograde signals (RS1 and RS2) in the nucleus (left panel) or in the
organelle (right panel). O1 and O2 refer to signals acting within the organelle.
Activating signals are indicated by arrows, repressing signals by blunt-ended
lines. Left panel (two retrograde signals, integration in the nucleus): gene a?,
induced by RS1 and RS2; gene b?, repressed by RS1 and induced by RS2;
gene c?, induced by RS1 and repressed by RS2; gene d?, repressed by RS1
and RS2. Right panel (two organellar signals, integration in the organelle): RS
induces gene a and represses gene b as in Figure 1A. (B) Signal integration
at the gene level. In this model each of the two retrograde signals can
accumulate to 0, 50, or 100% [depicted as black (RS1) or white (RS2) bars],
analogous to the three states discussed in Figures 1B,C, resulting in nine
different combinations, of which eight are shown in the top panels (the
combination RS1: 0%/RS2: 0% is not shown).The effects on NGE are
shown in WT and corresponding single (rs1 and rs2) and double (rs1 rs2)
signaling mutants in grayscale. In color scale, effects on differential gene
expression (mutant versus WT) are shown. In the left/right panel, the
expected impact of additive inducing/repressing effects of the retrograde
signals on genes a?and d?is shown. In the middle panel (b?/c?), the two
retrograde signals act antagonistically and the outcome for the expression of
genes b?and c?is very similar. (C) As in (B), the two organellar signals can act
additively with the same polarity (O1:+/O2:+) or act antagonistically
(O1:−/O2:+; O1:+/O2:−; upper panel).The net effect of OS1 and OS2 on RS
is shown in the second uppermost panel and can either induce gene
expression (the two left panels, gene a) or repress gene expression (the two
right panels, gene b).The resulting effects on NGE are again shown in
grayscale for absolute expression and in color scale for differential expression
(mutant versus WT).
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it will not be possible to discriminate between the two scenar-
ios on the basis of expression data from WT and mutant plants
defective in signaling under conditions in which both retro-
grade signaling factors or both organellar signaling factors act
In contrast, under conditions where the two retrograde sig-
nals or organellar factors act in opposite senses, differences in
differential expression between signaling mutants and WT plants
will emerge. In the case of two retrograde signals whose effects
are integrated in the nucleus, differential up-regulation for one
signaling mutant but down-regulation for the other signaling
mutant will be observed (Figure 3B). On the other hand, where
signal integration occurs already in the organelle, opposite regu-
lation of the two organellar signals will essentially neutralize their
effects on the generation of the retrograde signal (Figure 3C).
In the model presented here, five out of eight conditions fail
to produce a retrograde signal in the WT, and opposite dif-
ferential regulation in signaling mutants is found only in one
Overall,regulation of NGE by two independent retrograde sig-
nals appears to allow for more complex regulation of NGE, and
this should be detectable by opposite regulation of NGE in cor-
responding signaling mutants under appropriate conditions. Of
course, the crucial question is whether such complex regulatory
scenarios are actually realized and whether sufficient expression
data are available to decide whether signal integration occurs in
the organelle or the nucleus. In fact, two instances of synergis-
tic effects of two signaling mutants on NGE have been described
so far. The first example concerns the gun mutants and the sec-
ond involves mutants defective in OGE. The gun phenotype was
originally defined as derepressed LHCB1 expression after treat-
ment with norflurazon (NF). Then the double mutants gun1 gun4
and gun1 gun5 were shown to exhibit higher levels of LHCB1
mRNA after NF treatment than did the individual single mutants,
indicating that GUN1 affects a separate pathway from GUN4
and GUN5 (Mochizuki etal., 2001). However, the same group
showed in a later publication that the synergy between gun1 and
gun5 does not extend to the gun1–9 allele (Koussevitzky etal.,
2007). Nevertheless, the available data are not sufficient to decide
whether signal integration occurs in the chloroplast or at the
Changes in OGE affect the expression of nuclear genes, in
particular genes coding for photosynthetic proteins (reviewed
in Pesaresi etal., 2007). Pesaresi etal. (2006) showed that
simultaneous perturbation of OGE in both mitochondria and
chloroplasts down-regulates nuclear photosynthetic genes much
more drastically than do treatments that affect OGE in only
the one or the other. For this study, mutations affecting
mitochondrial or chloroplast ribosomes, as well as mutations
affecting a dual-targeted tRNA synthetase were analyzed. For
all nuclear photosynthesis genes studied, the double mutant
prpl11 mrpl11, in which ribosomal function was impaired in
both types of organelles, always produced fewer transcripts
than either of the single mutants prpl11 and mrpl11, indicat-
ing that both plastid OGE and mitochondrial OGE stimulate
NGE under the growth conditions employed in the study. In
this case also, it is not possible to discriminate between signal
integration in the organelle and the nucleus based on the
expression profiles. However, because the two mutations affect
separate organelles it is tempting to speculate that signal inte-
gration occurs in the cytosol or in the nucleus as outlined in
CONCLUDING REMARKS: SOLID CRITERIA TO DEFINE A
How can a candidate signaling molecule be unambiguously con-
firmed to be a real signal? In light of the different retrograde
signaling scenarios outlined above, it is not possible to draw
up a universal list of characteristics and experiments that could
unequivocally decide whether a putative retrograde signal is a
true retrograde signal. Nevertheless, a number of conclusions
can be drawn from the considerations set out above. A classical
retrograde signal should be detectable in all relevant compart-
ments (organelle, cytosol and nucleus), assuming that passage
through the cytosol is not rendered superfluous by the use of
stromules. So far, no putative classical signal has been unambigu-
ously detected in all relevant compartments (Table 2). Secondly,
for a signal which follows the free diffusion scenario a correla-
tion between overall signal level and NGE should exist, opening
the way for systematic approaches involving LC–MS or GC–MS
analyses. Finally, how can one show that a signaling molecule
is not only required but also sufficient to trigger NGE? Feeding
experiments in which PAP (the candidate most likely to con-
form to the “free diffusion” scenario) was supplied via the roots
have so far failed to confirm a signaling role for it (Estavillo
etal., 2011), whereas Mg-proto IX was reported to repress LHCB
expression when fed to protoplasts for 5 h at relatively high con-
centrations (Strand etal., 2003). These two examples highlight
the dilemma of feeding experiments. On the one hand, a nega-
tive result cannot conclusively disqualify a candidate retrograde
signal, unless one can monitor the accumulation of the added
molecule at its destination. Conversely, a positive result is only
valid if the feeding assays truly mimic physiological conditions.
Nevertheless,feeding experiments and in vitro assays with subcel-
lular fractions (in which target nuclear genes are directly exposed
to the signaling molecules), together with the transient activation
tems, are certainly needed to clarify the role of putative signaling
ergistic and antagonistic integration of diverse retrograde inputs
will be necessary for the knowledge-based modification of retro-
grade signaling. Studies, like the one of gene regulatory networks
involving AP2/EREB transcription factors (Dietz etal., 2010),
might serve as prototypes for systematic genome analysis of ret-
(see Figure 3) certainly represent simplifications of a much more
ing (grant FOR 804) and Paul Hardy for critical reading of the
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Conflict of Interest Statement: The
author declares that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 25May2012; accepted: 05June
2012; published online: 19 June 2012.
naling in plants: from simple to complex
scenarios. Front. Plant Sci. 3:135. doi:
This article was submitted to Frontiers in
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