Download Intracellular signalling: The chloroplast talks!

Dispatch
R307
Intracellular signalling: The chloroplast talks!
Paul Jarvis
Plant cells have a unique problem: the coordination of
three different genomes. While the dominance of the
nuclear genome is indisputable, it is now clear that
organellar signals can have profound effects, not just
on nuclear gene expression but, as the Arabidopsis laf6
mutant reveals, also on whole plant morphology.
Address: Department of Biology, University of Leicester, University
Road, Leicester LE1 7RH, UK.
Current Biology 2001, 11:R307–R310
0960-9822/01/$ – see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
It is now widely accepted that the ancestors of mitochondria and chloroplasts were free-living prokaryotic organisms capable of aerobic respiration and photosynthesis,
respectively [1]. These organisms were engulfed by a
plant cell progenitor in serial endosymbiotic events that
each occurred over a billion years ago. Since then, endosymbiont genes have gradually been transferred to the nuclear
genome so that, now, most genes encoding mitochondrial
and chloroplast proteins reside in the nucleus. Organellar
gene loss is incomplete, however, so both organelles retain
a fully functional, endogenous genetic system and, as a
consequence, a certain degree of autonomy. The existence within the same cell of three different genomes — in
the nucleus, mitochondria and chloroplasts — presents a
unique set of problems for plants and algae: the coordination of these genomes so that complex processes requiring
input from multiple compartments, such as photosynthesis, can be elaborated efficiently and effectively in response
to changing developmental and environmental cues.
The coordination of events in the nucleus and the
chloroplast — a type of plastid specialized for photosynthesis — necessarily involves the exchange of information, or
intercompartmental signalling. Most information exchange
between these organelles flows from nucleus to chloroplast,
rather than vice versa, not least because >90% of chloroplast
genes are in the nucleus. The import of nucleus-encoded
proteins into chloroplasts — from the cytosol where they
are made — constitutes a huge flow of information, and
elaborate machinery has evolved to ensure that it occurs
efficiently [2]. Although many of the proteins imported into
chloroplasts are structural components — particularly of
the photosynthetic apparatus and the chloroplast’s own
genetic system — a large number perform regulatory
functions, and serve to enforce nuclear control over the
organellar compartment. Post-transcriptional regulation
is prevalent in chloroplasts, and, in many cases, a single
nucleus-encoded factor is specifically required for a
particular step in the expression of a single chloroplastencoded gene — a kind of ‘gene-for-gene’ interaction [3].
Despite all of the above, a large body of evidence indicates
that the flow of information in a plant cell is not entirely
unidirectional, and it is clear that signals from the chloroplast wield significant influence in the nucleus and the
rest of the cell [4,5]. Early studies of chloroplast-tonucleus signalling used carotenoid-deficient mutant plants,
or plants grown on carotenoid biosynthesis inhibitors such
as the herbicide Norfluorazon [4]. Carotenoids prevent the
formation of reactive oxygen species by quenching excited,
triplet-state chlorophyll, and so chloroplasts deficient in
these photoprotective compounds suffer massive internal
photooxidative damage under intense light. The damage
appears to be restricted to chloroplasts, and yet a subset of
nuclear genes encoding chloroplast proteins is severely
and specifically repressed as a result. This indicates that
chloroplasts are able to communicate their functional
status to the nucleus — the elusive ‘plastid signal’ — and
that the nucleus responds by making appropriate changes
in gene expression [4].
Møller and colleagues [6] have now demonstrated that
the influence of the chloroplast extends far beyond the
regulation of nuclear genes encoding products for its own
direct use. In a genetic screen for Arabidopsis mutants with
defects in developmental responses to light — photomorphogenesis — they isolated a novel mutant with a Ds transposon inserted into the nuclear gene for a chloroplast
protein. The mutant is called long after far-red 6 (laf6) and,
as the name suggests, its main phenotype is impaired
hypocotyl growth inhibition in response to far-red light.
Such long hypocotyl mutants usually have defects in
photoreceptors — such as the phytochromes, which sense
red and far-red light — or associated signal transduction
components, and these all localize to the nuclear and/or
cytosolic compartments [7]. Although this is not the first
time that chloroplasts have been implicated in the transduction of light-signals controlling gene expression [8], it is the
first time that a chloroplast protein has been shown to be
required for the transduction of photomorphogenic signals.
The LAF6 gene encodes a previously uncharacterized
ATP-binding cassette (ABC) protein, called atABC1, with
strong homology to ABC proteins from prokaryotes and
lower eukaryotes, and an amino-terminal transit peptide
for targeting to the chloroplast compartment. ABC
proteins constitute a superfamily of solute transporters
with representatives in all kingdoms [9]. These transporters have four domains — two ABC domains and two
R308
Current Biology Vol 11 No 8
Figure 1
Glutamate
5-Aminolevulinate
Protoporphyrinogen IX
Protoporphyrinogen oxidase
Protoporphyrin IX
Fe-Chetalase
Haem
Haem
oxygenase
laf6
Mg-Chetalase
Mg-Protoporphyrin IX
hy1
Mg-Protoporphyrin IX
monomethyl ester
Biliverdin IXα
Phytochromobilin
synthase
3Z-Phytochromobilin
3E-Phytochromobilin
gun5
brs-1, brc-1
hy2
Protochlorophyllide
Chlorophyllide
Chlorophyll
Current Biology
The tetrapyrrole biosynthetic pathway of plants and algae. Chlorophyll
and haem are synthesized in chloroplasts from glutamate by different
branches of the same pathway. The branch point is defined by the
enzymes Mg-chelatase (for the chlorophyll branch) and Fe-chelatase (for
the haem branch). The phytochrome chromophore, 3E-phytochromobilin,
is synthesized from haem. Steps in the pathway disrupted by the
Arabidopsis hy1, hy2 and gun5 mutations, and the Chlamydomonas
brs-1 and brc-1 mutations, are indicated. The Arabidopsis laf6 mutation
is proposed to disrupt the indicated step indirectly.
membrane-spanning domains — that can exist within
single or multiple polypeptides. The atABC1 protein has a
single ABC domain and no membrane-spanning domain,
suggesting that it functions as an importer rather than as
an exporter — import systems tend to have ABC and
membrane domains in separate subunits, whereas the opposite is true for export systems [9]. Given the homology
between atABC1 and a cyanobacterial protein, it appears
that import in this case means transport into chloroplasts.
So what might atABC1 be importing into chloroplasts?
The pale-green phenotype of laf6, the involvement of
ABC proteins in the vacuolar transport of chlorophyll
catabolites [10], and the implication of chlorophyll
precursors in the signalling of light responses [11–13],
prompted Møller et al. [6] to investigate the levels of
chlorophyll precursors in laf6 mutant plants. Interestingly,
protoporphyrin IX (Figure 1) levels were found to be elevated two-fold in the mutant [6], and it was therefore proposed that atABC1 functions to import protoporphyrin IX
from the envelope — where it is synthesized — so that
chlorophyll biosynthesis can be completed inside the
chloroplast (Figure 2). In laf6 mutant plants, protoporphyrin IX accumulates — as it is not accessible to downstream enzymes of the chlorophyll biosynthetic pathway —
and leaks out across the outer envelope membrane.
How does the perturbation of chlorophyll biosynthesis in
laf6 mutant plants impair photomorphogenic responses to
far-red light? One possibility is that protoporphyrin IX acts
as a negative ‘plastid signal’ to attenuate the expression of
light-regulated nuclear genes. This hypothesis is supported
by the work of Johanningmeier and Howell [11] who,
working with the unicellular alga Chlamydomonas, observed
a negative correlation between factors presumed to cause
chlorophyll precursor accumulation and chlorophyll a/bbinding (Cab) protein transcript levels.
More recent advances in the plastid signalling field paint a
more complex picture, however. For example, Beck and
co-workers [12,13] have provided compelling evidence
that chlorophyll precursors actually act as positive signals
in the light-induction of nuclear gene expression. They
showed that the Chlamydomonas brown mutants brs-1 and
brc-1, which are defective in the synthesis of Mg-protoporphyrin IX, were unable to induce nuclear heat shock
protein (HSP70) gene expression in response to light. This
defect could be rescued by feeding Mg-protoporphyrin IX
or its methyl ester to mutant cells, suggesting roles for
Mg-protoporphyrin IX and/or its methyl ester as positive
‘plastid signals’. Unfortunately, no plastid signalling role
for protoporphyrin IX could be demonstrated, as feeding
protoporphyrin IX did not stimulate HSP70 gene expression in mutant cells [12] or downregulate HSP70 gene
expression in light-grown wild-type cells [13]. Further
work is necessary to resolve these apparent discrepancies — the use of non-photosynthetic genes in the latter
study may be significant — but it seems that the interpretation of the laf6 data given above is an oversimplification.
Recent work by Chory and colleagues [14] also points
towards tetrapyrrole involvement in chloroplast-to-nucleus
signalling. A genetic screen for Arabidopsis plastid signalling mutants was conducted back in 1993 [15]. The
screen exploited the earlier observation that photooxidative chloroplast damage after growth on Norfluorazon
results in the repression of nuclear genes for chloroplast
proteins [4]. Chory and colleagues [15] fused the Cab gene
promoter to an antibiotic resistance marker, introduced
the chimeric gene into Arabidopsis plants, and then
Dispatch
screened for antibiotic-resistant mutants after photooxidation in the presence of Norfluorazon. Several genomes
uncoupled (gun) mutants were isolated and, just recently,
one of the GUN genes was cloned [14].
R309
Figure 2
Proto
laf6
Another interesting development is the demonstration
that hy1 and hy2 mutants of Arabidopsis have gun
phenotypes — gun2 and gun3 are in fact alleles of hy1 and
hy2, respectively [14,16]. These mutants were originally
identified by their long hypocotyl phenotypes, and it is now
clear that they are both phytochrome-defective mutants.
Phytochromes each comprise two 120 kDa polypeptides —
of which there are five types in Arabidopsis — covalently
attached to an invariant linear tetrapyrrole chromophore,
phytochromobilin. Phytochrome A is the main photoreceptor for far-red responses, and so phyA apoprotein
mutants have long hypocotyl phenotypes in far-red light
[7]. Phytochrome B, on the other hand, is the dominant
photoreceptor in red and white light, and so phyB apoprotein mutants have long hypocotyl phenotypes in red and
white light, but not in far-red light [7].
The hy1 and hy2 mutants are defective in phytochromobilin synthesis — HY1 encodes a haem oxygenase [17,18],
and HY2 encodes a phytochromobilin synthase [19]
(Figure 1) — and so have phenotypes consistent with loss
of function of all five phytochromes. Why do mutations in
phytochromobilin synthesis disrupt plastid-to-nucleus
signalling? The pale-green phenotypes of hy1 and hy2
provide a clue. The tetrapyrrole biosynthetic pathway is
subject to extensive feedback regulation, and it seems that
disruption of downstream steps in the haem branch can
cause repression of very early steps in the pathway, so that
flux through the chlorophyll branch is also reduced [20].
Indeed, double mutant studies indicate that hy1 affects the
same plastid signalling pathway as gun5 [16], and so the
effects of these mutations are probably mediated through
the proposed Mg-chelatase ChlH sensor (Figure 3) [14].
So why does laf6, unlike gun5, affect nuclear gene expression and photomorphogenesis? Does the laf6 mutation
affect phytochrome A apoprotein or, like hy1 and hy2,
oto
Pr
Cytosol
M
PPO
atABC1
gen
ATP
Protogen
Pro
to
Remarkably, GUN5 turns out to encode the ChlH subunit
of Mg-chelatase, one of the enzymes at the branch point
of the tetrapyrrole biosynthetic pathway (Figure 1). Interpretation is once again not straightforward, however, as
Arabidopsis Mg-chelatase ChlI mutants surprisingly do not
have gun phenotypes, and Mg-protoporphyrin IX levels
are reduced in the gun5 mutant — rather than elevated, as
one might have expected given the results of Beck and
colleagues [12,13]. These data led to the formulation of
a model in which ChlH — located at the inner envelope
membrane — measures flux through the chlorophyll
biosynthetic pathway, and either sends a positive signal to
the nucleus or inhibits a negative signal (Figure 3).
Proto
ADP
Chlorophyll
Chloroplast
OE
IE
Current Biology
A model illustrating the role proposed for atABC1 in protoporphyrin IX
transport. Protoporphyrinogen IX (Protogen) is exported to the
chloroplast envelope where it is oxidized to protoporphyrin IX (Proto) by
protoporphyrinogen oxidase (PPO). Then, atABC1 — presumably acting
in conjunction with some membrane spanning protein (M) — mediates
the import of protoporphyrin IX into the stroma, where chlorophyll
biosynthesis proceeds. In the Arabidopsis laf6 mutant, which lacks
atABC1, protoporphyrin IX accumulates in the envelope and then leaks
out into the cytosol. OE denotes outer envelope membrane, and IE
denotes inner envelope membrane. (Adapted from [6].)
phytochromobilin biosynthesis? Apparently not. Phytochrome A apoprotein expression is normal in laf6 mutant
plants and, in any case, the laf6 mutation clearly lies
within the atABC1 gene [6]. What about the chromophore?
Any perturbation of tetrapyrrole biosynthesis might affect
phytochromobilin biosynthesis. But the laf6 phenotype is
specific to far-red light and, what is more, cannot be
rescued by feeding a chromophore substitute to mutant
plants [6]. The only logical conclusion is that laf6 disrupts
a plastid signalling pathway required for both photomorphogenic and gene-expression responses to far-red light.
An obvious question, then, is: does the laf6 mutant have a
gun phenotype? If it does, the relationship between the
LAF6 and GUN5 plastid signalling pathways can be tested
in double mutants studies.
Clearly, plastid-to-nucleus signalling is a far more complex
process than was previously assumed. Chlorophyll precursors are obviously involved in some way, but quite what
their role is remains to be determined. Moreover, there
appear to be multiple ways in which chlorophyll precursor
‘signals’ can be transduced to the nucleus: the laf6 mutation
disrupts a pathway that controls gene expression and photomorphogenesis, whereas gun5 affects another pathway — or
a branch pathway — that controls gene expression only. In
addition, double-mutant studies involving the gun and hy1
mutants suggest that there are even multiple signalling
R310
Current Biology Vol 11 No 8
Figure 3
References
Chloroplast
Chl
I D
ALA
MPM
Proto
MP
H
Y
X
?
MP(M) ?
Cab
Nucleus
Current Biology
A model illustrating the role proposed for the ChlH subunit of
Mg-chelatase in plastid signalling. Mg-chelatase comprises three
subunits — ChlH, ChlI and ChlD (H,I and D) — and catalyses the
formation of Mg-protoporphyrin IX (MP) from protoporphyrin IX (Proto).
Mutation of the ChlH subunit — as in the Arabidopsis gun5 mutant —
results in a plastid signalling defect. ChlI mutations do not affect plastid
signalling, indicating that the catalytic and plastid signalling roles of
Mg-chelatase can be uncoupled. ChlH may act as a sensor to monitor
flux through the chlorophyll biosynthetic pathway, relaying this information
to the nucleus via positive and/or negative plastid signalling pathway(s).
X and Y represent putative repressor and activator components
controlling Cab transcription. Chlamydomonas feeding experiments
suggest that Mg-protoporphyrin IX and its methyl ester derivative (MPM)
may act directly — perhaps in conjunction with an unknown factor — as
positive plastid signals. ALA denotes 5-aminolevulinic acid, and Chl
denotes chlorophyll. (Adapted from [14].)
pathways for controlling gene expression [14,16], and
other — perhaps entirely separate — signalling pathways
relay information on the redox status of the chloroplast to
the nucleus [21–23]. One clear message emerging from this
increasingly complex picture, however, is that chloroplasts
are not at all inert in their dealings with the nucleus — they
have a fair say in the running of the cell.
Acknowledgements
I am grateful to Joanne Chory, Enrique López-Juez, Simon Geir Møller and
Garry Whitelam for their helpful comments on the manuscript, and to the
Royal Society for support from the Rosenheim Research Fellowship. Work
in my laboratory on chloroplast biogenesis is supported by grants from the
Biotechnology and Biological Sciences Research Council (C12976 and
P12928) and the Nuffield Foundation (NAL/00026/G).
1. McFadden GI: Chloroplast origin and integration. Plant Physiol
2001, 125:50-53.
2. Keegstra K, Cline K: Protein import and routing systems of
chloroplasts. Plant Cell 1999, 11:557-570.
3. Rochaix J-D: Posttranscriptional control of chloroplast gene
expression. From RNA to photosynthetic complex. Plant Physiol
2001, 125:142-144.
4. Oelmüller R: Photooxidative destruction of chloroplasts and its
effect on nuclear gene expression and extraplastidic enzyme
levels. Photochem Photobiol 1989, 49:229-239.
5. Hedtke B, Wagner I, Borner T, Hess WR: Inter-organellar crosstalk
in higher plants: impaired chloroplast development affects
mitochondrial gene and transcript levels. Plant J 1999, 19:635-643.
6. Møller SG, Kunkel T, Chua N-H: A plastidic ABC protein involved in
intercompartmental communication of light signaling. Genes Dev
2001, 15:90-103.
7. Neff MM, Fankhauser C, Chory JC: Light: an indicator of time and
place. Genes Dev 2000, 14:257-271.
8. López-Juez E, Jarvis RP, Takeuchi A, Page AM, Chory J:
New Arabidopsis cue mutants suggest a close connection
between plastid- and phytochrome regulation of nuclear gene
expression. Plant Phys 1998, 118:803-815.
9. Holland BI, Blight MA: ABC-ATPases, adaptable energy generators
fuelling transmembrane movement of a variety of molecules in
organisms from bacteria to humans. J Mol Biol 1999, 293:381-399.
10. Lu YP, Li ZS, Drozdowicz YM, Hortensteiner S, Martinoia E, Rea PA:
AtMRP2, an Arabidopsis ATP binding cassette transporter able to
transport glutathione S-conjugates and chlorophyll catabolites:
functional comparisons with Atmrp1. Plant Cell 1998, 10:267-282.
11. Johanningmeier U, Howell SH: Regulation of light-harvesting
chlorophyll-binding protein mRNA accumulation in
Chlamydomonas reinhardtii. J Biol Chem 1984, 259:13541-13549.
12. Kropat J, Oster U, Rüdiger W, Beck CF: Chlorophyll precursors are
signals of chloroplast origin involved in light induction of nuclear
heat-shock genes. Proc Natl Acad Sci USA 1997,
94:14168-14172.
13. Kropat J, Oster U, Rüdiger W, Beck CF: Chloroplast signalling in
the light induction of nuclear HSP70 genes requires the
accumulation of chlorophyll precursors and their accessibility to
cytoplasm/nucleus. Plant J 2000, 24:523-531.
14. Mochizuki N, Brusslan JA, Larkin R, Nagatani A, Chory J: Arabidopsis
genomes uncoupled 5 (GUN5) mutant reveals the involvement of
Mg-chelatase H subunit in plastid-to-nucleus signal transduction.
Proc Natl Acad Sci USA 2001, 98:2053-2058.
15. Susek RE, Ausubel FM, Chory J: Signal transduction mutants of
Arabidopsis uncouple nuclear CAB and RBCS gene expression
from chloroplast development. Cell 1993, 74:787-799.
16. Vinti G, Hills A, Campbell S, Bowyer JR, Mochizuki N, Chory J,
López-Juez E: Interactions between hy1 and gun mutants of
Arabidopsis, and their implications for plastid/nuclear signalling.
Plant J 2000, 24:883-894.
17. Muramoto T, Kohchi T, Yokota A, Hwang I, Goodman HM:
The Arabidopsis photomorphogenic mutant hy1 is deficient in
phytochrome chromophore biosynthesis as a result of a mutation
in a plastid heme oxygenase. Plant Cell 1999, 11:335-348.
18. Davis SJ, Kurepa J, Vierstra RD: The Arabidopsis thaliana HY1
locus, required for phytochrome-chromophore biosynthesis,
encodes a protein related to heme oxygenases. Proc Natl Acad
Sci USA 1999, 96:6541-6546.
19. Kohchi T, Mukougawa K, Frankenberg N, Masuda M, Yokota A,
Lagarias JC: The Arabidopsis HY2 gene encodes
phytochromobilin synthase, a ferredoxin-dependent biliverdin
reductase. Plant Cell 2001, 13:425-436.
20. Terry MJ, Kendrick RE: Feedback inhibition of chlorophyll synthesis
in the phytochrome chromophore-deficient aurea and yellowgreen-2 mutants of tomato. Plant Physiol 1999, 119:143-152.
21. Escoubas JM, Lomas M, LaRoche J, Falkowski PG: Light intensity
regulation of cab gene transcription is signaled by the redox state
of the plastoquinone pool. Proc Natl Acad Sci USA 1995,
92:10237-10241.
22. Karpinski S, Escobar C, Karpinska B, Creissen G, Mullineaux PM:
Photosynthetic electron transport regulates the expression of
cytosolic ascorbate peroxidase genes in Arabidopsis during
excess light stress. Plant Cell 1997, 9:627-640.
23. Oswald O, Martin T, Dominy PJ, Graham IA: Plastid redox state and
sugars: interactive regulators of nuclear-encoded photosynthetic
gene expression. Proc Natl Acad Sci USA 2001, 98:2047-2052.