Chloroplast gene regulation: interaction of the nuclear and chloroplast genomes in the expression of photosynthetic proteins S.P. Mayfield Department
of Molecular
Biology,
Research
Institute
of Scripps Clinic,
La Jolla, California,
USA
Current Opinion in Cell Biology 1990, 2:509-513
Introduction The photosynthetic, chlorophyll-containing chloroplast is one developmental stage of the plastid organelle. Plastids are found in all plant cell types and contain their own genetic material as well as all the machinery necessary for DNA, RNA and protein synthesis. They have diverse structures, functions and protein contents dependent upon the plastid type, which is determined to a large degree by the tissue (leaf versus root, etc) in which the plastid resides. For example, chromoplasts are carotenoidrich, non-photosynthetic plastids (yellow, red or orange), often associated with flowers or fruit. Plastids found in roots or tubers are termed amyioplasts. Plastids other than chloroplasts lack many of the proteins associated with photosynthetic membranes, and have messenger RNA (mENA) populations which are distinct from those of chloroplasts [ 1 ] However, because each of these plastids is able to convert to one of the other plastid types (Kirk and Tinley-Bassett, The Plastih Elsevier/North Holland Press, 1978; Mayfield and Huff, Plant P&id 1986, 81:30-351, expression of chloroplast genes must be considered in relation to gene expression in plastid types other than chloroplasts. Of the hundreds of proteins involved in photosynthesis, less than half are encoded within the limited chloroplastic genome. It has become clear over the last few years that the nucleus encodes not only many photosynthetic proteins but also a number of functions necessary for the expression of chloroplast-encoded genes. It has also become clear that interactions between the nuclear and chloroplast genomes are responsible for coordinate expression of the photosynthetic apparatus. Photosynthetic membranes within chloroplasts consist of four major photosynthetic complexes (photosystem II, photosystem I, cytochrome b6/f, and ATPase), each of which contain proteins encoded in the chloroplast and nuclear genomes. The regulation of chloroplast gene expression, therefore, has three distinct aspects: gene expression during plastid development, gene expression in response to environmental signals, and gene expression in relation to
nuclear-chloroplast interactions. This review will summarize some of the recent progress made in understanding these regulatory aspects.
Development
of the chloroplast
Much of what is known about chloroplast gene expression comes from the study of the conversion of etioplasts (pre-chloroplasts lacking chlorophyll) to chloroplasts. This conversion is dependent upon light, which is necessary for the synthesis of chlorophyll. The conversion of etioplasts to chloroplasts involves a rapid accumulation of chlorophyll, photosynthetic membranes, and the associated photosynthetic proteins, both nuclear and chloroplast encoded. The accumulation of photosynthetic proteins correlates closely with the accumulation of chlorophyll (Harpster et al, Pkant Mol Bid 1984, 3:59-71). During this light-induced chloroplast biogenesis, there is a concomitant increase in the transcription and accumulation of several cytoplasmic mRNAs encoding photosynthesis-related proteins (reviewed in Tobin and Silvexthome, Ann Rev Plant Plysiol 1985, 36:569-593). Unlike the mRNAs for nuclear-encoded proteins, n-&N& for chloroplast-encoded photosynthesisrelated proteins accumulate to relatively high levels in plants grown in the dark (reviewed by Mullet, Ann Rev Plant Plysiol 1988, 39:47>502) and show little additional accumulation upon exposure to light Walnoe et al, J Cell Bioll988,106:609616). Etioplasts are capable of protein synthesis and accumulate several chloroplast and nuclear-encoded proteins. Their exposure to light results in a dramatic increase in the synthesis of proteins associated with photosynthetic membranes (Klein and Mullet, JBiolCbem 1986,261:1113&%11145; Klein eta& JC.211 Bill 1987, 106:28!+302; Malnoe et al, J CklI Biol1988, 106:603-616). Blocking plastid transcription during this transition has no effect on photosynthetic protein syn thesis, showing that existing mRNA pools are used for protein translation (Malnoe et al, 1988). Thus, the expression of photosynthetic genes is dependent upon the
Abbreviations Ill-inverted
@ Current
repeat;
Biology
mRNA-messenger
Ltd ISSN 0955-0674
RNA.
510
Nucleus
and gene expression
plastid type as well as upon light, the environmental stimulus. Several of the mechanisms which have been identified in this developmental and environmental regulation will be discussd in the following sections.
Transcription
of chloroplast
genes
Transcription promoter sequences identihed in the chloroplast genome appear quite similar to prokaiyotic consensus promoters (Han@Bowdoin and Chua, Trend Biocbem Sci 1987, 12:67-70) [ 11. Although several parameters (stage of development, environmental signals, DNA topology) a&t the transcription of chloroplast mIWAs (reviewed by Mullet, Ann Retl Plant P&v siol 1988, 39:47>502), there is a poor correlation between the expression of chloroplastic proteins and the transcription of their genes. Messenger RNAs of several abundant photosynthesisrelated proteins are transcribed at similar rates in plas tids of roots and immature and mature leaves, yet little or no accumulation of these mRNAs or their protein products is detected in any plastids except those of mature leaves (Deng and Gruissem, Cell 1987, 49:379-387; see [ 11 for review) [2]. Processing of these transcripts is identical in all three plastid types. Measurements of mFWA turnover in each of these plastids show differences which correlate with ~RNA accumulation, suggesting that stability rather than transcription is the determining factor for dilferential accumulation of mRNAs in these plastids [2]. There is little dilference between the transcription of these genes in chromoplasts and chloroplasts of bell pepper fruit [3]. However, in the conversion of chloroplasts to chromoplasts in ripening tomato fruit, there is a distinct change in the transcription of some, but not all, of these photosynthesis-related genes [4]. This reduction in transcription correlates with DNA methylation of the same genes. As discussed in the previous section there is little change in steady-state levels of plastid mRNAs during the conversion of etioplasts to chloroplasts. Direct measurement of plastid gene tmnscription during this transition shows no apparent new transcription of chloroplast genes [ 51, suggesting that new transcription within the plastid is not a requirement for chloroplast biogenesis. Viewed collectively, these data suggest that changes in plastid transcription are not a primary means of regulating the accumulation of chloroplast-encoded mRNAs for photosynthetic proteins. Changes in transcription do not correlate with the stage of plastid development either, even though the accumulation of mRNAs and photosynthetic proteins is very much dependent upon the stage of plastid development. Transcriptional regulation of chloroplast genes may indeed play a key role in chloroplast gene expression and in plastid development, but these roles appear to be centered on specific plastid genes and do not operate at times when large changes in chloroplast mRNA accumulation or protein synthesis are occurring.
Post-transcriptional mRNA accumulation
regulation
of chloroplastic
Several chloroplast genes are transcribed as polycistronic mRNAs which show diverse patterns of processing. This processing results in accumulation of mono and polycistronic mRNAs, both of which appear to be used for protein synthesis (Barkan, EMBO J 1988, 7:2637-2644). Splicing of rnRN~s occurs both inn-a- and intermoleculady in chloroplast transcripts, with some mature mRNAs formed by the splicing of portions of distinct unlinked transcripts (Kuck et al, EMBO J 1987,6:218+2195) [61. Many of these processing and splicing events require nuclear gene products, the loss of which can result in the absence of specihc mature rnKN& (Choquet et al, Cell 1988, 52903-913). It is still unclear exactly how this mRNA processing alfects chloroplast mRNA accumulation and translation, or to what degree ~RNA processing is used as a regulatory step in chloroplast gene expression. As discussed in the last section, mRN& for several photosynthesis-related proteins accumulate to different levels in green and non-green plastids, despite comparable levels of transcription in both plastid types. Gruissem and co-workers have suggested that this dilferential accumulation results from unequal turnover of specilic transcripts via a mechanism involving inverted repeats (IRS) found in the 3’ untranslated portion of many chloroplast mRNh (reviewed in [ 11). RNAS containing IRS are processed in chloroplast extracts to the correct size and are stable compared with similar sequences lacking IRs [ 11. SpeciIic proteins, which may be developmentally regulated, could interact with these IRs and act either to stabilize the transcript or to regulate mRNA turnover (Gruissem et al, Trend Genet 1988, 4:5863). Differential accumulation of plastid mRNAs would obviously have profound effects on plastid gene expression. Thus, the turnover of plastid mRNAs, via some mechanism involving 3’ IRs and specitic proteins that interact with them, is likely to play a key role in plastid gene expression in relation to plastid development. What role this diiferential accumulation of plastid mRNA has in the expression of photosynthetic proteins in chloroplasts has yet to be determined.
Translational expression
control
of chloroplast
gene
Analysis of photosynthetic mutants in the green alga CHumyabmonus has revealed a class of nuclear genes that are required for translation of specific chloroplast mRNAs (lensen et al., J Cell Biol 1986, 103:13151325; Kuchka et al., EMBO J 1988, 7:31!+324) [7]. Each of these nuclear-encoded genes effects the translation of only a single chloroplast ~RNA, and in some cases multiple nuclear genes appear to be required for the translation of a single mRNA [7]. Genetic analysis indicates that these nuclear genes encode factors that recognize stem-loop structures in the 5’ untranslated portion of
Chloroplast
the chloroplast mRNA and act to allow translation of the message. Chloroplast mutations in the stems of these structures, which either increase or decrease the stability of the stem, act to block translation of the message as well [7]. Although none of the nuclear genes have been cloned or characterized, it seems likely that these genes will encode proteins which interact with the stem-loop structures and act in translation initiation or elongation. The light-induced changes in translation of existing chloroplast mRNAs for photosynthetic proteins could account for many of the observed changes in the accumulation of photosynthetic proteins during chloroplast biogenesis. How translation of some chloroplast mRNAs is promoted over the translation of others, or by what mechanism light influences this process, remains unclear. Whether the factors responsible for light-regulated translation during chloroplast biogenesis are similar to factors identified by the nuclear mutants has not yet been determined. The identification and characterization of factors which control chloroplast mRNA translation should provide some insight into translational control as a mechanism for regulating chloroplast gene expression. The role of translational control in plastid gene expression in plas tids other than chloroplasts remains to be seen.
Post-translational photosynthetic
accumulation proteins
of
A mutation in a structural gene in either genome, resulting in the absence of any single protein from a photosynthetic complex, results in the loss of the entire complex (Chua and Bennoun, Proc Nat1 Acud Sci USA 1975, 72:2175-2179; Metz and Miles, Biochem Bioplys Actu 1982, 681:95-102; Mayfield et al, EMBO J 1987, 6313-318). Synthesis of other members of that complex continues at wild-type levels, but the newly synthesized proteins are quickly degraded and, thus, never accumulate within the mutant (Bennoun et al, Pfant Mol Bioll986, 6:151-160; Erickson et al., EMBO J 1986, 5:1745-1754) [7]. Within a single complex, photosyn thetic proteins can have different half lives, often determined by the growth conditions (light versus dark) of the plants. Plants must therefore have the ability to replace proteins within a complex without turning over the entire complex. How this is accomplished and what role specific protein turnover has in regulating the accumulation of photosynthetic proteins has yet to be determined. Clearly, there must be some regulation between protein synthesis and degradation but how this association is governed and under what mechanism it is functioning is not yet clear.
Chloroplast
effects on nuclear
gene expression
A final aspect of the regulation of chloroplast gene expression involves the interaction of the nuclear and chloroplast genomes in the coordinate expression of
gene regulation
Mayfield
photosynthesis-related proteins. Many of the above-mentioned regulatory steps involve nuclear encoded factors that exhibit some control over chloroplast gene expression, be it mRNA synthesis, processing, accumulation or translation. Each of these regulatory mechanisms indicates that the nuclear genome contains the information necessary to regulate the expression of chloroplast genes. It w-as first shown in 1984 that events within the chloroplast could alfect the accumulation of cytoplasmic mRNAs (Mayiield and Taylor, Eur J Biocbem 1984, 106:144:79+34), and has since been confirmed in many plant species [8-lo] ( reviewed in [ll]). Despite a large body of work showing that chloroplast photooxidation inhibits the expression of nuclear encoded photosynthesis-related proteins (reviewed in [ ll]), our understanding of this phenomenon remains perfunctory and the original hypothesis that a signal from the chloroplast might influence nuclear gene expression remains hypothetical (Mayfield and Taylor, Mol Gen Genet 1987, 208:30%314) [ll]. During the light-activated conversion of etioplasts to chloroplasts there is a concomitant increase in cytoplasn-tic mRNAs for photosynthetic proteins. As the biogenesis of the chloroplast and accumulation of a number of cytoplasmic mRNAs are closely associated, it is difficult to discriminate between light-regulated nuclear gene expression pe’ se and nuclear gene expression associated with chloroplast biogenesis. Many authors have concluded that light-activated transcription of nuclear genes is responsible for controlling the synthesis of the chloroplast-encoded photosynthetic proteins and for the development of the chloroplast. In systems where chloroplast biogenesis is not light dependent, synthesis of photosynthetic proteins proceeds in the dark and photosynthetic membranes accumulate; thus, it is possible to separate chloroplast biogenesis and light-modulated gene expression (Malnoe et al., J Cell Biol1988, 106:609-616). However, in these cells chloroplast mRNA translation remains responsive to fight as does the accumulation of photosynthetic-related cytoplasmic mRNAs. These data suggest that light-activated increases in cytoplasmic ~RNA accumulation and chloroplast protein synthesis are independent of chloroplast biogenesis. Thus, it is unlikely that light-regulated transcription of nuclear genes initiates chloroplast biogenesis. Kuhlemeier et al. [ 8] report that a promoter from an abundant nuclear encoded photosynthetic protein contains two distinct domains, one for light-regulated transcription and one for tissue specificity. As tissue specificity and the stage of plastid development are tightly correlated, it seems that these data would also argue for separation of light-regulated gene expression and plastid development. Chory and coworkers [ 121 have identified a mutant in the higher plant Ar&wsis tiliana that develops leaves containing pre-chloroplasts in the dark. These plants accumulate several cytoplasmic mRNAs for photosynthesisrelated proteins which are normally observed to be light regulated and thus found mainly in light-grown plants. This accumulation, and the appearance of pre-chloroplasts in these plants, show that the influence of light
511
512
Nucleus
and
gene
expression
can be uncoupled from plastid development. This observation also demonstrates that the accumulation of cytoplasmic mRNAs for photosynthetic proteins may be coupled to chloroplast development, as well as coupled to light exposure. It is therefore possible that much of the accumulation of cytoplasmic mFWA associated with light exposure is in fact a response to the development of the chloroplast, which is itself initiated by light exposure. Although these data provide no direct information on the identity of a chloroplastic factor or signal that may be influencing nuclear gene expression, they suggest that such a signal is present in cells containing pre-chloroplasts, and that it does not require light exposure for its activity.
Conclusions The regulation of photosynthetic gene expression involves a complex scheme in which mRNA translation and mRNA stability play key roles. Fig. 1 outlines the normal pathway of photosynthetic protein synthesis and identifies the specidc steps that have been characterized which regulate this gene expression. From the data available to date, it appears that transcriptional control of plastid genes has a limited role in determining mRNA accumulation in different plastid types, and perhaps none at all in regulating the rapid changes observed in photosynthetic protein accumulation during chloroplast biogenesis. The exact mechanisms used to regulate mRN4 stability and translation are only now begin-
ning to be characterized, but both of these regulatory mechanisms appear to require nuclear encoded factors and may be controlled to a large degree by the nucleus. Much of the variation observed in plastid gene expression in the tierent tissues of higher plants may be accounted for by mRNA stability while translational regulation appears to account for much of the variation in photosynthetic protein accumulation observed during biogenesis of the chloroplast. The use of translational control for chloroplast gene expression under various environmental conditions may be dictated by a requirement for rapid alteration in the level of photosynthetic protein synthesis. Under maximum photosynthetic conditions (bright light), synthesis of photosyn thetic proteins needs to be at relatively high levels to replace proteins that are turning over at an accelerated rate. Changes in light conditions can occur rapidly and the use of translational controls would allow protein synthesis rates to change rapidly in response to this. The use of transcriptional regulation would add an additional lag time for synthesis of new mRNA pools, a lag which may be too long to regulate efficiently the rapid changes required for photosynthetic protein synthesis. The closely coordinated expression of the chloroplast and nuclear genes is regulated by mechanisms that have yet to be characterized. Plastid development clearly has some bearing on nuclear gene expression and nuclear genes are clearly required for plastid development. It seems likely that the nuclear genome sets some developmental program that determines, to a large degree, fig. 1. Regulatory steps thesis of the photosynthetic tus. The open normal pathway
in
the synappara-
arrows illustrate for the synthesis
photosynthesis-related clear-encoded proteins
proteins. Nuare synthesized
from cytopfasmic messenger (mRNAs) (a) as soluble precursor peptides transported
(b)
velope encoded from with
and across
membranes proteins
cc). (e) are
(0.
coordinate chloroplast
ulated increase accumulation; crease plastic ferential
(d) and combine polypeptides to
Several
steps
expression genes: (I)
regulate of nuclear light-mod-
in cytoplasmic (2) light-modulated
mRNA in-
in translation of existing chloroand cytoplasmic mRNAs; (3) difstability of plastid mRNAs re-
sulting in unequal accumulation cific mRNAs; (4) rapid turnover tosynthetic of other
en-
Chloroplastsynthesized
multisubunit photosynthetic located within the thylakoid
membranes this and
RNAs poly-
post-translationally the chloroplast
plastid mRNAs nuclear-encoded
form the complexes
the of
proteins associated
in the proteins;
of speof phoabsence (5) feed-
back ‘signal’ from the chloroplast to the nucleus specifically affecting mRNA transcription and accumulation; and (6) regulation of chloroplast mRNA translation and stability by nuclear gene products. Ct DNA, choloroplast DNA.
Chloroplast
the stage of plastid development, and then reacts to this developing plastid to 6ne tune transcription of specific nuclear genes involved in photosynthesis. The challenge now is to characterize the specific mechanisms by which the nucleus and chloroplast interact. This interaction offers a very interesting model for understanding the cooperation of distinct genomes, as well as for examining the interactions of development and environmental signals in regulating photosynthetic gene expression.
Annotated reading: l l e
references
GRUSSEM
and recommended
l e
their
W: Chloroplast
plastids
on.
gene expression: Cell 1989, 56:161-170.
how
plants
l
X-W, GRLJISEM W: Constitutive transcription ulation of gene expression in non-photosynthetic of higher plants. EM30 / 1988. 7:3301-3308.
and reg. plastids
DENG
This paper shows that plastid genes are transcribed at similar rates in plastids of roots, immature and mature leaves. mRNA processing is identical in roots and leaves, and tierentiai accumulation of mRNAs for photosynthetic proteins in chloroplasts is due to stability of those mRNAs in plastids of leaves but not in roots or immature leaves. 3.
KUNIZ
l
pression of plastid and nuclear genes in bell pepper (Cupsicum annuum)
M, EVARD J.l
D’HARLINGUE
4
WEU
J-H,
KOBAYASHI
l e
regulation and tional conversion
and
H, NGERNPRASLRTSUU J, AKUAWA
DNA methylation of chloroplasts
KRUPINSKA K, APEL K: Light-induced
l
plasts to chloroplasts of barley control of plastid gene expression. 2 19:467-473.
HERRIND, SCHMIDTGW: Trans-splicing
of transcripts for the
chloroplast
psaA1 gene. J Biol Ckm 19813, 263~14601-14604. luclear mutants al&t splicing of the chloroplast psaAl mRNA which is transcribed from 3 distinct regions of the chloroplast genome. Addi.
tional evidence is provided that nuclear gene products are required for mRNA processing and expression of chloroplast genes. 7.
RGCW
l e
GIRMD-B,v?COU J, BENNOUN P: Nuclear tations tiect the synthesis or stahiity pshC gene product in Chlamydomonas
J-D, KUCHKA M, MAYFEUI S,
8.
KUHIEMEIEIER C, s TRITI%ATrER
l
rhcS-M promoter organ specificity.
%3IRMER-bHU@
M,
and chloroplast muof the chloroplast reiinhardtiL EMBO
sunflower
9. e
in plastids during transito chromoplasu. EM90 /
transformation of etiowithout transcriptional Mel Gen Genet 1989,
The transcription of plastid genes was compared in chloroplasts and etioplasts and found to be essentially identical. This suggests that the
C. CHUA
N-H:
responsiveness 1:471478.
The
Pea
but
not
STOCKHAUS J, SCHEU J, W-
of the exgene !X-LSl with / 1989, 8:2445-2451.
R L: COtTelatiOn
pression of the nuclear photosynthetic the presence of chloroplasts. EMBO
A nuclear gene, encoding a chloroplastic protein involved in photo. synthesis, is expressed only in cells with developed chloroplasts. This corroborates earlier experiments which show that the development of the chloroplast is a requirement for the expression of nuclear genes whose products are located in the chloroplast. 10.
HARKINS
l
G.UBRM~ DW: Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proc Nat1 Acud Sci USA 1989. 87:81-20.
(Heli-
T: Transcriptional
G. WARD
mediates light Pkant Cell 1989,
A promoter for a nuclear encoded photosynthesis-related protein was used to show that tissue specificity and light regulation are 2 separate phenomena that are likely located on distinct regions of the promoter.
CAMARA B: Ex-
1990, 9:307-313. The transcription of several photosynthesis-related genes was found to vary between chloroplasts and chromoplasts of tomato fruit. The loss of transcriptional activity was found to correlate with DNA methyiation. This report differs with that of Deng and Gruissem 12) for tmnsctiption in non-green plastids, suggesting that not alI plastids regulate mRNA accumulation in exactly the same manner. 5.
6.
during di5erentiation
anthus annuus). Mel Gen Gener 1989, 216:15&163. Messenger RNA for 2 nuclear encoded genes, involved in photosynthesis, accumulates in non-photosynthetic leaves. 2 chloroplast-encoded mRN& also involved in photosynthesis, showed little differences in transcription in green or non-green p&ids and accumulated equally in both plastid types. These data suggests that transcription and mRNA stability are not important variables in the conversion of chloroplasts to chromoplasts. 4.
light.dependent development of a chloroplast from an etioplast is not dependent upon activation (transcription) of novel genes.
turn
A review of chloroplast gene expression which discusses mRNA transcription, splicing and stability and also covers involvement of nuclear genes in mRNA stability and translation. It includes many refer. ences up to and including 1988. 2.
Mayfield
/ 1989, 8:101~1021. This paper SNdieS the characterization of nuclear and chloroplast mutants. These mutants defme a stem-loop region in the 5’ end of the psbc ~~RNA which interacts with nuclear encoded ‘factors’ to regulate p&C mRtU translation. Genetic evidence that a nuclear gene product controls translation of specific chloroplast mRNAs is provided.
Of interest Of outstanding interest
1.
gene regulation
rcR,
JEFFERSON
RA,
KAVANAGH
TA,
BEVAN
MW,
This Study demonstrates that the expression of chimeric genes in both ttansgenic and ttansfected protoplasts, under the control of photosynthetic gene promoters, is restricted to photosynthetic cells, corroborating, in transfected protoplasts, what several other laboratories have previously shown. 11. l e
R Photooxidative its effect on nuclear gene enzyme levels. Pbotocbemishy
OELMUUER
destruction expression
of chloroplasts and and extraplastidic
1989, 49:22%239. review of the effect of photo-oxidation of the chloroplast on the loss of cytoplasmic ~RNA encoding photosynthesis-related proteins. Photooxidative loss of the chloroplast reduces transcription and accumulation of nuclear encoded mRh% for photosynthetic proteins. This paper discusses chloroplast signals and their implication in nuclear gene regulation.
A
12.
CHORY J, PETO C, FEINEZAUM R, PRAWN I AUSLIESEI. F: Arubidop
l e
sfs tbafiana mutant that develops in the absence of light. Cell 1989,
as a light-grown 5899-999.
plant
This paper examines the characterization of a nuclear mutant which allows the expansion of leaves and the development of pre-chloroplasts in the absence of light. This mutant accumulates certain nuclear encoded mRNAs in the dark which normally accumulate only in light. It is strongly suggested that much of the ‘light-regulated’ nuclear gene expression may in fact be a response to chloroplast development, itself influenced by light.
513
of Molecular
Biology,
Research
Institute
of Scripps Clinic,
La Jolla, California,
USA
Current Opinion in Cell Biology 1990, 2:509-513
Introduction The photosynthetic, chlorophyll-containing chloroplast is one developmental stage of the plastid organelle. Plastids are found in all plant cell types and contain their own genetic material as well as all the machinery necessary for DNA, RNA and protein synthesis. They have diverse structures, functions and protein contents dependent upon the plastid type, which is determined to a large degree by the tissue (leaf versus root, etc) in which the plastid resides. For example, chromoplasts are carotenoidrich, non-photosynthetic plastids (yellow, red or orange), often associated with flowers or fruit. Plastids found in roots or tubers are termed amyioplasts. Plastids other than chloroplasts lack many of the proteins associated with photosynthetic membranes, and have messenger RNA (mENA) populations which are distinct from those of chloroplasts [ 1 ] However, because each of these plastids is able to convert to one of the other plastid types (Kirk and Tinley-Bassett, The Plastih Elsevier/North Holland Press, 1978; Mayfield and Huff, Plant P&id 1986, 81:30-351, expression of chloroplast genes must be considered in relation to gene expression in plastid types other than chloroplasts. Of the hundreds of proteins involved in photosynthesis, less than half are encoded within the limited chloroplastic genome. It has become clear over the last few years that the nucleus encodes not only many photosynthetic proteins but also a number of functions necessary for the expression of chloroplast-encoded genes. It has also become clear that interactions between the nuclear and chloroplast genomes are responsible for coordinate expression of the photosynthetic apparatus. Photosynthetic membranes within chloroplasts consist of four major photosynthetic complexes (photosystem II, photosystem I, cytochrome b6/f, and ATPase), each of which contain proteins encoded in the chloroplast and nuclear genomes. The regulation of chloroplast gene expression, therefore, has three distinct aspects: gene expression during plastid development, gene expression in response to environmental signals, and gene expression in relation to
nuclear-chloroplast interactions. This review will summarize some of the recent progress made in understanding these regulatory aspects.
Development
of the chloroplast
Much of what is known about chloroplast gene expression comes from the study of the conversion of etioplasts (pre-chloroplasts lacking chlorophyll) to chloroplasts. This conversion is dependent upon light, which is necessary for the synthesis of chlorophyll. The conversion of etioplasts to chloroplasts involves a rapid accumulation of chlorophyll, photosynthetic membranes, and the associated photosynthetic proteins, both nuclear and chloroplast encoded. The accumulation of photosynthetic proteins correlates closely with the accumulation of chlorophyll (Harpster et al, Pkant Mol Bid 1984, 3:59-71). During this light-induced chloroplast biogenesis, there is a concomitant increase in the transcription and accumulation of several cytoplasmic mRNAs encoding photosynthesis-related proteins (reviewed in Tobin and Silvexthome, Ann Rev Plant Plysiol 1985, 36:569-593). Unlike the mRNAs for nuclear-encoded proteins, n-&N& for chloroplast-encoded photosynthesisrelated proteins accumulate to relatively high levels in plants grown in the dark (reviewed by Mullet, Ann Rev Plant Plysiol 1988, 39:47>502) and show little additional accumulation upon exposure to light Walnoe et al, J Cell Bioll988,106:609616). Etioplasts are capable of protein synthesis and accumulate several chloroplast and nuclear-encoded proteins. Their exposure to light results in a dramatic increase in the synthesis of proteins associated with photosynthetic membranes (Klein and Mullet, JBiolCbem 1986,261:1113&%11145; Klein eta& JC.211 Bill 1987, 106:28!+302; Malnoe et al, J CklI Biol1988, 106:603-616). Blocking plastid transcription during this transition has no effect on photosynthetic protein syn thesis, showing that existing mRNA pools are used for protein translation (Malnoe et al, 1988). Thus, the expression of photosynthetic genes is dependent upon the
Abbreviations Ill-inverted
@ Current
repeat;
Biology
mRNA-messenger
Ltd ISSN 0955-0674
RNA.
510
Nucleus
and gene expression
plastid type as well as upon light, the environmental stimulus. Several of the mechanisms which have been identified in this developmental and environmental regulation will be discussd in the following sections.
Transcription
of chloroplast
genes
Transcription promoter sequences identihed in the chloroplast genome appear quite similar to prokaiyotic consensus promoters (Han@Bowdoin and Chua, Trend Biocbem Sci 1987, 12:67-70) [ 11. Although several parameters (stage of development, environmental signals, DNA topology) a&t the transcription of chloroplast mIWAs (reviewed by Mullet, Ann Retl Plant P&v siol 1988, 39:47>502), there is a poor correlation between the expression of chloroplastic proteins and the transcription of their genes. Messenger RNAs of several abundant photosynthesisrelated proteins are transcribed at similar rates in plas tids of roots and immature and mature leaves, yet little or no accumulation of these mRNAs or their protein products is detected in any plastids except those of mature leaves (Deng and Gruissem, Cell 1987, 49:379-387; see [ 11 for review) [2]. Processing of these transcripts is identical in all three plastid types. Measurements of mFWA turnover in each of these plastids show differences which correlate with ~RNA accumulation, suggesting that stability rather than transcription is the determining factor for dilferential accumulation of mRNAs in these plastids [2]. There is little dilference between the transcription of these genes in chromoplasts and chloroplasts of bell pepper fruit [3]. However, in the conversion of chloroplasts to chromoplasts in ripening tomato fruit, there is a distinct change in the transcription of some, but not all, of these photosynthesis-related genes [4]. This reduction in transcription correlates with DNA methylation of the same genes. As discussed in the previous section there is little change in steady-state levels of plastid mRNAs during the conversion of etioplasts to chloroplasts. Direct measurement of plastid gene tmnscription during this transition shows no apparent new transcription of chloroplast genes [ 51, suggesting that new transcription within the plastid is not a requirement for chloroplast biogenesis. Viewed collectively, these data suggest that changes in plastid transcription are not a primary means of regulating the accumulation of chloroplast-encoded mRNAs for photosynthetic proteins. Changes in transcription do not correlate with the stage of plastid development either, even though the accumulation of mRNAs and photosynthetic proteins is very much dependent upon the stage of plastid development. Transcriptional regulation of chloroplast genes may indeed play a key role in chloroplast gene expression and in plastid development, but these roles appear to be centered on specific plastid genes and do not operate at times when large changes in chloroplast mRNA accumulation or protein synthesis are occurring.
Post-transcriptional mRNA accumulation
regulation
of chloroplastic
Several chloroplast genes are transcribed as polycistronic mRNAs which show diverse patterns of processing. This processing results in accumulation of mono and polycistronic mRNAs, both of which appear to be used for protein synthesis (Barkan, EMBO J 1988, 7:2637-2644). Splicing of rnRN~s occurs both inn-a- and intermoleculady in chloroplast transcripts, with some mature mRNAs formed by the splicing of portions of distinct unlinked transcripts (Kuck et al, EMBO J 1987,6:218+2195) [61. Many of these processing and splicing events require nuclear gene products, the loss of which can result in the absence of specihc mature rnKN& (Choquet et al, Cell 1988, 52903-913). It is still unclear exactly how this mRNA processing alfects chloroplast mRNA accumulation and translation, or to what degree ~RNA processing is used as a regulatory step in chloroplast gene expression. As discussed in the last section, mRN& for several photosynthesis-related proteins accumulate to different levels in green and non-green plastids, despite comparable levels of transcription in both plastid types. Gruissem and co-workers have suggested that this dilferential accumulation results from unequal turnover of specilic transcripts via a mechanism involving inverted repeats (IRS) found in the 3’ untranslated portion of many chloroplast mRNh (reviewed in [ 11). RNAS containing IRS are processed in chloroplast extracts to the correct size and are stable compared with similar sequences lacking IRs [ 11. SpeciIic proteins, which may be developmentally regulated, could interact with these IRs and act either to stabilize the transcript or to regulate mRNA turnover (Gruissem et al, Trend Genet 1988, 4:5863). Differential accumulation of plastid mRNAs would obviously have profound effects on plastid gene expression. Thus, the turnover of plastid mRNAs, via some mechanism involving 3’ IRs and specitic proteins that interact with them, is likely to play a key role in plastid gene expression in relation to plastid development. What role this diiferential accumulation of plastid mRNA has in the expression of photosynthetic proteins in chloroplasts has yet to be determined.
Translational expression
control
of chloroplast
gene
Analysis of photosynthetic mutants in the green alga CHumyabmonus has revealed a class of nuclear genes that are required for translation of specific chloroplast mRNAs (lensen et al., J Cell Biol 1986, 103:13151325; Kuchka et al., EMBO J 1988, 7:31!+324) [7]. Each of these nuclear-encoded genes effects the translation of only a single chloroplast ~RNA, and in some cases multiple nuclear genes appear to be required for the translation of a single mRNA [7]. Genetic analysis indicates that these nuclear genes encode factors that recognize stem-loop structures in the 5’ untranslated portion of
Chloroplast
the chloroplast mRNA and act to allow translation of the message. Chloroplast mutations in the stems of these structures, which either increase or decrease the stability of the stem, act to block translation of the message as well [7]. Although none of the nuclear genes have been cloned or characterized, it seems likely that these genes will encode proteins which interact with the stem-loop structures and act in translation initiation or elongation. The light-induced changes in translation of existing chloroplast mRNAs for photosynthetic proteins could account for many of the observed changes in the accumulation of photosynthetic proteins during chloroplast biogenesis. How translation of some chloroplast mRNAs is promoted over the translation of others, or by what mechanism light influences this process, remains unclear. Whether the factors responsible for light-regulated translation during chloroplast biogenesis are similar to factors identified by the nuclear mutants has not yet been determined. The identification and characterization of factors which control chloroplast mRNA translation should provide some insight into translational control as a mechanism for regulating chloroplast gene expression. The role of translational control in plastid gene expression in plas tids other than chloroplasts remains to be seen.
Post-translational photosynthetic
accumulation proteins
of
A mutation in a structural gene in either genome, resulting in the absence of any single protein from a photosynthetic complex, results in the loss of the entire complex (Chua and Bennoun, Proc Nat1 Acud Sci USA 1975, 72:2175-2179; Metz and Miles, Biochem Bioplys Actu 1982, 681:95-102; Mayfield et al, EMBO J 1987, 6313-318). Synthesis of other members of that complex continues at wild-type levels, but the newly synthesized proteins are quickly degraded and, thus, never accumulate within the mutant (Bennoun et al, Pfant Mol Bioll986, 6:151-160; Erickson et al., EMBO J 1986, 5:1745-1754) [7]. Within a single complex, photosyn thetic proteins can have different half lives, often determined by the growth conditions (light versus dark) of the plants. Plants must therefore have the ability to replace proteins within a complex without turning over the entire complex. How this is accomplished and what role specific protein turnover has in regulating the accumulation of photosynthetic proteins has yet to be determined. Clearly, there must be some regulation between protein synthesis and degradation but how this association is governed and under what mechanism it is functioning is not yet clear.
Chloroplast
effects on nuclear
gene expression
A final aspect of the regulation of chloroplast gene expression involves the interaction of the nuclear and chloroplast genomes in the coordinate expression of
gene regulation
Mayfield
photosynthesis-related proteins. Many of the above-mentioned regulatory steps involve nuclear encoded factors that exhibit some control over chloroplast gene expression, be it mRNA synthesis, processing, accumulation or translation. Each of these regulatory mechanisms indicates that the nuclear genome contains the information necessary to regulate the expression of chloroplast genes. It w-as first shown in 1984 that events within the chloroplast could alfect the accumulation of cytoplasmic mRNAs (Mayiield and Taylor, Eur J Biocbem 1984, 106:144:79+34), and has since been confirmed in many plant species [8-lo] ( reviewed in [ll]). Despite a large body of work showing that chloroplast photooxidation inhibits the expression of nuclear encoded photosynthesis-related proteins (reviewed in [ ll]), our understanding of this phenomenon remains perfunctory and the original hypothesis that a signal from the chloroplast might influence nuclear gene expression remains hypothetical (Mayfield and Taylor, Mol Gen Genet 1987, 208:30%314) [ll]. During the light-activated conversion of etioplasts to chloroplasts there is a concomitant increase in cytoplasn-tic mRNAs for photosynthetic proteins. As the biogenesis of the chloroplast and accumulation of a number of cytoplasmic mRNAs are closely associated, it is difficult to discriminate between light-regulated nuclear gene expression pe’ se and nuclear gene expression associated with chloroplast biogenesis. Many authors have concluded that light-activated transcription of nuclear genes is responsible for controlling the synthesis of the chloroplast-encoded photosynthetic proteins and for the development of the chloroplast. In systems where chloroplast biogenesis is not light dependent, synthesis of photosynthetic proteins proceeds in the dark and photosynthetic membranes accumulate; thus, it is possible to separate chloroplast biogenesis and light-modulated gene expression (Malnoe et al., J Cell Biol1988, 106:609-616). However, in these cells chloroplast mRNA translation remains responsive to fight as does the accumulation of photosynthetic-related cytoplasmic mRNAs. These data suggest that light-activated increases in cytoplasmic ~RNA accumulation and chloroplast protein synthesis are independent of chloroplast biogenesis. Thus, it is unlikely that light-regulated transcription of nuclear genes initiates chloroplast biogenesis. Kuhlemeier et al. [ 8] report that a promoter from an abundant nuclear encoded photosynthetic protein contains two distinct domains, one for light-regulated transcription and one for tissue specificity. As tissue specificity and the stage of plastid development are tightly correlated, it seems that these data would also argue for separation of light-regulated gene expression and plastid development. Chory and coworkers [ 121 have identified a mutant in the higher plant Ar&wsis tiliana that develops leaves containing pre-chloroplasts in the dark. These plants accumulate several cytoplasmic mRNAs for photosynthesisrelated proteins which are normally observed to be light regulated and thus found mainly in light-grown plants. This accumulation, and the appearance of pre-chloroplasts in these plants, show that the influence of light
511
512
Nucleus
and
gene
expression
can be uncoupled from plastid development. This observation also demonstrates that the accumulation of cytoplasmic mRNAs for photosynthetic proteins may be coupled to chloroplast development, as well as coupled to light exposure. It is therefore possible that much of the accumulation of cytoplasmic mFWA associated with light exposure is in fact a response to the development of the chloroplast, which is itself initiated by light exposure. Although these data provide no direct information on the identity of a chloroplastic factor or signal that may be influencing nuclear gene expression, they suggest that such a signal is present in cells containing pre-chloroplasts, and that it does not require light exposure for its activity.
Conclusions The regulation of photosynthetic gene expression involves a complex scheme in which mRNA translation and mRNA stability play key roles. Fig. 1 outlines the normal pathway of photosynthetic protein synthesis and identifies the specidc steps that have been characterized which regulate this gene expression. From the data available to date, it appears that transcriptional control of plastid genes has a limited role in determining mRNA accumulation in different plastid types, and perhaps none at all in regulating the rapid changes observed in photosynthetic protein accumulation during chloroplast biogenesis. The exact mechanisms used to regulate mRN4 stability and translation are only now begin-
ning to be characterized, but both of these regulatory mechanisms appear to require nuclear encoded factors and may be controlled to a large degree by the nucleus. Much of the variation observed in plastid gene expression in the tierent tissues of higher plants may be accounted for by mRNA stability while translational regulation appears to account for much of the variation in photosynthetic protein accumulation observed during biogenesis of the chloroplast. The use of translational control for chloroplast gene expression under various environmental conditions may be dictated by a requirement for rapid alteration in the level of photosynthetic protein synthesis. Under maximum photosynthetic conditions (bright light), synthesis of photosyn thetic proteins needs to be at relatively high levels to replace proteins that are turning over at an accelerated rate. Changes in light conditions can occur rapidly and the use of translational controls would allow protein synthesis rates to change rapidly in response to this. The use of transcriptional regulation would add an additional lag time for synthesis of new mRNA pools, a lag which may be too long to regulate efficiently the rapid changes required for photosynthetic protein synthesis. The closely coordinated expression of the chloroplast and nuclear genes is regulated by mechanisms that have yet to be characterized. Plastid development clearly has some bearing on nuclear gene expression and nuclear genes are clearly required for plastid development. It seems likely that the nuclear genome sets some developmental program that determines, to a large degree, fig. 1. Regulatory steps thesis of the photosynthetic tus. The open normal pathway
in
the synappara-
arrows illustrate for the synthesis
photosynthesis-related clear-encoded proteins
proteins. Nuare synthesized
from cytopfasmic messenger (mRNAs) (a) as soluble precursor peptides transported
(b)
velope encoded from with
and across
membranes proteins
cc). (e) are
(0.
coordinate chloroplast
ulated increase accumulation; crease plastic ferential
(d) and combine polypeptides to
Several
steps
expression genes: (I)
regulate of nuclear light-mod-
in cytoplasmic (2) light-modulated
mRNA in-
in translation of existing chloroand cytoplasmic mRNAs; (3) difstability of plastid mRNAs re-
sulting in unequal accumulation cific mRNAs; (4) rapid turnover tosynthetic of other
en-
Chloroplastsynthesized
multisubunit photosynthetic located within the thylakoid
membranes this and
RNAs poly-
post-translationally the chloroplast
plastid mRNAs nuclear-encoded
form the complexes
the of
proteins associated
in the proteins;
of speof phoabsence (5) feed-
back ‘signal’ from the chloroplast to the nucleus specifically affecting mRNA transcription and accumulation; and (6) regulation of chloroplast mRNA translation and stability by nuclear gene products. Ct DNA, choloroplast DNA.
Chloroplast
the stage of plastid development, and then reacts to this developing plastid to 6ne tune transcription of specific nuclear genes involved in photosynthesis. The challenge now is to characterize the specific mechanisms by which the nucleus and chloroplast interact. This interaction offers a very interesting model for understanding the cooperation of distinct genomes, as well as for examining the interactions of development and environmental signals in regulating photosynthetic gene expression.
Annotated reading: l l e
references
GRUSSEM
and recommended
l e
their
W: Chloroplast
plastids
on.
gene expression: Cell 1989, 56:161-170.
how
plants
l
X-W, GRLJISEM W: Constitutive transcription ulation of gene expression in non-photosynthetic of higher plants. EM30 / 1988. 7:3301-3308.
and reg. plastids
DENG
This paper shows that plastid genes are transcribed at similar rates in plastids of roots, immature and mature leaves. mRNA processing is identical in roots and leaves, and tierentiai accumulation of mRNAs for photosynthetic proteins in chloroplasts is due to stability of those mRNAs in plastids of leaves but not in roots or immature leaves. 3.
KUNIZ
l
pression of plastid and nuclear genes in bell pepper (Cupsicum annuum)
M, EVARD J.l
D’HARLINGUE
4
WEU
J-H,
KOBAYASHI
l e
regulation and tional conversion
and
H, NGERNPRASLRTSUU J, AKUAWA
DNA methylation of chloroplasts
KRUPINSKA K, APEL K: Light-induced
l
plasts to chloroplasts of barley control of plastid gene expression. 2 19:467-473.
HERRIND, SCHMIDTGW: Trans-splicing
of transcripts for the
chloroplast
psaA1 gene. J Biol Ckm 19813, 263~14601-14604. luclear mutants al&t splicing of the chloroplast psaAl mRNA which is transcribed from 3 distinct regions of the chloroplast genome. Addi.
tional evidence is provided that nuclear gene products are required for mRNA processing and expression of chloroplast genes. 7.
RGCW
l e
GIRMD-B,v?COU J, BENNOUN P: Nuclear tations tiect the synthesis or stahiity pshC gene product in Chlamydomonas
J-D, KUCHKA M, MAYFEUI S,
8.
KUHIEMEIEIER C, s TRITI%ATrER
l
rhcS-M promoter organ specificity.
%3IRMER-bHU@
M,
and chloroplast muof the chloroplast reiinhardtiL EMBO
sunflower
9. e
in plastids during transito chromoplasu. EM90 /
transformation of etiowithout transcriptional Mel Gen Genet 1989,
The transcription of plastid genes was compared in chloroplasts and etioplasts and found to be essentially identical. This suggests that the
C. CHUA
N-H:
responsiveness 1:471478.
The
Pea
but
not
STOCKHAUS J, SCHEU J, W-
of the exgene !X-LSl with / 1989, 8:2445-2451.
R L: COtTelatiOn
pression of the nuclear photosynthetic the presence of chloroplasts. EMBO
A nuclear gene, encoding a chloroplastic protein involved in photo. synthesis, is expressed only in cells with developed chloroplasts. This corroborates earlier experiments which show that the development of the chloroplast is a requirement for the expression of nuclear genes whose products are located in the chloroplast. 10.
HARKINS
l
G.UBRM~ DW: Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proc Nat1 Acud Sci USA 1989. 87:81-20.
(Heli-
T: Transcriptional
G. WARD
mediates light Pkant Cell 1989,
A promoter for a nuclear encoded photosynthesis-related protein was used to show that tissue specificity and light regulation are 2 separate phenomena that are likely located on distinct regions of the promoter.
CAMARA B: Ex-
1990, 9:307-313. The transcription of several photosynthesis-related genes was found to vary between chloroplasts and chromoplasts of tomato fruit. The loss of transcriptional activity was found to correlate with DNA methyiation. This report differs with that of Deng and Gruissem 12) for tmnsctiption in non-green plastids, suggesting that not alI plastids regulate mRNA accumulation in exactly the same manner. 5.
6.
during di5erentiation
anthus annuus). Mel Gen Gener 1989, 216:15&163. Messenger RNA for 2 nuclear encoded genes, involved in photosynthesis, accumulates in non-photosynthetic leaves. 2 chloroplast-encoded mRN& also involved in photosynthesis, showed little differences in transcription in green or non-green p&ids and accumulated equally in both plastid types. These data suggests that transcription and mRNA stability are not important variables in the conversion of chloroplasts to chromoplasts. 4.
light.dependent development of a chloroplast from an etioplast is not dependent upon activation (transcription) of novel genes.
turn
A review of chloroplast gene expression which discusses mRNA transcription, splicing and stability and also covers involvement of nuclear genes in mRNA stability and translation. It includes many refer. ences up to and including 1988. 2.
Mayfield
/ 1989, 8:101~1021. This paper SNdieS the characterization of nuclear and chloroplast mutants. These mutants defme a stem-loop region in the 5’ end of the psbc ~~RNA which interacts with nuclear encoded ‘factors’ to regulate p&C mRtU translation. Genetic evidence that a nuclear gene product controls translation of specific chloroplast mRNAs is provided.
Of interest Of outstanding interest
1.
gene regulation
rcR,
JEFFERSON
RA,
KAVANAGH
TA,
BEVAN
MW,
This Study demonstrates that the expression of chimeric genes in both ttansgenic and ttansfected protoplasts, under the control of photosynthetic gene promoters, is restricted to photosynthetic cells, corroborating, in transfected protoplasts, what several other laboratories have previously shown. 11. l e
R Photooxidative its effect on nuclear gene enzyme levels. Pbotocbemishy
OELMUUER
destruction expression
of chloroplasts and and extraplastidic
1989, 49:22%239. review of the effect of photo-oxidation of the chloroplast on the loss of cytoplasmic ~RNA encoding photosynthesis-related proteins. Photooxidative loss of the chloroplast reduces transcription and accumulation of nuclear encoded mRh% for photosynthetic proteins. This paper discusses chloroplast signals and their implication in nuclear gene regulation.
A
12.
CHORY J, PETO C, FEINEZAUM R, PRAWN I AUSLIESEI. F: Arubidop
l e
sfs tbafiana mutant that develops in the absence of light. Cell 1989,
as a light-grown 5899-999.
plant
This paper examines the characterization of a nuclear mutant which allows the expansion of leaves and the development of pre-chloroplasts in the absence of light. This mutant accumulates certain nuclear encoded mRNAs in the dark which normally accumulate only in light. It is strongly suggested that much of the ‘light-regulated’ nuclear gene expression may in fact be a response to chloroplast development, itself influenced by light.
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