Gerhard Link

Summary
Chloroplasts and other plastids are plant cell organelles that account for major
biochemical functions. They contain their own gene expression system but are
integrated into the signaling network of the entire cell. Both nuclear and plastid
genes are involved in chloroplast biogenesis, and the gene expression pathways
both inside and outside the organelle are subject to developmental and
environmental control. The plastid transcription apparatus reflects this general
scheme, with a basic organelle-encoded enzymatic machinery surrounded by
factors that may be encoded by nuclear genes. Among the transcription
regulatory mechanisms thought to play a role during plastid development are: (1)
differential usage of promoter elements; (2) phosphorylation of transcription
factors by a protein kinase, which is itself subject to phosphorylation and redox
control; (3) dynamic changes in the composition of the transcription apparatus. In
etioplasts, the dominating polymerase ‘6’ is a bacterial-type enzyme, whereas
the major chloroplast polymerase ‘A’ is a much larger enzyme reminiscent of
those in the nucleus. These two enzyme forms may share common components Accepted
and recruit others during development. 17 January 1996

Introduction Plastids contain multiple copies of circular double-

Plant cells are unique in nature in having three gene-con- stranded DNA molecules of a size that varies from species
taining compartments, i.e. the nucleus, the mitochondria to species but is typically 150 kbp ( 1 ~ 1 0 Da)(5).
~ Despite
and the plastids(’P2). The latter comprise a group of cell species-specific differences in certain details, the general
type-specific organelles, each surrounded by an envelope arrangement of chloroplast DNA is conserved, with more
that consists of an inner and outer membrane. Plastids can than 100 functional genes for rRNAs, tRNAs and proteins.
be viewed as major ‘chemical factories’ of the plant cell, The encoded proteins can be classified into those involved
being responsible at least in part for the synthesis of com- in photosynthesis-relatedfunctions and others that are part
pounds such as carbohydrates, amino acids, fatty acids, of the plastid gene expression system. This latter group
galactolipids, isoprenoids, chlorophylls and pyrimidines, as
comprises proteins of chloroplast ribosomes as well as sub-
well as informational macromolecules, i.e. nucleic acids
units of organellar RNA p o l y m e r a ~ e ( ~ > ~ ) .
and proteind3). The best-known plastid type is the green
Although plastids contain a complete enzymatic appara-
chloroplast, which is the site of photosynthesis and assimi-
tus for gene expressiod7), this does not mean that all the
lation of carbon, sulphur and nitrogen. In addition, several
proteins involved are products of organelle genes. On the
‘nongreen’ plastid forms exist, including red or yellow chro-
moplasts, colourless leucoplasts and amyloplasts, and yel- contrary, plastids, like mitochondria, are genetically ‘semi-
lowish etioplasts, each form being restricted to certain autonomous’, i.e. they require nuclear gene products for
types of specialized cells and developmental situationd4). their biogenesis and function. Well-known examples of
They all develop either from small undifferentiated proplas- nuclear-encodedchloroplast proteins are the small subunit
tids or by conversion from a specialized mature plastid of the carbon dioxide-fixing enzyme ribulose-l,5-bisphos-
type. A widely studied example of the latter route is the con- phate carboxylase/oxygenase (Rubisco) and many proteins
version of etioplasts into chloroplasts, which takes place of the photosynthetic apparatus. The same dual-coding
when dark-grown seedlings of higher land plants are principle seems to apply to the enzymatic complexes that
exposed to light. carry out reactions of organelle gene expression(8~9).
 Multi-step regulation of plastid gene expression cotranscribed with frnK precursor sequence^('^,^^). This
How is the flow of genetic information inside the organelle long cotranscript may then be cut by a sequence-specific
regulated during development, and how does the mature endoribonuclease termed p54, which separates the psbA
chloroplast respond to variable environmental conditions portion from the frnKportion of the transcript(24).The result-
such as light intensity? These questions have been ing psbA RNA molecules differ from those being transcribed
addressed in numerous attempts to describe the molecular from the proximal psbA promoter by a longer 5’-untrans-
changes that take place at the various levels of gene lated region (UTR).
expression. Depending on the technical arsenal available, 5’-UTR sequences were shown to serve as cis-elements
the focus has changed over the years, with emphasis on dif- for RNA binding proteins that determine both the stability
ferent aspects of this complex scenery. For instance, many and translation efficiency of the mRNA. Different 5’-UTR
(but not all) plastid proteins accumulate to higher levels in sequences may recruit different sets of RNA binding factors,
light-grown as compared to dark-grown plant tissue@).With which would be expected to affect the amounts of the trans-
the availability of cloned chloroplast genes as specific lation p r o d ~ c t ( ~In~ addition,
~ ~ ~ ) . if the UTR-binding com-
hybridization probes it became possible to measure steady- plexes differ with respect to associated protein kinase
state transcript levels. This work indicated that the pool and/or phosphatase activities(16),this may result in differen-
tial modification of the D1 protein. A minor D1-related pro-
sizes of many plastid transcripts are unaffected by light ver-
tein form termed D1* was previously detected, which differs
sus dark growth conditions, while a relatively small group of
from the major D1 form in its higher degree of phosphoryla-
transcripts reveals a positive response(’0.11).
tion(26!27).It is tempting to speculate that the D1” form may
In organellar run-on experiments, i.e. RNA synthesis in
originate by an alternative expression pathway, e.g. by
isolated plastids followed by hybridization of newly synthe-
cotranscription from the distal frnK promoter, followed by
sized RNA to specific gene probes, have helped clarify the
endonuclease cleavage of the primary transcript(22).The
extent of transcriptional versus post-transcriptional regula-
transcripts driven by the distal (trnK) promoter are not
tion at RNA In general, the relative transcription
lightldark-regulated and accumulate transiently during
intensity of many plastid genes was found to be constant in
seedling development, whereas the major psbA transcript
different tissues and developmental stages, despite global
driven by the proximal promoter accumulates further during
changes in transcription rates. This might reflect the require-
seedling development in the light but not in the dark(21).It is
ment for the synthesis of ‘house-keeping’ products during
hence possible that a developmental stage-specific D1 pro-
plastid biogenesis, as well as their stoichiometric assembly tein exists, which may or may not be related to D1* but could
with nuclear-encoded components. Superimposed on this fulfil a special role in the biogenesis of the photosynthetic
global transcription activity is the differential transcription of apparatus.
a small number of organelle genes. However, the observed
changes often did not fully reflect those at the level of stable
transcript accumulation, which emphasizes the role of post- Transcription during plastid development: the psbA
transcriptional regulation(7,9,14-17). story
One of the most widely studied plastid genes, the psbA The psbA gene gives rise to enhanced levels of transcripts
gene for the D1 reaction center protein of photosystem I I in light-grown seedlings as compared to dark-grown
(PSIl)(l8), can serve as a paradigm. This gene, which in seedlings(’ I). Transcript levels are higher in both absolute
most plants exists as a single copy and is transcribed from and relative terms, i.e. based on equal cell or plastid num-
its own (proximal) promoter, gives rise to an abundant tran- bers as well as on equal amounts of plastid DNA or RNA(21).
script(I0).The mRNA is efficiently translated into a precursor This means there is a selective increase in the transcript
protein, which is processed to give the mature D1 pro- pool size of the psbA gene versus that of other plastid genes
tein(lg). After integration into the reaction center complex, in chloroplasts as compared to etioplasts, i.e. the typical
this protein undergoes rapid turnover as a result of photo- plastid type of dark-grown seedlings.
damage of PSII, which leads to multiple cycles of degrada- It was shown that DNNprotein-complexes termed ‘tran-
tion, resynthesis and replacement of PSI1 core proteins(20). scriptionally active chromosomes’ (TAC)(”) prepared from
This requirement for continuous replacement of the D1 chloroplasts were several-fold more active in synthesizing
gene product is thought to reflect, and its turnover to pro- psbA RNA than were equivalent preparations from etio-
voke, the active expression of the psbA gene. The promoter p l a s t ~ ( ~Likewise,
~). organellar run-on experiments indi-
in front of this gene is particularly strong, giving rise to abun- cated a higher transcription rate in chloroplasts than in etio-
dant transcripts at levels that are several times higher in pla~ts(~~,~ InOboth
). the TAC transcription and run-on
light-grown seedlings than in dark-grown seedlings(’0z21).A experiments, the RNA synthesis measured mainly repre-
fraction of psbA transcripts, however, originates from a dif- sents elongation of pre-existing chains. However, in order to
ferent promoter that is located further upstream in front of a study possible regulatory mechanisms in more detail, plas-
neighbouring gene termed trnK (for tRNALyS), i.e. it is tid in vitro systems that are capable of faithful initiation at a
defined promoter are required. Several such systems, could each be subject to regulation by additional factors in a
involving crude and fractionated plastid extracts, have been cell type- and developmental stage-specific way and in
successfully developed and used in studies to define the response to environmental cues. In both the ‘prokaryotic’
role of sequence elements and protein factors involved in and ‘eukaryotic’ scenarios, two types of factors would be
basic and regulated t r a n ~ c r i p t i o n ( ~
(Table
~ - ~ ~1).
) expected to play a role: (1) sequence-specific DNA-binding
The psbA promoter contains sequence elements that proteins that directly interact with regulatory regions on the
resemble the ‘-10’ and ‘-35’ sequence elements of sigma70- DNA and serve as positive or negative regulators; and (2)
type bacterial promoters, which are present in front of many (sigma-type) factors that do not bind DNA by themselves
plastid gened5). It was shown by functional assays using in but confer specificity on the holoenzyme upon their associa-
vitro transcription that both these elements are required for tion with the core RNA polymerase. There are numerous
efficient psbA t r a n s ~ r i p t i o n ( ~ ~However,
3~~). unlike most
examples for both DNA-binding and sigma-type specificity
other chloroplast genes, the psbA promoter contains an factors in the prokaryotic world. Considering the various fea-
interspersed AT-rich element that resembles the TATA-box tures shared by plastids and bacteria, including the typical
of many nuclear genes transcribed by RNA ‘prokaryotic’ organization of many chloroplast promoters, it
polymerase ll(36).This latter element was shown to be is therefore tempting to consider this scenario first.
required for basic transcription levels in extracts from both Nucleotide sequence analysis has revealed open reading
chloroplasts and etioplasts. Using chloroplast extracts, frames in chloroplast DNA which are highly homologous to
however, enhanced transcription was observed only in the the bacterial rpoA, rp0B and rpoC genes, except that in
presence of an intact upstream region that included the ‘-35’ plastids the latter is typically split into two genes termed
sequence element(31).This same region did not have an rpOC7 and rp0C2(~~). These genes all seem to be functional
activating effect on psbA transcription when etioplast since the RNA and protein products were detected in vivo by
extracts were used. Furthermore, there was evidence for DNNRNA hybridization and immunochemical techniques,
differential DNNprotein interaction in this region with respectively. The encoded polypeptides termed a, p, p’and
chloroplast versus etioplast extracts, as revealed by methyl- are functional subunits of purified plastid RNA poly-
ation interference and DNAse I footprint experiments(37). merase preparations(39340).
Taken together, these data suggest that there is differential Several DNA-bindingproteins were identified and at least
usage of the psbA promoter by chloroplast versus etioplast some of them seem to act as sequence-specific regulators
proteins (Fig. 1). This raises the question of which proteins in vitr0(~2~l).Likewise, plastid extracts contain sigma-type
might be involved in transcriptional regulation during initiation factor activity and this activity could be resolved
organelle development. into three different sigma factors designated SLF67, SLF52
and SLF29(42).
Comparison of these highly purified polypeptides from
Proteins involved in differential plastid transcription chloroplasts and etioplasts showed that both plastid types
In principle, two different scenarios could be en~isaged(~6). appear to contain a set of sigma factors of equivalent sizes.
In a ‘prokaryotic’ situation, a single RNA polymerase core Nevertheless, the corresponding factors from chloroplasts
enzyme might be associated with protein factor(s) that con- differ from their etioplast counterparts in both their functional
fer transcription of certain genes in a developmental stage- properties and their physical association with the core poly-
specific manner. Alternatively, there could be more than a merase. The latter tend to be more tightly bound within the
single class of RNA polymerase, as is the case in the nuclei transcription complex and to confer efficient, but less selec-
of eukaryotic cells. Multiple polymerases could be responsi- tive, promoter binding than the chloroplast factors(43).When
ble for the transcription of certain classes of genes and the isolated factors were first treated with protein kinase or

Table 1. Plastid in vitro transcription systems

Type of system Characteristics Purpose References
Broken (‘lysed’) plastids Mainly elongation Organellar run-on 13
(transcription rates) 12,14
DNA-protein complexes Mainly elongation Run-on 28, 29, 63, 64
(TAC; nucleoids) Polypeptide analysis
Soluble extracts DNA-dependent Promoter strength 32, 35-37, 65-67
(cruddfractionated (initiation) Promoter dissection
lysates) Factor assays
Purified DNA-dependent Subunit composition 49, 40, 57, 68-70
RNA polymerase(s) (initiation) Enzymatic properties
Factor assays
Genes
Regulation
 Promoter EZement Switch use the chloroplast sigma factors efficiently as substrates for
phosphorylation (K. Tiller, S. Baginsky and G. Link, unpub-
lished data). Hence, this transcription complex-associated
kinase is a candidate for being the enzyme responsible for
phosphorylation control during plastid transcription.
It was proposed that chloroplast transcription might be
controlled by the photosynthetic apparatus via a two-com-
ponent signaling system(45)consisting of a ‘redox sensor’
kinase and ‘redox response’ regulator(46).However, PTK
appears to be a serinehhreonine kinase rather than a ‘two-
component’ histidine k i n a ~ e ( ~ which
~ ) , makes it unlikely that
-35 TATA PTK is the proposed ’redox sensor’(46).Furthermore, this
Fig. 1. Promoter element switch: a model showing differential usage of transcription kinase is itself subject to phosphorylation con-
promoter regions as a regulatory mechanism in plastid gene expression. The trol in vifro, which might indicate that PTK is the endpoint of
horizontal line indicates the plastid DNA region containing the psbA promoter
a cascade-type signaling chain that connects transcription
with the -35-and TATA-box-like elements (boxed), the transcription starl site
(vertical arrow), as well as a portion of the psbA gene. Transcripts are shown photosynthesis-related metabolic functions of the chloro-
as wavy horizontal arrows. The TATA-element is required for basic plast.
transcription in both chloroplasts and etioplasts, and the upstream -35-like The phosphorylation and redox control of the chloroplast
region confers high-level transcription. The latter region is required in
chloroplast but not in etioplast t r a n ~ c r i p t i o n ( ~ ’ , ~ ~ ) .
transcription apparatus are reminiscent of the situation
described for a post-transcriptional regulatory system con-
sisting of a set of proteins that assemble at the 5’ UTR of
phosphatase and then tested in binding reactions contain- plastid mRNAs and are thought to be involved translation
ing €. coli core RNA polymerase and chloroplast promo- control in vivo(I6). In vitro, the resulting RNAP complex
tors, the DNA-binding specificity in this assay was related to formed with psbA 5’ RNA sequences was shown to be
their phosphorylation state. The etioplast SLFs, which are affected by both phosphorylation/dephosphorylation(47) and
more highly phosphorylated, could be converted into the redox reagents(48).For instance, both dithiothreitol (DTT), a
‘chloroplast’-type form by phosphatase treatment and, con- vicinal dithiol reductant, and the enzyme thioredoxin from E.
versely, the chloroplast SLFs were altered by kinase treat- coli promoted the RNA binding activity of the complex,
ment into a form that revealed ‘etiop1ast’-type SLF proper- whereas the monothiol reductant J3-mercaptoethanol did
t ies(44). not. It was concluded that a signaling chain involving trans-
These results suggest that phosphorylation is a major fer of reducing power by the chloroplast thioredoxin system
determinant of plastid type-specific transcription via a sig- might connect light via photosynthesis with psbA mRNA
naling pathway that affects the properties of the SLF fac- translation(48).At closer view, however, the details of this
t o r ~ ( ~In~the
) . chloroplast, transcription of the psbA gene is proposed translational signaling are different from those
efficiently initiated by a sigma factor that has a low degree of that control transcription by sigma factor modification. First,
phosphorylation and associates loosely with the core a different protein kinase activity appears to be involved,
enzyme. Following the completion of the initiation step, this with the translation-related enzyme being ADP-depen-
factor is easily released and recycled, which results in effi- dent(47),whereas the transcriptional PTK activity is not. Sec-
cient chain elongation by the core enzyme. In the etioplast, ond, the latter is neither affected by DTT nor by E. coli
the more highly phosphorylated factor is capable of tight t h i o r e d ~ x i n ( ~but
~ ) ,responds specifically to the reduced or
complex formation with the core polymerase, resulting in oxidized forms of glutathione (K. Tiller, S. Baginsky and G.
strong promoter binding; however, subsequent release of Link, unpublished data). Therefore, it appears that more
SLF from this complex is inhibited, which leads to an overall than a single signaling pathway is responsible for the modu-
lower efficiency of RNA chain elongation(44). lation of plastid gene expression at various control sites
This model is based on in vifro experiments using heter- (Fig. 2).
ologous animal protein kinase (catalytic subunit of bovine A number of nuclear transcription factors in animal sys-
heart PKA) and phosphatase (calf intestinal alkaline phos- tems are known to be regulated by redox reagents such as
phatase) for phosphorylation/dephosphorylationof the SLF g l u t a t h i ~ n e ( ~The ~ ~enzyme
~ ~ ) . glutathione reductase, which
factor preparations. A question that arises, therefore, is is responsible for the intracellular balance of the oxidized
whether specific plastid enzymes do exist that fulfil this role and reducedforms, is a cytoplasmic protein in animals but is
in vivo. Indeed, a protein kinase activity was found to copurify mostly plastid-located in plant cells(51).In view of the data
with chloroplast RNA polymerase and could be separated suggesting glutathione regulation of psbA transcription, it is
from the transcription complex during subsequent chro- conceivable that this enzyme might be an essential part of
matography. This ‘free’ kinase form (termed PTKf), as well the signaling chain that activates organelle transcription and
as the polymerase-bound form (PTKb), were each found to connects it to photosynthesis.
 the larger nuclear RNA polymerases of eukaryotic cells.
Furthermore, the B enzyme is sensitive to rifampicin, a
known inhibitor of prokaryotic RNA polymerase, whereas
the A enzyme is resistant to this drug. Although each poly-
merase form is detectable in both chloroplasts and etio-
plasts, their relative levels in these two plastid types differ
considerably.The B enzyme is the predominantform in etio-
plasts and the A enzyme in chloroplasts. During light-
dependent etioplast -+ chloroplast differentiation, there is
an inverse switch from one enzyme form towards the other,
Fig. 2. Possible connections between photosynthesis and chloroplast with a decrease in activity and protein level of the B enzyme
t r a n s ~ r i p t i o n ( ~This
~ - ~ ~model
). gives a simple schematic view of the ‘outlets’
for redox poise, i.e. ferredoxin (Fd) on the stromal side of photosystem I (I)
by almost one order of magnitude and a concomitant
and a hypothetical redox sensor (Sensor) between photosystem I1(11) and the increase in the A
cytochrome b6f complex (bf)(46).Ferredoxin selves as electron donor for the A still unresolved question is whether these changes are
reduction of thioredoxin(s) (Trx) (via ferredoxin-thoredoxin reductase, not entirely due to synthesis and degradation, respectively, or
shown) and of glutathione reductase ( G R ) , each of which is capable of
converting disulphide groups into thiol groups at regulatory sites of target whether the two enzyme forms are interconverted during
proteins. The latter may include components involved in various stages in plastid differentiation. This would mean that the A and B
plastid gene expression, ranging from transcription via RNA processing to polymerases share common subunits and, in addition, each
tran~lation(~~ Such
. ~ ~ )proteins
. could be under direct redox control (SH) or,
alternatively, the redox signal could affect the activity of signaling
enzyme form might recruit unique subunits and additional
components such as protein kinases (P) and phosphatases, which in turn factors (Fig. 3). It will be important to resolve the role and
regulate the activity of the target proteins via their phosphorylation state. A intracellular genetic origin of all the polypeptides involved.
somewhat similar mechanism could play a role in the case of the redox Several types of ‘unusual’ chloroplast promoters have
sensor on the left, which itself might act as a protein k i n a ~ e c ~There
~ ) . may be
cascades of more than one intermittent kinase, and both common and been reported, which completely lack, or show considerable
distinct components may be involved in the different signaling pathways that deviation from, the canonical ‘-35/-10’ sequence elements
connect photosynthesis with transcription and translation. (e.9. refs 58 and 59). These findings would be consistent
with the idea that even more than two RNA polymerase
forms may exist in the chloroplast, and biochemical support
Multiple plastid RNA polymerases for this has been obtained by the purification of a single sub-
If both phosphorylation and redox control are key mecha- unit-type enzyme preparation(56).Moreover, evidence for a
nisms for the modulation of plastid gene expression, one number of sequence-specific binding factors has recently
might ask whether this is all it takes to make chloroplast emerged, indicating that the regulation of plastid transcrip-
development possible and to ensure the proper function of tion is even more complex. One of these factors, CDF2,
the organelle. Plastids can be considered as ‘relics’ of seems to act as a repressor of rRNA transcription in an in
prokaryotic endosymbionts that still share many prokaryotic vifro system from spinach chloroplasts(60).Another DNA
features. Nevertheless, their biogenesis and function binding factor, termed AGF, contacts AAG-rich sequences
requires multiple interactions with the surrounding nucleo- of one of the three promoters of the psbD operon from bar-
cytoplasmic compartment. This is most evident by the many ley@’).This promoter is ‘unusual’ in its structural organisa-
polypeptides that are imported and assembled along with
those being made inside the ~ r g a n e l l e ( ~A~pertinent
~~~~). Etio plast Chloroplast
question therefore is whether a second, nuclear-encoded,
RNA polymerase might exist within the chloroplast. Work
carried out with plastid ribosome-deficient plants(53)and
with the parasitic plant E p i p h a g ~ d ~ ~ ) , lacks plastid
which
rpo genes, suggests that this might indeed be the case,
since plastid transcripts have been detected in these defec-
tive systems. Furthermore, there is biochemical evidence
for the presence of more than a single class of plastid RNA Fig. 3. Possible interconversion of RNA polymerase forms in etioplasts and
polymerases and these forms have been characterized in chloroplasts. This model shows the two forms of RNA polymerase, each of
further detai1(55-57). which consists of multiple subunits(57).Form B is the more abundant enzyme
in the etioplast and the (larger) form A that one in the chloroplast. The B
Two plastid polymerase forms, termed A and B, were iso- enzyme is more highly phosphorylated in etioplasts than in chloroplasts (blue
lated from mustard (Sinapis alba) and were shown to differ circles). This model assumes that upon dephosphorylation of the B enzyme,
substantially in their structural and functional properties(57). the intermediate (B) recruits other polypeptides to give the A form. These
additional components may include both polymerase A-specific subunits and
Both are multi-subunit enzymes, with the B polymerase
associated factors (AAFs). Conversely, the known sigma factors (SLFS)(~’-~~)
resembling bacterial enzymes with respect to size and sub- are thought to act with the B enzyme. As a result, each enzyme complex may
unit composition, whereas the A enzyme is reminiscent of have components that are shared and others that are unique.
tion by lacking conserved -39-10 regions and containing and mRNA levels vary over 300-fold; predicted mRNA stabilities vary 30-foid. J.
Biol. Chem. 267,21404-2141 1.
other sequence elements not normally found upstream of 16 Mayfield, S. P., Yohn, C. B., Cohen, A. and Danon, A. (1995). Regulation of
other plastid genes. Moreover, this promoter, termed LRP, chloroplast gene expression. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 147-
was found to confer (blue) light control of psbD gene 166.
17 Link, G. (1991). Photoregulated development of chloroplasts. In The
expression in vivo,which could be a consequence of regula- PhotosyntheticApparatus: Molecular Biology and Operation(ed. L. Bogorad and I.
tory DNA-protein i n t e r a c t i ~ n d ~Although
~). a direct role of K. Vasil), pp. 365-394. Academic Press, San Diego.
18 Nanba, 0.and Satoh, K. (1987). Isolationof the photosystem I1reactioncenter
AGF in the light activation of LRP was not established, this consisting of D-1 and D-2 polypeptides and cytochrome b-559. Proc. Natl Acad.
factor is essential for transcription from this promoter(61).It S C USA84,
~ 109-112.
19 Grebanier, A. E., Coen, D. M., Rich, A. and Bogorad, L. (1978). Membrane
is likely, however, that other factors contribute to the pho- proteins synthesized but not processed by isolated maize chloroplasts. J. Cell
toregulated transcription of the psbD o p e r ~ n ( ~ This ~ ! ~ ~ ) .Biol. 78,734-746.
20 Mattoo, A. K., Hoffman-Falk, H., Marder, J. B. and Edelman, M. (1984).
again illustrates that, despite many advances in our knowl- Regulation and protein metabolism: coupling of photosynthetic electron transport
edge of chloroplast gene expression, the dissection of the to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the
chloroplast membranes. Roc. Natl Acad. Sci. USA 81, 1380-1384.
plastid transcription machinery remains an ambitious task 21 Hughes, J. E., Neuhaus, H. and Link, G. (1987). Transcript levels of two
for the years to come. The combination of solid biochemistry adjacent chloroplast genes during mustard (Sinapis alba L.) seedling
with powerful genetic d i s s e ~ t i o n ( and ~ ~ ~ ~ ~ ) development are under differential temporal and light control. Plant Mol. Biol. 9,
~ ~ ~transgenic
355-364.
t e c h n i q ~ e s (can
~ ~be ~ ~ ~ ~ ~to) providethe answers.
~ expected 22 Nickelsen, J. and Link, G. (1991). RNA-protein interactions at transcript 3'
ends and evidence for tmK-psbA cotranscription in mustard chloroplasts. Mol.
Gen. Genet. 228,89-96.
23 Lidholm, J. and Gustafsson, P. (1992). A functional promoter shift of a
Acknowledgements chloroplast gene: a transcriptionalfusion betweena novel psbA gene copy and the
trnK(UUU) gene in Pinus contorta. Plant J. 2, 875-886.
Because of space limitations I apologize to all colleagues 24 Nickelsen, J. and Link, G. (1993). The 54 kDa RNA-binding protein from
whose work has not been cited. I am indebted to the mem- mustard chloroplasts mediates endonucleolytictranscript 3' end formation in vitro.
Plant J. 3,537-544.
bers of my group for comments and discussion, in particular 25 Gillham, N. W., Eoynton, J. E. and Hauser, C. R. (1994). Translational
to Kai Tiller and Sacha Baginsky, who contributed unpub- regulation of gene expression in chloroplasts and mitochondria. Annu. Rev.
lished results. This work was funded by the Deutsche Genet. 28,71-93.
26 Callahan, F. E., Ghirardi, M. L., Sopory, S. K., Mehta, A. M., Edelman, M.
Forschungsgemeinschaft and the Fonds der Chemischen and Mattoo, A. K. (1990). A novel metabolic form of the 32 kDa-D1 protein in the
Industrie, FRG. grana-localized reaction center of photosystem II. J. Biol. Chem. 265, 15357-
15360.
27 Elich, T. D., Edelrnan, M. and Mattoo, A. K. (1992). Identification,
characterization and resolution of the in vivo phosphorylated form of the D1
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