Regulation of nuclear genes encoding chloroplast proteins in transgenic plants

DEVELOPMENTAL GENETICS 11:197-204 (1990)

Regulation of Nuclear Genes Encoding Chloroplast Proteins in Transgenic Plants ROBERT FLUHR Department of Plant Genetics, The Weizmann Institute of Science, Rehouot, Israel

ABSTRACT Transgenic plants have been particularly useful in studying nuclear genes encoding for photosynthetic functions. The expression of these genes and their chimeric constructs in transgenic plants faithfully mimics their natural counterparts. The use of sensitive chimeric reporter genes has enabled localizing the activity of genes encoding photosynthetic proteins to individual cells. Cab and rbcS transgenes have been shown to retain sensitivity to light quality, which is modulated by phytochrome. Conditional light activation under the influence of a circadian rhythm has been shown for Cab transgenes. Transgenic plants containing truncated promoters have helped delineate cis-regulatory positive and negative elements involved in light-mediated transcriptional induction and tissue specificity.

Key words: Light-regulated genes, transgenic plants, enhancer, silencer, regulatory elements, trans-acting factors

INTRODUCTION The synthesis of chloroplast-targeted polypeptides in

the cytoplasm shows a marked degree of synchronism with the chloroplast developmental and physiological state. The harmonious state of affairs can be achieved in a variety of ways. They include regulation at the level of gene transcription, message stability, rate of message translation, and fine tuning by polypeptide product turnover. Critical analysis of these steps necessitates work at the whole plant level. Our ability to achieve easy regeneration of transformed plants containing single copy mutant or chimeric genes has enabled great strides forward in the understanding of gene regulation. In the last 5 years transgenic plants have been used as experimental tools for the study of nuclear genes concerned with photosynthesis. These studies have been carried out in the mature plant organ and during plant development. They have focussed on two very abundant transcripts. One of them is the nuclear-coded small subunit of ribulose bisphosphate carboxylase (rbcS), which is part of a hetero-16-mer enzyme complex that is located in the chloroplast and plays a key role in photosynthetic carbon assimilation.

The second gene codes for the chlorophyll binding protein (Cab). Cab polypeptides bind chlorophyll a and b and exist as a complex in the thylakoid membranes of chloroplasts. This review summarizes some of our cur- rent knowledge about the regulation of these and other genes, as has been elucidated in transgenic plants. The emphasis will be on those reports in which transgenic plants have provided new conceptual experimental frameworks.

THE TRANSGENIC PLANT SYSTEM While the isolated plant protoplast has been fre-

quently used to transiently express certain plant genes, it has not provided productive information about the regulation of genes involved in photosynthesis. De- spite the fact that freshly prepared protoplasts contain well-developed chloroplasts, this state is ephemeral. In the time it takes to measure transient expression of introduced genes the regulatory machinery of the protoplast has been reorganized into a state of dedif- ferentiation, which is characteristic of totipotent cell systems. In that state photosynthetic genes are inacti- vated. An alternative experimental system is stable transformation of cell cultures. This procedure is much less time consuming than regenerating transgenic plants. However, it has many drawbacks. Cell cultures are maintained with the help of exogenously supplied hormones, which by themselves modify gene activation [Flores and Tobin, 19881. In addition to the relatively mixed bag of chloroplast development profiles present in cell cultures, no fast light-mediated gene activation or phytochrome regulation of photosynthetic genes is observed. Instead, light-dependent activation in cell culture is defined by comparing light-grown calli to dark-grown calli. Thus there are very strong reserva- tions against this experimental tool as well.

Transgenic plants are the experimental system of choice, but if they are to be used effectively it is of

Itrccivcd for priblicntion November 14, 1989; accepted November 26, 1989.

Address reprint requests to Robert Fluhr, Department of Plant Ge- netics, P.O. 26, Rehovot, 76100, Israel.

0 1990 WILEY-LISS, INC.

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prime importance to establish the system’s fidelity. The activity of transgenes should be a reflection of their precise endogenous activity. Thus the specific activity of any potential transgene that is part of a multigene family must initially be characterized. In the case of rbcS which is composed of a small multigene family, measurements of endogenous gene activity were carried out by using gene specific probes [for a summary see Dean et al., 19891. Many interspecific differences in endogenous expression were detected. In pea, the light- regulation of all members of the gene family is quali- tatively similar in the leaf, but in some organs only subsets of the gene family are expressed [Fluhr et al., 1986al. In tomato some members show weak dark down-regulation in leaves or are constitutively expressed in certain orgns [Manzzara and Gruissem, 19881. In general transgenes have been found to qual- itatively mimic their natural activity. As an example, the 10-fold difference in endogenous expression between pea rbcS-3A and rbcS-E9 is faithfully repro- duced when both genes are co-transformed into tobacco [Kuhlemeier et al., 1988al. Moreover, judging by the activity of transgenes, the controlling elements for light-induced genes are well conserved within dicotyledonous transfers. However, attempts to express rbcS from the monocot wheat in dicots were unsuccessful, but the wheat Cab transgene was highly expressed in a regulated manner in the dicot tobacco [Nagy et al., 19851.

Whereas transgenes are generally expressed cor- rectly, there is considerable variation in absolute transcript abundance between individual transformed plants [Nagy et al., 1985; Jones et al., 19851. Although the variance has been ascribed to localized influence of flanking host sequences, this needs to be rigorously proved in plants. Experimentally, when quantitative comparisons between different constructs are desirable the plant- to-plant variance becomes a serious problem. One straightforward solution is simply averaging the results of many transgenic plants. Another method which has proved very useful in the study of rbcS pea transgenes is the construction of special cis-linked reference genes [Kuhlemeier et al., 1988aI. When both the test and reference gene contain similar promoter elements, their expression was found to vary from plant to plant in a similar fashion. Afiisng 15 independent regenerated plants containing full-length test rbcS promoters a t least 10-fold variation in transgene transcript accumulation was detected between the plants. However, the ratio between the activity of the test and reference genes was constant [Kuhlemeier et al., 1988al. The utility of this system in analysis of pea rbcS-3A is shown in Figure 1. The test and reference genes both contained the rbcS promoter elements. Their activity was differentiated by the use of one com- mon hybridization probe and S-1 nuclease transcript analysis. The quantitative results are shown in Figure 1 (lanes 1 and 2). Signals of equal strength relative to

1 2 3 nnn

} Test

) Ref.

} Ref.

L D L D L D Fig. 1. S-1 nuclease analysis of mature leaves from transgenic

plants in the light (L) or dark (D). Czs-linked reference and deleted pea rbcS promoters were constructed and used to transform tobacco plants. The reference gene contained the polyadenylation site of the rbcS-3A 3‘ end, while the test constructs contained the rbcS-E9 3’ end. Analysis was by S-1 protection using a single-stranded probe derived from the 3’ end of rbcS-E9. This makes it possible to analyze both reference and test genes with a single probe. The probe protects 160 nucleotides of the reference gene transcript (Ref) and 230 nucleotides of the test gene transcript (Test). Lane 1-3 are deletions of -327, -166, and -100 relative to the transcription start, respec- tively.

the reference gene are obtained for deletion mutants -327 and - 166 but not - 100. Whether cis-linked cotransformation will be generally applicable for the quantitative analysis of other photosynthetic transgenes remains to be seen.

LIGHT-INDUCED EXPRESSION OF GENES ENCODING CHLOROPLAST POLYPEPTIDES The steady-state level of the photosynthetic machin-

ery is maintained by a constant input of nascently synthesized components. Light, perceived by the photoreceptor phytochrome, is necessary for this maintenance in very complex ways. The induction of rbcS in immature etiolated pea cotyledons is motivated by the phytochrome photoreceptor under regimes of low fluence red light [Kaufmann et al., 19841. Yet in mature green leaves a constant source of blue light together with phytochrome activation is necessary to maintain de- tectable levels of rbcS transcript [Fluhr and Chua, 1986bl. The induction of Cab by light is more sensitive to red light than rbcS by a t least three orders of mag- nitude. How the light perception by phytochrome or the blue light receptor is transduced to gene activation is

TRANSGENES OF NUCLEAR ENCODED CHLOROPLAST PROTEINS 199

unknown, but the use of transgenes has illuminated some of the complexities.

Light-activation of rbcS and Cab is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis, but not to chloramphenicol, an inhibitor of or- ganellar protein synthesis [Lam et al., 19891. Light- activation of the transgene Cab-1 in tobacco was inhibited by cycloheximide but when the 35s CaMV viral promoter was fused to the Cab-1 coding region no inhibition was observed. Thus, cycloheximide exerts its effect at the transcriptional level by preventing the light-activated response. In contrast, the down-regulation of a light-deactivated gene, protochlorophyllide reductase, which is also controlled by phytochrome, was unaffected by cycloheximide. The data suggest that phytochrome regulation in the case of Cab and rbcS is mediated by a labile protein factor synthesized on cytoplasmic ribosomes.

Cab regulation displays dual light and circadian rhythm control in mature leaves of many plant species [Giuliano et al., 1988a; Piechulla, 19881. Circadian control implies that an endogenous clock, continuously entrained by diurnal events, is responsible for the precise timing of biological events. Its effect on Cab expression in tomato is especially prominent and RNA levels were found to oscillate over 20-fold. The oscillations, in both RNA accumulation and transcription rate, continued with diminishing amplitude in darkness for at least 3 days [Giuliano et al., 1988al. This indicates that the circadian clock is capable of inducing light-independent activation of Cab. Wheat plants also exhibited circadian control of the Cab-1 gene. Furthermore, when the wheat Cab-1 gene was transferred to tobacco the circadian regulation is maintained [Nagy et al., 19881. Fusion of the Cab-1 upstream region to the bacterial chloramphenicol acetyl transferase gene also resulted in oscillating amounts of transcripts. Con- versely, the chimeric gene containing the constitutively expressed 35s promoter joined to the Cab-1 coding region was not under circadian control. This indicates that the circadian clock is exerting its influence a t the transcriptional level. The Cab-1 transgene included 1,800 bp of the upstream region thus delin- eating within that region the cis-regulatory elements responsible for circadian control.

DEFINING CIS-REGULATORY DNA ELEMENTS WITH TRANSGENES

At the molecular level the most detailed work in elu- cidating cis-regulatory DNA elements has focused on the nuclear gene families encoding Cab and rbcS. Chi- meric rbcS transgenes have demonstrated regulation at the transcriptional level. A 280 bp enhancer-like element (-330 to -50 relative to the transcription start) conferred both light-induction and organ-specificity [Fluhr et al., 1986~1. Two highly conserved sequences within this region (boxes I1 and I11 around -150)

DARK nn nn

Ill' I I' II 1 Ill w w ww

Fig. 2. Model for activation of rbcS-3A genes. Box I1 is GTGTG- GTTAATATG between -151 and -138. Box I11 is ATCATWTCACT between -125 and -114. Box III* is CAnTACACT between -257 and -248. Box 11* is GTGAGGTAATAT between -224 and -213. Negative elements symbolized as circles bind to the LREs in the dark. An activation complex of which the nuclear factor GT-1 is a part can bind in the light when the negative elements are removed. Full regulated activation requires that the complex interacts with more than one LRE and probably with TATA elements.

which show strong homology to the constitutive enhancer elements from simian virus 40 and adenovirus were shown to bind a nuclear factor called GT-1 (Fig. 2) [Green et al., 19871. This factor also binds to farther upstream region boxes 11* and III*, which show homology to boxes I1 and 111. In addition, the farther upstream regions act as conditionally redundant enhanc- ers and will be discussed later. Another nuclear protein-binding factor isolated from seedlings of tomato and Arabidopsis binds to pea, tomato, and Ara- bidopsis rbcS upstream regions in an area called the G box [Giuliano et al., 1988bl. The concensus sequence is TCTTACACGTGGCAYY where Y is a pyrimidine. In- terestingly, the factor showed tissue-specificity and was not detected in roots. It also showed light-dependent alteration in its migration in gel retardation experiments. The area of binding is distinct from GT-1 and falls in a region (around -220 of rbcS-3A) that has redundant enhancer capacity [Kuhlemeier et al., 1988bl.

Detailed transgene analysis showed that the core enhancer sequences were capable of depressing the expression of the 35s constitutively expressed gene [Kuhlemeier et al., 19871. Hence these light-regulatory elements (LREs) can function as negative elements. Analysis of 5' deletion mutants showed a steep drop in transgene activity when sequences between - 166 and - 149 were removed (compare test and reference genes in Fig. 1, lanes 2 and 3) [Kuhlemeier et al., 19871. In addition, site-specific mutagenesis within the core enhancer motif showed that base pair substitutions in boxes I1 and I11 abolished transcriptional activity in a 175-size promoter fragment, implying that negative and positive transcriptional elements physically over- lap [Green et al., 1988; Kuhlemeier et al., 1988bl. Al- though the core sequence by itself binds GT-1 and its

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integrity is essential for gene activation it cannot mo- tivate transcription as an independent unit. The core region needs flanking upstream and downstream sequences to be operational, at least in the positive sense. As mentioned above, the core unit itself does exhibit activity in the negative sense and confers light-induced down-regulation.

The fact that GT-1 is present, judged by in vitro binding assays, in both light and dark grown extracts has led to the following model hypothesis (Fig. 2). GT-1 might be an activator that is present in light- and dark- treated leaves. However, in vivo i t binds I1 and I11 only in the light. In the dark GT-1 binding is blocked by a repressor. Hence it is repressor binding that modulates light sensitivity. This hypothetical repressor has never been detected in vitro. An additional part of the model maintains that GT-1 should interact with an another factor, as GT-1 binding and gene activation are not synonymous. One candidate for this interaction would be a TATA factor. As will be elaborated on latter, there is ample evidence that rbcS TATA box functions can complement upstream rbcS regions [Kuhlemeier et al., 19891. Finally, among those additional factors should be labile protein components, which as discussed in the previous section are critical for light activation.

Cab transgenes, isolated from pea and wheat [Simp- son et al., 1986; Nagy et al., 19871, were shown to contain positive light-regulatory sequences within the first 400 bp. A silencer region was also defined for the pea gene [Simpson et al., 19861. More extensive analysis of the Cab-E gene from N . plumbaginifolia localized three positive and one negative cis-acting elements [Castresana et al., 19881. Sequences similar to pea rbcS boxes I1 and I11 are present immediately upstream of the G box in the tobacco Cab-E gene, just as they are present immediately upstream of the G box in rbcS genes as well. In Cab-E this region was found to be essential for transgene expression. The data suggest that the G box sequences may be functionally equivalent. However, if these homologous sequences do play a part in light control, it should be kept in mind that additional factors are involved, as Cab and rbcS genes show very different light requirements for activation. A novel type of negative regulatory element was localized farther upstream in an AT-rich region (between -1182 and -747). It differs from the other regulatory elements in that it depresses the amount of transgene transcript in the light as well [Castresana et al., 19881. Analysis of elements close to the TATA region revealed a repeating GATA motif in Cab genes from many dicotyledonous plants. Mutagenesis of this motif caused a fivefold decrease in petunia Cab transgenes [Gidoni et al., 19891.

POST-TRANSCRIPTIONAL REGULATION OF NUCLEAR PHOTOSYNTHETIC GENES

The complex biosynthetic pathway by which a nuclear gene product is finally expressed in the chloro-

plast invites ample opportunities for regulation. When greening Amaranth (A. hypochondriacus) seedlings are placed in the dark there is an abrupt cessation of rbcS polypeptide synthesis. The presence of functional RNA remains unchanged [Berry et al., 19881. Trans- gene analysis has contributed much to our understanding of these and other post-transcriptional processes as well.

The role of RNA stability in the regulation of nuclear genes involved in photosynthesis has been recently described in tobacco transgenic plants containing a pea ferrodoxin 1 gene [Fed-1, Elliot et al., 1989a,bl. Trans- genes that contain the light-insensitive 35s constitutive viral promoter fused to the Fed-1 coding region exhibit light-induced accumulation. The light effect on the absolute levels of expression and the ratio of light- to-dark activity was indistinguishable from transgenic plants carrying the isolated full-length pea Fed-1 gene (-2,000 to + 1,600). The reciprocal construct which contained the Fed-l upstream region linked to the GUS marker showed constitutive accumulation. The simplest explanation hypothesizes that light-regulation can be accounted for by a constitutive rate of Fed-l transcription modulated by post-transcriptional stability. This possibility is consistent with the photo- physiology and promoter structure of Fed-1 as exem- plified in the following experiments. “Run-on” transcripts from isolated pea nuclei showed light-activation of both rbcS and Cab genes but not Fed-1 whose level remained unchanged [Sagar et al., 19881. In addition, Fed-1 mRNA showed accumulation earlier than other light-regulated genes following phytochrome activation, which is consistent with constitutive gene expression [Kaufman et al., 19851. Despite a certain amount of sequence homology in the 5‘ upstream region between Fed-1 and other light-induced genes (especially pea rbcS-SA), the binding of nuclear extracts from light-grown pea seedlings to Fed-1 could not be effectively competed by rbcS promoter sequences [Elliot et al., 1989al. I t remains to be seen whether other aspects of Fed-1 gene regulation, such as cellular and tissue specificity, show similar post-transcriptional control.

NUCLEAR AND PLASTID GENOME INTERACTION DURING DEVELOPMENT

It is generally accepted that the developmental state of the cell, as dictated by nuclear gene activity, can determine the developmental state of the plastid. How- ever, the influence that plastid development can have on nuclear gene expression is more obscure. The fact that pleiotropic chloroplast-based mutations which completely abolish chloroplast function have little effect on general plant morphology argues for limited interaction during the early stages of leaf development [Taylor, 19891. The following mutant analysis illus- trates this point. The maize nuclear mutation iojap which induces maternally inherited plastid defects

TRANSGENES OF NUCLEAR ENCODED CHLOROPLAST PROTEINS 201

yields plants with undeveloped proplastid-like organelles devoid of ribosomes. In certain iojap mutations the phenotypic effect is limited to leaf stripping with no apparent morphological aberrations in the leaves. However, in plants homozygous for iojap aborted embryos accumulate. This observation may re- flect the participation of organelles in embryogenesis [Coe et al., 19881. In dicotyledonous plants such as tobacco, pleiotropic chloroplast mutations induced by mutagens contain undeveloped proplastids and result in albino leaves of normal morphology [Fluhr et al., 19851.

The phenotypes described above refer to nucleus- plastid interaction at early stages of organ development. Nuclear genes that code for photosynthetic components are generally active a t later stages of leaf development. Their activity is correlated with plastid differentiation to chloroplasts which takes place after basic leaf morphology has been determined. To account for this synchronism a regulatory feedback loop might exist between the mature chloroplast and the nucleus. Another possibility is that nuclear gene expression runs out a pre-programmed course of events. Both mutant plants and herbicides have been used to resolve this point. Mutations in barley plants that effect carotenoid accumulation and subsequently bleach chloroplasts, in a reversible way, caused a depression of Cab gene activation as measured directly in nuclear “run- on” experiments. Other photosynthetic genes such as rbcS or the protochlorophyllide reductase were relatively unaffected [Batschauer et al., 19861. Similar results were obtained in carotenoid mutants of maize, but not in maize double mutants containing both carotenoid and chlorophyll deficiencies, indicating the part chlorophyll photooxidation plays in the process [Bur- gess and Taylor, 19881. Application of the herbicide Norflurozon mimics the mutants of carotenoid biosyn- thesis and similarly results in Cab gene inactivation in many plant species [Schuster et al., 1988; Batschauer et al., 19861. These results imply the existence of a plastidic factor, destroyed during photooxidation, whose activity is necessary for the maintenance of Cab expression [Taylor, 19891.

The existence of plastidic factors has been examined in transgenic plants. Transformed N . tabacum shoots containing the chimeric constructs of Cab and rbcS promoter regions and the bacterial neomycin phos- photransferase I1 gene (NPT 11) show light-regulated accumulation of NPT(I1) enzymatic activity [Simpson et al., 19861. In the presence of Norflurozon the enzymatic activity was greatly diminished. The photooxidative damage generated in the presence of Norfluro- zon was reversible, and during a period of 10 days after the removal of the herbicide, chloroplasts recovered their normal morphology and function. However, the detection of NF’T(I1) activity preceded the plastid recovery demonstrating that fully mature chloroplasts are not essential to trigger activation of the two pho-

tosynthetic genes. The fact that rbcS activity as well as Cab was affected may be a result of species-specific differences in gene regulation between barley and tobacco. In this respect the transcription of tomato rbcS genes in a tomato carotenoid deficient mutant was similarly blocked by photooxidation [Giuliano and Scolnik, 19881.

Due to the nature of plant development, growing plants offer the complete milieu of developmental steps. The chloroplast matures from a poorly defined proplastid stage in the apical meristem to the fully differentiated form in the mature leaf. Examination of chimeric constructs in the various tissues enables cor- relative conclusions a t each developmental stage. Ac- tivity of photosynthetic transgenes has confirmed their tissue specificity down to the cellular level. The pea rbcS-3A and tobacco Ntss23 small subunit promoter was shown to confer cell-specific expression on chimeric reporter genes [Jefferson et al., 1987; Aoyagi et al., 19881. In these cases all cells with chloroplasts expressed the reporter gene including chloroplast- containing guard cells situated in the leaf epidermis which is otherwise devoid of all photosynthetic functions. The photosynthetic gene ST-LS1 encodes a 10 kD polypeptide component of the water-oxidizing complex of photosystem 11 [Lautner et al., 19881. Upstream promoter regions of ST-LS1 motivated light-activation of P-glucoronidase reporter gene in green potato tissue [Stockhaus et al., 19891. However, P-glucuronidase activity was not only limited to tissue that normally contains chloroplasts but was also present in green roots. These results emphasize that the developmental stage of the plastid is the primary determinant in ST-LS1 expression and not the final morphological state of the plant cell. They also demonstrate the plasticity of plant gene regulation which parallels the plasticity in plant development. The activity of photosynthetic genes is associated with developed leaves, but is not subject to irreversible inactivation during the plant’s differentiation to other tissues. In some ways this phenomenon parallels recent observations on the regulation of patatin. Patatin, which is a tuber-specific storage protein, has been found to accumulate in leaves under certain metabolic conditions [Rocha-Sosa et al., 19891. The gen- erality of this phenomenon with respect to the regulation of other plant genes remains to be seen.

CONDITIONAL ENHANCER REDUNDANCY

The use of transgenic plants has underlined the existence of conditional redundancy in the pea rbcS genes. Normally, constructs containing deletions of promoter upstream regions to position - 166 are indistinguishable in their light-regulation from those containing full-length promoters (Fig. 1). This implies that the areas downstream from -166 are sufficient for light regulation in leaves. However, when the sequences downstream from - 166 were deleted the gene

AND TISSUE-SPECIFICITY OF LREs

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remained fully reactive to light as long as upstream regions between -166 and -410 were also present. This observation implied redundancy of light-regulatory units between areas upstream and downstream from position -166 [Kuhlemeier et al., 1987, 1988bI. Gene activity in transgenic tobacco plants containing the -166 construct and a normal pea rbcS reference gene was systematically examined in other leaf developmental stages [Kuhlemeier et al., 1988bl. The expression of the deleted -166 promoter relative to the full-length promoter was markedly lower in young leaves and greening cotyledons. Taken together the data show that redundancy is not an absolute inherent property but is influenced by the stage of development. In mature leaves this deletion is fully active and the distal region is functionally redundant. In immature leaves deletion of the distal regions severely retards gene expression. A possible explanation for this phenomenon is based on the fact that both promoter regions contain areas of sequence homology (Fig. 2). A certain amount of in vitro binding cooperativity exists between the light-regulatory elements [Green et al., 1987, 19881. In the developing leaf and greening coty- ledon these factors may be limiting and the presence of additional cis-regulatory elements would offer an ad- vantage in the competition for scarce nuclear factors.

The complexity of the rbcS promoter was further il- lustrated in transgenic plants containing the - 166 rbcS-3A mutant promoter. As noted above this deletion retains high-level light-regulated expression in mature leaves. However, when additional regions comprising the TATA box area (-50 to + 15) were mutated to con- form to the TATA box sequence present in the 35s promoter, the expression of the -166 deletion was severely reduced in mature leaves. A deletion of -189 that contained the same mutant 35s-like TATA box retained wild-type activity [Kuhlemeier et al., 19891. It follows that the cognate rbcS-3A TATA region can complement a function of the upstream region (Fig. 2). The regulatory contribution of the rbcS-3A TATA area was highlighted when it was fused to what is consid- ered a weak light-insensitive test enhancer, the heat- shock element. The chimeric gene, i.e., heat shock element rbcS-3A TATA box and CAT reporter gene, without any additional light-regulatory upstream regions, displayed a high degree of light-activation [Kuhlemeier et al., 19891, a result which extends conclusions drawn from previous experiments conducted on transformed calli [Timko et al., 1985; Morelli et al., 19851. Unexpectedly this construct was found to be active in the root organs of plants grown in sterile tip culture. It was thus possible to separate light regulation from organ specificity. A similar construct containing the complete upstream rbcS-3A region fused to the heat-shock element retained both light-dependent response and organ-specificity [Strittmatter and Chua, 19871. This leads to a hypothesis that two types of light-regulatory elements exist, one that confers both

light- and organ-specificity (LREs in the upstream region), and another that confers only light-regulation (LRE in the TATA box region).

ORGANELLE TARGETING AND ASSEMBLY OF NUCLEAR PHOTOSYNTHETIC GENES IN

TRANSGENIC PLANTS A key development that allowed significant ad-

vances in our understanding of the transport of cytoplasmically synthesized polypeptides into chloroplasts and their intraorganellar targeting was the demon- stration that import could be reconstructed in vitro [Highfield and Ellis, 1978; Chua and Schmidt, 19781. Targeting of polypeptides to any of the six potential chloroplast compartments comprising membrane and membrane-enclosed aqueous interiors may use different strategies. Sequence information for stromal transport of rbcS has been localized to the N-terminal por- tion of the rbcS precursor [Van den Broeck et al., 1985; Lubben et al., 19881. N-terminal sequences mediate Cab chloroplast import, but intraorganellar sorting of the Cab protein is determined by sequences from within the mature protein [Hand et al., 19891. In contrast, targeting of the 33 kD oxygen evolving protein to the thylakoid lumen seems to involve sequences in a bipartite transit peptide [KO and Cashmore, 19891.

For the large part the analysis of protein import has been done in isolated chloroplasts. However, when in vivo and in vitro data are compared the conclusions are not always identical. Wasmann et al. [1986] found that the N-terminal rbcS transit sequence was sufficient for transport of the NptII protein in isolated chloroplasts. However, highly efficient transport was achieved by inclusion of an additional 23 amino acids of the mature rbcS polypeptide. When transgenic plants containing the identical constructs were analyzed the opposite results were obtained [Kuntz et al., 19861. A possible explanation of this discrepancy may lie in the different relevant time frames of the two experimental ap- proaches. It is possible that due to the longer period of time in which these experiments are carried out en- hanced turnover of an unstable fused polypeptide (23 amino acids of rbcS and the coding sequence for NptII) would critically reduce the steady-state amounts of import protein in transgenic plants. Import necessitates states of folding and unfolding during transport. In addition the proper assembly of polypeptides into multi- meric structures is critical for their stabilization. It is recognized that auxiliary scaffolding proteins, called molecular chaperones, are essential for proper function of these processes. In the case of rbcS the chaperones are themselves cytoplasmically coded and imported into the chloroplast [Ellis and Hemmingsen, 19891. The dependency of other imported chloroplast proteins on these functions is unknown. However, the utility of in vitro systems will be limited by the stability and cat- alytic turnover of these components.

TRANSGENES OF NUCLEAR E

CONCLUSIONS Meaningful study of nuclear genes involved in pho-

tosynthesis necessitates work in transgenic plants. This is mainly due to the fact that expression of these genes is well correlated with chloroplast development. The transgene system has uncovered some of the re- markable complexity of plant promoters. They include tissue-specificity, light-regulatory elements, light- independent circadian control, conditional redundancy, and silencers all finely tuned by developmental processes. Undoubtedly there is a need to broaden the types of gene families studied so that a general picture of their regulation can emerge. However, the next ma- jor challenge will focus on the interrelationship of cis elements and their trans-acting factors. The study of these elements and factors demands the development of novel cell-free systems that can faithfully tease apart the complexities of gene activation.

ACKNOWLEDGMENTS The author gratefully acknowledges the support of

the Jack Goodman Career Development Chair.

REFERENCES Aoyagi K, Kuhlemeier C, Chua N-H (1988): The pea rbcS-3A en-

hancer-like element directs cell-specific expression in transgenic tobacco. Mol Gen Genet 213:179-185.

Batschauer A, Mosinger E, Kreuz K, Dorr I, Ape1 K (1986): The im- plication of a plastid-derived factor in the transcriptional control of nuclear genes encoding the light-harvesting chlorophyll aib protein. Eur J Biochem 154:625-634.

Berry JO, Cam JP, Klessig DF (1988): mRNAs encoding ribulose- 1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness. Proc Natl Acad Sci USA 85:4190-4194.

Burgess DG, Taylor WC (1988): The chloroplast affects the transcription of a nuclear gene family. Mol Gen Genet 214:89-96.

Castresana C, Garcia-Luque, Alonso E, Malik VS, Cashmore AR (1988): Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbugin- ifolia. EMBO 7:1929-1936.

Chua N-H, Schmidt GW (1978): Post-translational transport into in- tact chloroplasts of a precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase. Proc Natl Acad Sci USA 75: 6110-6114.

Coe EH J r , Thompson D, Walbot V (1988): Phenotypes mediated by the iojap genotype in maize. Am J Bot 75:634-644.

Dean C, Pichersky E, Dunsmuir P (1989): Structure, evolution and regulation of rbcS genes in higher plants. Annu Rev Plant Physiol Plant Mol Biol 40:415-439.

Elliot RC, Pederson TJ, Fristensky B, White MJ, Dickey LF, Thompson WF (1989a): Characterization of a single copy gene encoding ferredoxin 1 from pea. Plant Cell 1581-690.

Elliott RC, Dickey LF, White MJ, Thompson WF (1989b): Cis-acting elements for light regulation of pea ferredoxin 1 gene expression are located within transcribed sequences. Plant Cell 1591-698.

Ellis RJ, Hemmingsen SM (1989): Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures. TIBS 14:339-342.

Flores S, Tobin EM (1988): Cytokinin modulation of LHCP mRNA levels: the involvement of post-transcriptional regulation. Plant Mol Bio 11:409-415.

NCODED CHLOROPLAST PROTEINS 203

Fluhr R, Aviv D, Galun E, Edelman M (1985): Efficient induction and selection of chloroplast-encoded antibiotic-resistant mutants in Nicotzana. Proc Natl Acad Sci USA 82:1485-1489.

Fluhr R, Moses P, Morelli G, Coruzzi G, Chua N-H (1986a): Expres- sion dynamics of the pea rbcS multigene family and organ distri- bution of the transcripts. EMBO J 52063-2071.

Fluhr R, Kuhlemeier C, Nagy F, Chua NH (1986~): Organ-specific and light-induced expression of plant genes. Science 232: 1106-1112.

Fluhr R, Chua N-H (1986b): Developmental regulation of two genes encoding ribulose-bisphosphate carboxylase small subunit in pea and transgenic petunia plants: Phytochrome response and blue- light induction. Proc Natl Acad Sci USA 83:2358-2362.

Gidoni D, Brosio P, Bond-Nutter D, Bedbrook J , Dunsmuir P (1989): Novel cis-acting elements in Petunia Cab gene promoters. Mol Gen Genet 215337-344.

Giuliano G, Hoffman NE, KO K, Scolnik PA, Cashmore AR (1988a): A light-entrained circadian clock controls transcription of several plant genes. EMBO 7:3635-3642.

Giuliano G, Pichersky E, Malik BS, Timko MP, Scolnik PA, Cash- more AR (1988b): An evolutionarily conserved protein-binding sequence upstream of a plant light-regulated gene. Proc Natl Acad Sci USA 85:7089-7093.

Giuliano G, Scolnik P (1988): Transcription of two photosynthesis- associated nuclear genes families correlates with the presence of chloroplasts in leaves of the variegated tomato ghost mutant. Plant Physiol 86:7-9.

Green PJ, Kay SA, Chua N-H (1987): Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene. EMBO J 622.543-2549.

Green PJ, Yong MH, Cuozzo M, Kano-Murakami Y, Silverstein P, Chua N-H (1988): Binding site requirements for pea nuclear protein factor GT-1 correlate with sequences required for light-dependent transcriptional activation of the rbcS-3A gene. EMBO J 7:4035- 4044.

Hand JM, Szabo LJ, Vasconcelos AC, Cashmore AR (1989): The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal targeting sequence. EMBO J 8:3195- 3206.

Highfield PE, Ellis R J (1978): Synthesis and transport of the small subunit of chloroplast ribulose bisphosphate carboxylase. Nature 271:420-424.

Jefferson RA, Kavanagh TA, Bevans MN (1987): GUS fusions: p- glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 63901-3908.

Jones JDG, Dunsmuir P, Bedbrook J (1985): High-level expression of introduced chimeric genes in regenerated transformed plants. EMBO J 4:2411-2418.

Kaufman LS, Thompson WF, Briggs WR (1984): Different red light requirements for phytochrome accumulation of cab RNA on rbc S RNA. Science 226:1447-1449.

Kaufman LS, Briggs WR, Thompson WF (1985): Phytochrome control of specific mRNA levels in developing pea buds. Plant Physiol 78: 388-393.

KO K, Cashmore AR (1989): Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33kd oxygen-evolving protein. EMBO J 8:3187-3194.

Kuhlemeier C, Fluhr R, Green PJ, Chua N-H (1987): Sequences in the pea rbcS-3A gene have homology to constitutive mammalian en- hancers but function as negative regulatory elements. Genes Dev 1:247-255.

Kuhlemeier C, Fluhr R, Chua N-H (1988a): Upstream sequences determine the difference in transcript abundance of pea rbc S genes. Mol Gen Genet 212:405-411.

Kuhlemeier C, Cuozzo M, Green PJ, Goyvaerts E, Ward K, Chua N-H (198813): Localization and conditional redundancy of regulatory elements in rbcS-3A, a pea gene encoding the small subunit of ribulose-bisphosphate carboxylase. Proc Natl Acad Sci USA 854662- 4666.

Kuhlemeier C, Strittmatter G, Ward K, Chua N-H (1989): The pea

204 FLUHR

rbcS-3A promoter mediates light-responsiveness but not organ- specificity. Plant Cell 1:471-478.

Kuntz M, Simons A, Schell F, Schreier, PH (1986): Targeting of protein to chloroplasts in transgenic tobacco by fusion to mutated transit peptide. Mol Gen Genet 205:454-460.

Lam E, Green PJ, Wong M, Chua N-H (1989): Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis. EMBO J 8:2777-2783.

Lautner A, Klein R, Ljungberg U, Reilander H, Bartling D, Anders- son B, Reinke H, Beyreuther K, Herrmann RG (1988): Nucleotide sequence of cDNA clones encoding the complete precursor for the “10-kDa” polypeptide of photosystem I1 from spinach. J Biol Chem 263:10077-10081.

Lubben TH, Theg SM, Keegstra K (1988): Transport of proteins into chloroplasts. Photosyn Res 17:173-194.

Manzzara T, Gruissem W (1988): Organization and expression of the genes encoding ribulose-1,5-bisphosphate carboxylase in higher plants. Photosyn Res 16:117-139.

Morelli G, Nagy F, Fraley RT, Rogers SG, Chua N-H (1985): A short conserved sequence is involved in the light inducibility of a gene encoding ribulose 1,5-bisphosphate carboxylase small subunit of pea. Nature (London) 315:200-204.

Nagy F, Morelli G, Fraley RT, Rogers SG, Chua N-H (1985): Photo- regulated expression of a pea rbc S gene in leaves of transgenic plants. EMBO J 4:3063-3068.

Nagy F, Boutry M, Hsu M-Y, Wong M, Chua N-H (1987): The 5’- proximal region of the wheat Cab-1 gene contains a 268-bp enhancer-like sequence for phytochrome response. EMBO J 6:2537- 2542.

Nagy F, Kay SA, Chua N-H (1988): A circadian clock regulates transcription of the wheat Cab-1 gene. Genes Dev 2:376-382.

leaves-diurnal and circadian rhythms during extended dark and light periods. Plant Mol Biol 11:345-353.

Rocha-Sosa M, Sonnewald U, Frommer W, Stratmann M, Schell J, Willmitzer L (1989): Both developmental and metabolic signals ac- tivate the promoter of a class I protein gene. EMBO J 8:23-29.

Sagar AD, Horwitz BA, Elliott RC, Thompson WF, Briggs WR (1988): Light effects on several chloroplast components in norflurazon- treated pea seedlings. Plant Physiol 88:340-347.

Schuster C, Oelmiiller R, Bergfeld R, Mohr H (1988): Recovery of plastids from photooxidative damage: Significance of a plastidic factor. Planta 174:289-297.

Simpson J, Van Montagu M, Herrera-Estrella L (1986): Photosynthe- sis-associated gene families: differences in response to tissue-specific and environmental factors. Science 233:34-38.

Stockhaus J , Schell J , Willmitzer L (1989): Correlation of the expression of the nuclear photosynthetic gene ST-LS1 with the presence of chloroplasts. EMBO J 8:2445-2451.

Strittmatter G, Chua N-H (1987): Artificial combination of two cis- regulatory elements generates a unique pattern of expression in transgenic plants. Proc Natl Acad Sci USA 84:8986-8990.

Taylor WC (1989): Reglatory interactions between nuclear and plastid genomes. Annu. Rev Plant Physiol Plant Mol Biol 40:211-233.

Timko MP, Kausch AP, Castresana C, Fassler J , Herrera-Estrella L (1985): Light regulation of plant gene-expression by an upstream enhancer-like element. Nature 318:579-582.

Van den Broeck G, Timko MP, Kausch AP, Cashmore AR, Van Mon- tagu M, Herrera Estrella L (1985): Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. Nature 313:358-363.

Wasmann CC, Reis B, Bartlett SG, Bohnert HS (1986): The importance of the transit peptide and the transport protein for protein import into chloroplasts. Mol Gen Genet 205446-453. Piechulla B (1988): Plastid and nuclear mRNA fluctuations in tomato

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Regulation of nuclear genes encoding chloroplast proteins in transgenic plants