Alfred Batschauer

Molecular Interaction between COP1 and HY5 Defines a Regulatory Switch for Light Control of Arabidopsis Development

Arabidopsis COP1 acts as a light-inactivable repressor of photomorphogenic development, but its molecular mode of action remains unclear. Here, we show that COP1 negatively regulates HY5, a bZIP protein and a positive regulator of photomorphogenic development. Both in vitro and in vivo assays indicate that COP1 interacts directly and specifically with HY5. The hyperphotomorphogenic phenotype caused by the over-expression of a mutant HY5, which lacks the COP1-interactive domain, supports the regulatory role of HY5-COP1 interaction. Further, HY5 is capable of directly interacting with the CHS1 minimal promoter and is essential for its light activation. We propose that the direct interaction with and regulation of transcription factors by COP1 may represent the molecular mechanism for its control of gene expression and photomorphogenic development.

Cloning and characterization of a chalcone synthase gene from mustard and its light-dependent expression.

Genomic DNA from mustard was cloned in EMBL4 and screened for chalcone synthase (CHS) genes using a heterologous cDNA probe from parsley. Two clones which hybridized with the parsley cDNA probe were isolated. They showed different restriction patterns. One clone was sequenced and identified as a CHS gene by sequence comparison with published CHS sequences. The sequence of the coding region is 1188 bp, and encodes a protein of 43 kDa. The startpoint oas determined by primer extension. The sequence of 0.9 kbp at the 5′ end of the transcription start and part of the noncoding 3′ of this gene were also determined. The coding sequence is interrupted by a single intron of 523 bp. The coding and the noncoding 5′ sequence of this gene was compared with CHS genes from other species. A very high homology was found with the Arabidopsis CHS coding region. A sequence motif (CACGTGT) which is present in most rbcS and all CHS upstream regions, and which specifically binds a protein factor from plant nuclear extracts, is also present in the upstream region of the mustard CHS gene.Measurements of CHS transcript levels show that phytochrome controls expression of this gene in cotyledons of mustard seedlings; however, blue/UV-light photoreceptors control expression in later stages of development.

Promoter elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants

The expression of chalcone synthase (CHS) genes, which encode the first enzyme of the flavonoid pathway, is under developmental control as well as affected by external stimuli such as light. Varying fragments of the 1 kb upstream region of the CHS1 gene from white mustard (Sinapis alba L.) were fused to the GUS-coding region, and the light-regulated expression of these constructs was analysed in transgenic Arabidopsis and tobacco plants. Studies performed with Arabidopsis seedlings indicate the presence of two elements within the CHS1 promoter mediating light responses via different photoreceptors. One element, located about 150 bp upstream of the transcription start site, is homologous to Unit 1 of the parsley CHS gene, the second, far more upstream element carries sequences similar to Unit 2 of the same gene. Detailed studies on Unit 1-driven expression indicate that this element transfers the expression characteristics of the original gene to both Arabidopsis and tobacco. Although the expression characteristics of Unit 1 are indistinguishable from those of the full-length promoter within the same species, we observed differences in mustard CHS promoter regulation between Arabidopsis and tobacco plants transgenic for the identical construct. The difference in photoreceptor usage by the same promoter element in different transgenic species (Unit 1 from mustard in Arabidopsis vs. tobacco) was also observed for different but homologous promoter elements in the same transgenic species (Unit 1 from mustard and parsley in tobacco). We therefore conclude that the same promoter and even the same promoter element (Unit 1) can mediate different spatial patterns of expression and modes of light regulation in different transgenic species.

Coaction of blue light and light absorbed by phytochrome in control of glutamine synthetase gene expression in Scots pine (Pinus sylvestris L.) seedlings

The level of plastidic glutamine synthetase (GS; EC 6.3.1.2) in the cotyledonary whorl of the Scots pine (Pinus sylvestris L.) seedling was previously reported to be regulated by light. In the present paper we report on the control by light of the GS transcript level. A full-length GS cDNA clone of Scots pine was isolated (pGS1), sequenced and employed to measure GS transcript levels. Using dichromatic light treatments it was found that the transcript level is regulated by phytochrome. The strong specific effect of blue light is to be attributed to an increase of the responsiveness to phytochrome. Since no direct correlation between the transcript level and the rate of GS protein synthesis was observed, it was concluded that GS gene expression is only coarsely regulated at the level of transcript accumulation. Synthesis of GS protein is by itself light-dependent (light-mediated fine tuning of gene expression). This control at the translational level is also exerted via phytochrome with blue light determining the reponsiveness of the process toward phytochrome. If the level of the far-red absorbing form of phytochrome (Pfr) is kept very low, blue light is not capable of bringing about synthesis of GS protein.

The molecular biology of photoregulated genes

In the last two decades a great deal of data has accumulated which shows that gene expression is very often under phytochrome control during de-etiolation and re-etiolation of young seedlings (for recent reviews see chapters in: Thomas and Johnson 1991). It is also well documented that some photomovements (polarotropism of some ferns and mosses and chloroplast orientation in the alga Mougeotia) are under phytochrome control (Chapters 7.2, 9.4 and 9.8). In these cases an action dichroism can be demonstrated which has been taken as an indication that phytochrome may act as a membrane effector. Wagner and his colleagues (1984) and Serlin and Roux (1984) have demonstrated the involvement of Ca2+, calmodulin and actin filaments in the signal transduction process between the far-red light (FR)-absorbing form of phytochrome (Pfr) and chloroplast turning. Phytochrome localization (Chapter 4.5), photomovement (Chapter 9.4), and phytochrome and membranes (Chapter 4.6), will be discussed elsewhere in this book. Here we will concentrate on the question as to what is known about the mode of phytochrome action on gene expression. Before treating this problem, some general kinetic properties of phytochrome, signal transduction and underlying developmental programs will be discussed.

Cis-acting elements of the CHS1 gene from white mustard controlling promoter activity and spatial patterns of expression

Chalcone synthase (CHS) catalyses the first regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. External stimuli such as stress, light and wounding induce CHS expression that is both tissue-specific and under developmental control. In order to identify cis-acting elements involved in organ and tissue specifity, we fused varying parts of the CHS1 promoter of white mustard (Sinapis alba L.) to the GUS-coding region and analysed the expression of these constructs in stably transformed Arabidopsis plants. Two different stages of development were examined, seedlings as an early stage and flowers as the final stage of development. In seedlings, the full-length promoter showed expression in all organs except the hypocotyl; in flowers expression could be observed in all whorls. Unit 1 of the mustard CHS1 promoter, an element conserved in several CHS genes, which has been recently identified as a light responsive element, is able to mediate a tissue-specific expression pattern similar to that obtained with the full-length promoter in seedlings as well as in flowers. Other elements enhance or repress expression in combination with Unit 1, or mediate defined spatial expression independently of Unit 1. One such element, located between-907 and -655, directs expression similar to that of the full-length promoter in flowers but not in seedlings and differs therefore in function to Unit 1. Our data suggest a dominant regulation of CHS1 expression by Unit 1. Other elements within this promoter might interact with Unit 1 or confer a subset of spatial expression patterns when Unit 1 is deleted.

Gene and cDNA for plant cytochrome P450 proteins (CYP72 family) from Catharanthus roseus, and transgenic expression of the gene and a cDNA in tobacco and Arabidopsis thaliana

Abstract Genomic and cDNA libraries from Catharanthus roseus (Madagascar periwinkle) cell cultures were screened with cDNA Cros2 (CYP72 family) which codes for an abundant cytochrome P450 in the cells. A nearly full-length cDNA clone for the previously characterized partial cDNA Crosl and a gene ( Cros3 , the third member of the family) were characterized. The gene contains five exons and four introns. The last two exons encode the four regions A to D which are conserved in many P450 proteins, and region B is split by an intron. Gene Cros3 and cDNA Cros2 , both under control of the 35S CaMV promoter, were introduced into tobacco (by direct gene transfer to protoplasts) and into Arabidopsis thaliana (by Agrobacterium -mediated gene transfer). Immunoblots showed that proteins of the correct size were expressed. Enzyme assays performed to identify the function of the proteins failed to detect hydroxylase activity with eleven substrates for P450-dependent reactions which are present in C. roseus cell cultures.

Unit 1 of the mustard chalcone synthase promoter is sufficient to mediate light responses from different photoreceptors

Abstract Chalcone synthase (CHS) expression in mustard is light regulated and depends on the action of different photoreceptors (i.e. phytochrome, blue and UV-light photoreceptors). It has been shown previously [1] that approximately 1 kbp of the promoter of the chs1 gene from mustard [2] is sufficient for light-induced expression. In this paper, we focus on which sequences mediate the light response and which photoreceptors are involved. With transient expression studies with parsley protoplasts, we have identified a single light-responsive element (LRE) within the mustard chs promoter with high similarity to Unit 1, one of two LREs of the parsley chs gene [3]. We have also demonstrated that Unit 1 of the mustard chs gene, when fused to the minimal 35S promoter from cauliflower mosaic virus (CaMV), responds to different photoreceptors (UV, blue, phytochrome), a response indistinguishable from that of the full-length promoter. Unit 1 therefore is sufficient to mediate light responses from three photoreceptors. Although Unit 1 from mustard and parsley chs genes show striking similarity in sequence and function, our studies indicate different in vitro binding to proteins from parsley cells. We conclude that the activation of different chs genes may occur by the use of different DNA-binding activities.

The presence and synthesis of the NADPH-protochlorophyllide oxidoreductase in barley leaves with a high temperature-induced deficiency of plastid ribosomes

High-temperature-induced deficiency of plastid ribosomes in barley plants (Hordeum vulgare L.) was used as a system for studying the role of the cytoplasm in the synthesis of the NADPH-protochlorophyllide oxidoreductase. The enzyme is present in 33° C-grown plants. The failure of high-temperature-grown plants to accumulate chlorophyll during illumination is not caused by the absence of the protochlorophyllide-reducing enzyme. The synthesis of the NADPH-protochlorophyllide oxidoreductase was studied by feeding [35S]methionine to the seedling and by following the incorporation of the radioactively labeled amino acid into plastid proteins. The NADPH-protochlorophyllide oxidoreductase was labeled in high-temperature-grown barley plants to the same extent as in control plants grown at 25° C. It is concluded that the 36,000-Mr polypeptide of the NADPH-protochlorophyllide oxidoreductase is synthesized outside the plastid on cytoplasmic 80S ribosomes.

Isolation and characterization of a gene encoding a chlorophyll a/b-binding protein from mustard and the targeting of the encoded protein to the thylakoid membrane of pea chloroplasts in vitro

Three independent clones carrying a mustard gene coding for the chlorophyll a/b-binding protein were isolated by screening a genomic library of mustard with a heterologous cDNA probe from pea. All of them encode the same CAB gene, which, as shown by sequence analysis and comparison with published CAB sequences, belongs to the family of type I PSII CAB genes, encoding a precursor protein of 266 amino acids. Several conserved sequence motifs are observed in the 5′ and 3′ non-coding region of the gene. The putative transcription start site could be localized to 60 bp upstream of SA-CAB1 initiator codon by S1 mapping.Plasmids were constructed which allow in vitro transcription and translation of the whole chlorophyll a/b-binding protein and of truncated species which lack increasing portions of the C-terminus. Whereas the in vitro import into pea chloroplasts is not affected by these C-terminal deletions, targeting to the thylakoid membrane is abolished by the removal of the C-terminal helical domain. Accordingly, the 54 amino acids which contain the C-terminal membrane-spanning helix and flanking regions is an essential component of the thylakoid targeting signal.