Transcription and somatic transposition of the maize En/Spm transposon system in rice.

Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision products and isolation of flanking genomic sequences in T0 and T1 progeny plants. Since the transposition efficiency appeared to be considerably lower than that observed in maize or in other heterologous systems like Arabidopsis, we examined En/Spm transcription and splicing in the transgenic rice plants. Northern analysis revealed the presence of transcripts encoding the active TnpA and TnpD transposases, with the latter predominating; this is the reverse of what is seen in maize and Arabidopsis. RT-PCR analysis confirmed the occurrence of correct splicing and the formation of the two other alternatively spliced transcripts (TnpB and TnpC), as previously described for maize. Two alternative splice donor sites at the end of exon 1 were identified in maize at positions 578 and 704. We observe that rice is similar to maize in that TnpA is preferentially spliced at position 578. We also show that in Arabidopsis splicing occurs preferentially at position 704, as in other dicots like tobacco. These observations indicate differences in the splicing of transcripts of the maize En/Spm element between dicot and monocot hosts. Nevertheless, the ratio in which the transcripts for the active transposases are produced seems to determine the efficiency of transposition, irrespective of the host considered. A limiting amount of TnpA might therefore be responsible for the lower transposition activity of En/Spm in rice. Alternatively, reduced mobility of the modified I/dSpm element used may have resulted from the absence of critical sequences necessary for transposition. The influence of endogenous, autonomous, En/Spm -related elements present in the rice genome on the transposition behaviour of the exogenous maize element is also considered.

Glucocorticoid-inducible gene expression in rice.

We have studied the use of a glucocorticoid receptor-based inducible gene expression system in the monocotyledonous model plant rice (Oryza sativa L.). This system, originally developed by T. Aoyama and N.-H. Chua [(1997) Plant J 11: 605-612], is based on the chimaeric transcriptional activator GVG, consisting of the yeast Gal4 DNA-binding domain, the VP16 activation domain and the glucocorticoid receptor domain. For application in rice, we designed an optimized binary vector series (pINDEX) and tested this with the beta-glucuronidase (gusA) reporter gene. GUS expression was tightly controlled and relatively low concentrations (1-10 microM) of the glucocorticoid hormone dexamethasone (DEX) were able to induce GUS activities to levels comparable to those conferred by the strong cauliflower mosaic virus (CaMV) 35S promoter. DEX was taken up efficiently by the roots of tissue-cultured plantlets or mature plants in hydroponic culture, and induced GUS activity throughout the whole plant. DEX-induced GUS expression patterns were consistent in all lines and their T1 progeny. The phenotype of tissue-cultured rice plantlets was not affected when inductions with 10-100 microM DEX were limited to 1-4 days or when 2-week inductions were performed with 1 microM DEX, which was already sufficient to reach near-maximal GUS activity. However, 2-week inductions with 10 microM DEX caused growth retardation and developmental defects. As the severity of these effects varied between different lines, we could select lines with a mild phenotype for future use as activator lines in crosses with 'target' plants.

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis.

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.

The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation.

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.

A role for the rice homeobox gene Oshox1 in provascular cell fate commitment.

The vascular tissues of plants form a network of interconnected cell files throughout the plant body. The transition from a genetically totipotent meristematic precursor to different stages of a committed procambial cell, and its subsequent differentiation into a mature vascular element, involves developmental events whose molecular nature is still mostly unknown. The rice protein Oshox1 is a member of the homeodomain leucine zipper family of transcription factors. Here we show that the strikingly precise onset of Oshox1 gene expression marks critical, early stages of provascular ontogenesis in which the developmental fate of procambial cells is specified but not yet stably determined. This suggests that the Oshox1 gene may be involved in the establishment of the conditions required to restrict the developmental potential of procambial cells. In support of this hypothesis, ectopic expression of Oshox1 in provascular cells that normally do not yet express this gene results in anticipation of procambial cell fate commitment, eventually culminating in premature vascular differentiation. Oshox1 represents the first example of a transcription factor whose function can be linked to specification events mediating provascular cell fate commitment.

Overexpression of Arabidopsis thaliana SKP1 homologues in yeast inactivates the Mig1 repressor by destabilising the F-box protein Grr1.

The timed destruction of cell cycle regulatory proteins is of key importance in controlling cell cycle progression in eukaryotes. Recently, Skp1 from yeast (Saccharomyces cerevisiae) was shown to play an important role in the ubiquitin-mediated proteolysis of these proteins via the Skp1-Cdc53-F-box (SCF) pathway. Here we describe the fortuitous cloning of cDNAs for two Skp1 homologues from the plant Arabidopsis thaliana on account of their ability to activate reporter gene expression in yeast directed by the cyt-1 element from the promoter of the Agrobacterium tumefaciens T-cyt gene, which is essential for expression of the gene in plants. This element is strikingly similar in sequence to the binding site for the yeast Migl protein, a transcriptional repressor of genes involved in the utilisation of carbohydrates other than glucose. We report that Mig1 protein binds to the cyt-1 element with similar specificity as a previously described plant nuclear protein factor, and that the cyt-1 element is a target for an unknown yeast transcriptional activator when Mig1 itself is inactivated. Interestingly, our data further indicate that A. thaliana Skp1 inactivates Mig1 by destabilising the yeast F-box protein Grr1, which is required for cyclin degradation and is thus involved in control of the cell cycle, and for glucose-regulated gene repression. Our results suggest that the plant counterpart of yeast Skp1 is probably also instrumental in ubiquitin-mediated proteolysis of specific proteins via an SCF-like pathway.

HD-Zip proteins of families I and II from rice: interactions and functional properties.

Proteins of the closely related homeodomain-leucine zipper (HD-Zip) families I and II in plants are putative transcription factors that interact with similar pseudopalindromic DNA recognition sites. We have previously described the Oshox1 gene from rice, which encodes an HD-Zip II protein. To identify further rice HD-Zip proteins, one-hybrid screens were performed in yeast strains containing a HIS3 reporter gene with upstream HD-Zip recognition sites. This resulted in the isolation of six new cDNAs encoding HD-Zip proteins belonging to family I (Oshox4, -5, -6) or family II (Oshox2, -3, -7). In transient assays, using rice suspension-cultured cells transformed by particle bombardment, we showed previously that Oshox1 can transcriptionally repress the activity of reporter gene constructs with upstream HD-Zip binding sites. Here, we confirm the repression properties of Oshox1 by showing that the repression function can be conferred on a heterologous DNA-binding domain. This portable functional domain (residues 1-155) is located proximal to the HD-Zip domain. In yeast, the same region of the Oshox1 protein was found to confer transcriptional activation instead of repression, pointing to the possibility that cell type-specific factors may determine the functional properties of the Oshox1 protein in rice. Like Oshox1, another HD-Zip family II protein (Oshox3) was also found to function as a transcriptional repressor in rice cells. In contrast, two HD-Zip I family proteins (Oshox4 and -5) appeared to act as activators in both rice and yeast cells. Results of two-hybrid assays and electrophoretic mobility shift assays strongly suggest that all HD-Zip proteins of families I and II can form homodimers and also heterodimers with all HD-Zip proteins of the same family. Heterodimerization across the HD-Zip families I and II apparently does not to occur.

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis.

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severely affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.

Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast.

We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice.

This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein. This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain.

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding.

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells.

A promoter region that controls basal and elicitor-inducible expression levels of the NADPH:cytochrome P450 reductase gene (Cpr) from Catharanthus roseus binds nuclear factor GT-1.

NADPH:cytochrome P450 reductase (CPR) is essential for the activation of cytochrome P450 enzymes, which are involved in a wide variety of metabolic pathways in plants, including those related to defence responses. In the subtropical plant Catharanthus roseus several cytochrome P450 enzymes operate in the biosynthesis of defence-related terpenoid indole alkaloids (TIAs). In agreement with the importance of CPR in defence, Cpr mRNA levels in C. roseus were found to be enhanced by fungal elicitor preparations that also induce TIA biosynthesis and P450 gene expression. Here we describe the isolation of a C. roseus genomic DNA clone covering the 5' part of the Cpr gene and 1.6-kb of upstream sequences. Mapping of the transcription start site showed the untranslated leader sequence is approximately 280 bp long. To study the control of gene expression by the Cpr promoter, transcriptional fusions between Cpr promoter fragments and the gusA reporter gene were generated and their expression was analyzed in stably transformed tobacco plants. The Cpr promoter fragment extending from -1510 to -8, with respect to the ATG start codon, conferred basal and elicitor-inducible expression on the gusA reporter gene, strongly indicating that the Cpr gene of C. roseus is indeed controlled by this promoter region. Progressive deletion from the 5' end of the promoter to position -632 had little effect on gusA expression. However, deletion to position -366 resulted in a complete loss of basal activity and largely eliminated elicitor-induced expression, indicating that the region from -632 to -366 contains the main transcription-enhancing cis-regulatory sequences. Electrophoretic mobility shift assays with tobacco nuclear extracts showed that binding sites for nuclear factor GT-1 are redundant in the Cpr promoter, but absent from the downstream part of the leader sequence. The presence of strong GT-1 binding sites in the main enhancer region (-632 to -366), is suggestive of a functional role for this factor in basal expression and elicitor responsiveness of the Cpr promoter.

Novel members of a family of AT hook-containing DNA-binding proteins from rice are identified through their in vitro interaction with consensus target sites of plant and animal homeodomain proteins.

The AT hook is an AT-rich DNA-binding domain that occurs three times in mammalian high-mobility-group I/Y chromosomal proteins and has recently also been identified in DNA-binding proteins from plants. We unexpectedly isolated three rice cDNA clones encoding AT hook-containing proteins in an attempt to isolate homeobox cDNA clones by south-western screening of an expression library with known binding sites for Arabidopsis and animal homeodomain proteins. One of these clones (Os-PF1) has previously been identified due to the binding of its encoded protein to PE1, a cis-acting element from the oat phytochrome promoter. The other two clones represent newly described cDNA clones, designated Os-AT1 and Os-AT2. The Os-AT1 and Os-AT2 proteins were found to have the same specificities as Os-PF1 with respect to in vitro binding of wild-type and mutant PE1 versions. However, all three proteins appeared to bind much stronger in south-western assays to two of the rather AT-rich sequences used in our screening than to the PE1 element. In none of the AT hook proteins clear homologies to transcriptional activation domains could be identified, but the N-terminal regions of Os-AT1 and Os-PF1 were found to show similarity to histone H1 chromosomal proteins. Given their structural characteristics it is conceivable that the rice AT hook proteins bind to gene promoter regions as accessory proteins that may alter the accessibility of chromatin to other nuclear factors. Their predominant expression in young and meristematic tissues suggests that the presence of the AT hook proteins may affect the expression of genes that determine the differentiation status of cells.

Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing the tdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.

Nucleotide sequence of the tryptophan decarboxylase gene of Catharanthus roseus and expression of tdc-gusA gene fusions in Nicotiana tabacum.

The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine. In Catharanthus roseus and other plants capable of producing terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). Here we report that TDC is encoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene expression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5' deleted promoter derivatives were joined in translational fusion to a beta-D-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemical and fluorimetric analysis of transgenic plants revealed that 1938 bp of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cell type specificity was noted. Promoter deletions up to nucleotide -398 directed lower levels of gusA expression but conferred the same pattern of staining for GUS activity as the -1938 construct. Further deletion of the tdc promoter up to nucleotide -232 resulted in drastically reduced GUS activity levels and loss of GUS staining in all parts of the transgenic plants. In contrast to stable transformation, the -232 tdc-gusA construct gave rise to GUS activity levels comparable to those of the -398 construct in an assay system for transient expression in protoplasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and regulation of tobacco extensin.

A tobacco cDNA clone (pCNT1) was characterized that encodes an extensin apoprotein almost entirely composed of the repeats Ser-Pro4(-Lys2), Pro-Tyr2-Pro2-His and Thr-Pro-Val-Tyr-Lys. In healthy plants extensin transcripts are abundant in the roots, less prevalent in the stem and rare in the leaves. In leaves extensin mRNA is induced by wounding, ethylene or virus infection. Tobacco was transformed with pCNT1 cDNA coupled in sense or antisense orientation to the CaMV 35S promoter. Analysis of transgenic plants that over- or underexpressed pCNT1 mRNA demonstrated that the encoded protein constituted the majority of hydroxyproline-rich glycoproteins in roots, stems and leaves. The pCNT1-encoded protein contained at least 50% of total hydroxyproline present in these organs and was abundant in the soluble protein fraction of stems and roots as well as in the cell wall of stem vascular bundles. Analysis of transgenic plants expressing sense or antisense extensin gene constructs showed no correlation between total hydroxyproline concentration or soluble HRGP content and plant development.

Interaction between the tobacco DNA-binding activity CBF and the cyt-1 promoter element of the Agrobacterium tumefaciens T-DNA gene T-CYT correlates with cyt-1 directed gene expression in multiple tobacco tissue types.

A novel DNA-binding activity, designated CBF, has been identified in nuclear extracts from tobacco leaf, stem and root tissue. CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt-1, of the Agrobacterium tumefaciens T-DNA cytokinin (T-cyt) gene. The T-cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt-1 element is located within a region that is essential for T-cyt promotor activity in leaf, stem and root cells of tobacco plants. Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element. Site-directed mutagenesis of the CBF binding site within the T-cyt promoter by using PCR resulted in an almost complete loss of T-cyt promoter activity in transgenic tobacco plants. In a gain-of-function experiment a hexamer of cyt-1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing -55 derivative of the T-cyt promoter. A mutant cyt-1 hexamer, defective in CBF binding, did not show activity above background levels. These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directed gene expression, suggesting that CBF might act as a transcriptional activator. Apart from the ASF-1 binding site of the CaMV 35S promoter, which is also present in the T-DNA nopaline and octopine synthase genes, the cyt-1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.

A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells.

A novel selection system for plant genetic transformation was developed based on the enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus. This enzyme converts the toxic tryptophan analogue 4-methyl tryptophan (4-mT) into the non-toxic compound 4-methyl tryptamine. Expression of tdc in transgenic plants that have no endogenous TDC-activity allows selection on 4-mT. A vector was constructed containing a tdc cDNA clone under control of the constitutively expressed cauliflower mosaic virus 35S promoter. This vector was used in Agrobacterium-mediated tobacco leaf disc transformation experiments. The optimal concentration for selection with 4-mT was found to be 0.1 mM. The transformed nature of shoots obtained after tdc gene transfer and subsequent selection on 0.1 mM 4-mT was confirmed by northern blot analysis.