Transpositional behaviour of an Ac/Ds system for reverse genetics in rice.

A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.

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.

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.

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.

Auxin rapidly down-regulates transcription of the tryptophan decarboxylase gene from Catharanthus roseus.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) converts tryptophan into tryptamine, and thereby channels primary metabolites into indole alkaloid biosynthesis. The production of these secondary metabolites in suspension cells of Catharanthus roseus depends on medium composition. Of the possible variables, we investigated the effect of hormones on the expression of the tdc gene in cell cultures. Omission of NAA from the growth medium resulted in accumulation of tdc mRNA. The addition of 1-naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) rapidly reduced the enhanced tdc transcript level. Cytokinin was unable to suppress the enhanced transcript level. Hairy roots transformed by Agrobacterium rhizogenes also showed a reduction of the tdc mRNA level after NAA addition. Run-off transcription experiments showed that the down-regulation takes place at the transcriptional level within 15 minutes and independent of de novo protein synthesis. Thus one of the mechanisms which control the activity of terpenoid indole alkaloid biosynthesis in C. roseus cell cultures is the negative regulation by auxin of the gene involved in the first committed step.

Plant expression signals of the Agrobacterium T-cyt gene.

Within the 5' and 3' non-coding regions of the T-cyt gene from the octopine T-DNA of Agrobacterium tumefaciens sequences required for expression of this gene in plant cells were identified by deletion mutagenesis. The results show that 184 bp of the 5' non-coding region and 270 bp of the 3' non-coding region are sufficient for wild-type expression. Within the 5' non-coding region two essential expression signals were identified: (1.) an activator element located between -185 and -129 with respect to the ATG start codon and (2.) one out of two TATA boxes. Deletions of the activator element or the two TATA boxes resulted in nonfunctional genes. Deletion of the upstream TATA box and both putative CAAT boxes did not significantly affect expression. Within the 3' non-coding region, the polyadenylation box most distal to the stop codon was not essential for expression, but sequences more upstream, including a second polyadenylation box were found to be required for wild-type expression.

Effects of mutations in the TATA box region of the Agrobacterium T-cyt gene on its transcription in plant tissues.

We have generated mutations in the promoter region of the octopine type cytokinin gene of Agrobacterium tumefaciens, and studied their effects on mRNA formation in different plant species. The promoter region of this gene contains several putative TATA boxes. Phenotypic expression and Northern blot hybridization showed that TATA boxes are essential for expression, but that one TATA box leads to wild-type transcript levels. Analysis of the 5' ends of T-cyt transcripts by primer extension using RNA from T-cyt gene transformed tobacco shoots revealed two major cap site clusters and one minor cap site. TATA box consensus sequences can be found approximately 30 bp upstream from each cap site cluster. Deletion of a TATA box results in loss of the corresponding cap sites. An insertion of 7 bp between the right TATA box and corresponding cap sites results in a shift of the position of the cap sites, so that the original distance of TATA box to cap sites is conserved as much as possible.