NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) A is the only known example thus far of a nucleus-encoded plastid protein that is imported to its final destination in a substrate-dependent, Pchlide-regulated manner. Previous work has shown that the cytosolic PORA precursor (pPORA) does not utilize the general import site but uses a distinct translocon designated the Pchlide-dependent translocon complex. Here we demonstrate that a pentapeptide motif, threonine-threonine-serine-proline-glycine (TTSPG) in pPORA's transit peptide (transA), is involved in Pchlide-dependent transport. Deletion of this motif from the COOH-terminal end of transA abolished both Pchlide binding and protein import. Incorporation of the TTSPG motif into normally non-Pchlide-responsive transit sequences conferred the pigment binding properties onto the engineered chimeric precursors but was insufficient to render protein import substrate dependent. An additional motif was identified in the NH(2)-terminal part of transA that was needed for binding of the precursor to the Pchlide-dependent translocon complex. Point mutations of the TTSPG motif identified glycine as the Pchlide binding site. By analogy to the major light-harvesting chlorophyll a/b binding protein of photosystem II, we propose that the peptidyl carbonyl oxygen of glycine may bind directly or via a water molecule to the central Mg atom of the pigment.

Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28,952 predicted proteins in Arabidopsis, the actual subcellular localization has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesized on membrane-bound polysomes. Therefore, the isolation and characterization of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as 'cellular component unknown', and potentially encode previously unrecognized secreted or membrane-associated proteins.

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.

Transcription of the Arabidopsis thaliana bZIP transcription factor ATB2 is induced by light and sugars. Elevated concentrations of sucrose repress gene expression though a posttranscriptional mechanism. Thus, sucrose induces steady state mRNA levels and represses translation. It has been shown that deletion of the 5’UTR of the ATB2 gene abolishes the translational repression by sucrose. To investigate if the leader sequence is sufficient to confer sucrose repression it was assayed alone in a reporter gene assay. These experiments confirmed the necessity and sufficiency of the leader sequence for sucrose repression. To gain further insight into the regulatory mechanism of sucrose regulation a non-destructive assay has been developed. This non-destructive assay utilizes a dual reporter construct (GUS and LUC genes) and enables the identification of regulatory mutants. Several lines, expressing the dual reporter construct, have been isolated and tested for their response to physiological concentrations of sucrose, a prerequisite for future use in mutagenesis experiments.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500Arabidopsisgenes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in theArabidopsisgenome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silencedArabidopsislines. These versatile GST resources provide novel and powerful tools for functional genomics.

We reported earlier that dephosphorylated nascent 4)X174 viral strand DNA molecules were less extensively degraded from the 5' end by spleen exonuclease than were non-nascent molecules. Experiments described here revealed that the insensitivity to the 5'-OH end-specific nuclease was more evident among the longer molecules in the population than among the shorter, all of the molecules being less than unit length in size. The smallest molecules in the population were about as sensitive to the enzyme as the control molecules and hence must possess unblocked 5'-terminal nucleotides. Degradation of the nascent DNA with the 3' end-specific snake venom phosphodiesterase revealed only a small enrichment for [3H]thymidine near the 3' end, seemingly insufficient to account completely for the apparent insensitivity of the longer molecules to spleen exonuclease. When the nascent molecules were isolated without the use of proteolytic enzymes, some pronase-sensitive material was found associated with the DNA, particularly the longer molecules. We suggest that the resistance of the longer nascent (pronasetreated) molecules to spleen exonuclease occurs because they have remnants of the viral gene A or A* protein covalently bound to the 5' end.

The Complete Arabidopsis Transcriptome Micro Array (CATMA) database contains gene sequence tag (GST) and gene model sequences for over 70% of the predicted genes in the Arabidopsis thaliana genome as well as primer sequences for GST amplification and a wide range of supplementary information. All CATMA GST sequences are specific to the gene for which they were designed, and all gene models were predicted from a complete reannotation of the genome using uniform parameters. The database is searchable by sequence name, sequence homology or direct SQL query, and is available through the CATMA website at http://www.catma.org/.

In plant meristems, dividing cells interpret positional information and translate it into patterned cell differentiation. Here we report the molecular identification of the Arabidopsis HOBBIT gene that is required for cell division and cell differentiation in meristems. We show that it encodes a homolog of the CDC27 subunit of the anaphase-promoting complex (APC). HOBBIT partially complements a yeast nuc2/cdc27 mutant. Unlike other CDC27 homologs inArabidopsis, its transcription is cell cycle regulated. Furthermore, hobbit mutants show a reduction inDR5 :: GUS auxin reporter gene expression and accumulate the AXR3/IAA17 repressor of auxin responses. HOBBIT activity may thus couple cell division to cell differentiation by regulating cell cycle progression in the meristem or by restricting the response to differentiation cues, such as auxin, to dividing cells.

A light-regulated Arabidopsis thaliana homeobox 1 gene (ATH1) was identified in a transcription factor gene collection. Primer extension analysis of this gene showed the presence of two major transcripts with unusually long 5' untranslated leader sequences. The leader sequence of the isolated cDNA clone contains several small open reading frames upstream of the initiation codon of the largest open reading frame coding for the homeodomain protein. This leader sequence was found to affect the translation efficiency negatively in an in vitro translation system. The expression of the ATHl gene is dependent on the presence of light. After illuminating etiolated or dark-adapted Arabidopsis seedlings, the ATHl mRNA level increased rapidly. Expression of ATHl does not require the presence of active chloroplasts because photooxidative destruction of the chloroplast by norflurazon treatment did not influence the ATH7 mRNA level. In dark-grown seedlings of the photomorphogenic constitutiwe photomorphogenic 1 (copl) and deetiolated 1 (detl) mutants, the ATHl mRNA level was elevated. This shows that the gene products of these loci directly or indirectly repress ATH1 expression in etiolated wild-type seedlings. A correlation between the strength of the copl allele and the ATHl mRNA level was found. This relationship suggests a role for the ATHl protein in the signal transduction pathway downstream of COPl. Brusslan and for the protocol for the RNase protec- tion assay and for the gift of pAB140; Dr. P. Timko for his gift of pATS22; Dr. for the gift of p3-1500-18s; Dr. Pilon for the assistance with in vitro translation procedures; Dr. Angenent for the construction of the Arabidopsis cDNA library; of the European Union BRIDGE Arabidopsis project for the distribution of the genomic IGEM-11 libraryconstructed by Dr. John Mulligan and Dr. Ron Davis and Dr. Oscar Vorst for critical reading of the manuscript. Sequence analysis supported by the ESSA (European Scientists Sequencing Arabidopsis) project of European Union.

Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety. Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions. In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene. Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning. This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each. ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans. It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358. PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P. aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P. aeruginosa. It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.

Departments of Molecular Plant Physiology and Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands (N.G., S.C.H.J.T., P.J.W., S.C.M.S.); Advanta Seeds, P.O. Box 1, 4410 AA Rilland, The Netherlands (K.P.M.v.D.); Agrotechnological Research Institute, P.O. Box 17, 6700 AA Wageningen, The Netherlands (S.H.D.H.); and Laboratory of Plant Breeding, Wageningen Agricultural University, P.O. Box 386, 6700 AJ Wageningen, The Netherlands (R.G.F.V.)

In plants, sugars act as signalling molecules that control many aspects of metabolism and development. Arabidopsis plants homozygous for the recessive sucrose uncoupled-6 (sun6) mutation show a reduced sensitivity to sugars for processes such as photosynthesis, gene expression and germination. The sun6 mutant is insensitive to sugars that are substrates for hexokinase, suggesting that SUN6 might play a role in hexokinase-dependent sugar responses. The SUN6 gene was cloned by transposon tagging and analysis showed it to be identical to the previously described ABSCISIC ACID INSENSITIVE-4 (ABI4) gene. Our analysis suggests the involvement of abscisic acid and components of the abscisic acid signal transduction cascade in a hexokinase-dependent sugar response pathway. During the plant life cycle, SUN6/ABI4 may be involved in controlling metabolite availability in an abscisic acid- and sugar-dependent way.

Tulip (Tulipa gesneriana L.) is a bulbous plant species that requires a period of low temperature for proper growth and flowering. The mechanism of sensing the low temperature period is unknown. The study presented in this paper shows that the essential developmental change in tulip bulbs during cold treatment is an increase in sensitivity to the phytohormone auxin. This is demonstrated using a model system consisting of isolated internodes grown on tissue culture medium containing different combinations of the phytohormones auxin and gibberellin. Using mathematical modelling, equations taken from the field of enzyme kinetics were fitted through the data. By doing so it became apparent that longer periods of low temperature resulted in an increased maximum response at a lower auxin concentration. Besides the cold treatment, gibberellin also enhances the response to auxin in the internodes in this in vitro system. A working model describing the relationship between the cold requirement, gibberellin action and auxin sensitivity is put forward. Possible analogies with other cold-requiring processes such as vernalization and stratification, and the interaction of auxin and gibberellin in the stalk elongation process in other plant species are discussed.

Root formation in plants involves the continuous interpretation of positional cues. Physiological studies have linked root formation to auxins. An auxin response element displays a maximum in the Arabidopsis root and we investigate its developmental significance. Auxin response mutants reduce the maximum or its perception, and interfere with distal root patterning. Polar auxin transport mutants affect its localization and distal pattern. Polar auxin transport inhibitors cause dramatic relocalization of the maximum, and associated changes in pattern and polarity. Auxin application and laser ablations correlate root pattern with a maximum adjacent to the vascular bundle. Our data indicate that an auxin maximum at a vascular boundary establishes a distal organizer in the root.

In Arabidopsis, the root meristem originates from the hypophyseal cell and from an adjoining cell tier that is distinct at the heart stage of embryogenesis. We have analysed mutations in the HOBBIT (HBT) gene that is essential for root meristem formation. hbt embryos display incorrect hypophyseal cell development from the quadrant stage onward. At the heart stage, the adjoining cell tier of hbt embryos develops abnormally, in that the activation of cell division and the formation of a lateral root cap layer are disturbed. Strong hbt mutants give rise to seedlings that lack an anatomically recognisable quiescent centre and differentiated columella root cap cells, the cell types derived from the wild-type hypophysis. Furthermore, they have no mitotically active root meristem and lack a differentiated lateral root cap. Secondary roots of hbt mutants and roots obtained from cultured cells of hbt mutants have similar defects. Therefore the HBT gene is required for root meristem formation in different developmental contexts.

Post-embryonic development in plants is mainly achieved by its meristems. Within the Arabidopsis root meristem, both the fate and origin of its cells can be predicted with high accuracy. Mutants defective in the determination of root cell fates show that the corresponding genes are first required during embryogenesis. The sub-specification of cell fates, such as during epidermal root hair formation, involves transcription factors and phytohormones. In the Arabidopsis root, initial cell fate specification events must be followed by position-dependent reinforcement of cellular identity. A major question remains as to whether the signals that are involved in initiating the cellular pattern are the same or different from the signals used to reinforce it. The integrity of the root meristem is kept by balancing cell proliferation and cell differentiation, and differentiation-inhibiting signals originating from the quiescent centre are involved.

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.