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Small GTP-binding proteins belonging to the Ras superfamily have been found in evolutionarily divergent organisms. Here, WC report the isolation and analysis of a cDNA encoding a putative small GTP-binding protein, designated Rhnl. from the plant, Nicoriutlap~atnbagginijblia. The 21 .I-kDa protein I*:Is 60% amino acid similarity with the mammalian RabS proteins. The Rhnl protein is encoded by a small multigene family. Northern analysis shows the highest steudy-state mRNA levels to be in roots and flowers. Furthermore, the Rhnl protein has 80% amino acid similarity with an Arubidopsis small GTP-binding protein, designated Rhal. GTP-binding protein; Protein secretion; ruc~s gene expression; Nicotiuna phmrbugitttfi~ia

As part of the European Union program of European Scientist Sequencing Arabidopsis (ESSA), the DNA sequence of a 24.053-bp insert of cosmid clone CC17J13 was determined. The cosmid is located on chromosome 1 at the PFL locus (position 30 cM). Analysis of the sequence and comparison to public databases predicts seven genes in this area, thus approximately one gene every 3.3 kb. Three cDNAs corresponding to genes in this region were also sequenced. The homologies and/or possible functions of the (putative) genes are discussed. Proteins encoded by genes in this region include a polyadenylatebinding protein (PAB-3) and a GTP-binding protein (Rab7) as well as a novel protein, possibly involved in double-stranded RNA unwinding and apoptosis. Intriguingly, the gene encoding the PAB-3 protein, which is very specifically expressed, is flanked by putative matrix attachment regions.

As a contribution to the European Scientists Sequencing Arabidopsis (BIOTECH ESSA) project, a contig of almost 40 kb has been sequenced at the extreme top of chromosome 1, around the Arabidopsis thaliana gene coding for a member of the 1-aminocyclopropane-1-carboxylate synthesis gene family. The region contains, besides the ACS1 gene itself, 10 putative genes, all new for Arabidopsis. Among these are three genes encoding kinases, a late embryogenesis-abundant protein, a MADS boxcontaining protein, a dehydrogenase, and a Myb-related transcription factor. In addition, six cDNAs have been sequenced that correspond to this region.

Grain legumes play a crucial role in the sustainability of agricultural systems and in food protein supply in developing countries. Several constraints that limit crop production or quality have been addressed by conventional breeding and enhanced management, but there are situations where the existing germplasm lacks the required traits. Genetic transformation could help provide solutions to certain constraints, thus improving food security in developing countries. The potential benefits of this form of genetic improvement have not yet been realised, mainly because efficient and reproducible gene transfer systems are not available. We review the state of the art of gene technology for genetic improvement of those grain legumes of major importance to developing countries. Protocols are evaluated for their reproducibility, efficiency and robustness.

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosomeassociated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and proteinsorting mechanisms in the secretory pathway.

The rhal gene from Arabidopsis encodes a small GTP binding protein belonging to the YpVRab family. Transgenic Arabidopsis plants containing the promoter region of the rhal gene fused to the P-glucuronidase (gus) reporter gene revealed gus expression limited mainly to the guard cells of stomata, the stipules, and the mot tip of young plants. In flowering plants, expression was found predominantly in the receptacle and in guard cells of the different flower organs. High GUS activity could also be seen in callus tissue and developing seeds. No detectable activity was present in other plant tissues; activity could not be induced by various treatments. GUS activity was visualized histochemically using both 5-bromo-4-chloro-3-indolyl P-D-glucuronide and a newly developed GUS substrate: Sudan Il-8-glucuronide. The latter precipitates as red crystals at the site of GUS activity. Results obtained by the gus analysis were confirmed by wholemount mRNA in situ hybridization. A hypothesis for the function of the Rhal protein is discussed.

Helianthinin is the major 11S seed storage protein of sunflower (Helianthus annuus). Like most seed proteins, helianthinin is encoded by a small gene family; two members of this gene family, HaG3-A and HaG3-D, have been isolated and characterized. Tobacco was transformed with a 6 kb fragment of HaG3-A containing the helianthinin coding region flanked by 3.8 kb upstream and 0.4 kb downstream sequence. Expression of helianthinin was developmentally regulated in seeds of transgenic tobacco plants; furthermore, helianthinin polypeptides were proteolytically processed and targeted to the protein bodies of transgenic tobacco. A fragment of HaG3-A from-2376 to + 24 was fused to the fl-glucuronidase (GUS) reporter gene and transferred to tobacco. GUS expression driven by this helianthinin upstream region was developmentally regulated in seeds. Germinating seedlings of the same transformant exhibited a time-dependent decrease in GUS activity with none detected by 6 days post imbibition (DPI). Histochemical analysis of GUS activity in embryos and 2 to 5 DPI seedlings showed expression restricted to the cotyledons and upper embryonic axis with none detected at the radicle end. No GUS activity was found in cotyledons, hypocotyls, leaves, and roots of 18 day seedlings or in leaves of an 8 week F1 plant. These results indicate that the cis-regulatory elements required for developmental control of the HaG3-A helianthinin gene are located in a 2.4 kb upstream region of this gene. This region was sequenced together with the upstream region of the HaG3-D helianthinin gene.

Members of the Arabidopsis Genome Initiative, including the European Union Arabidopsis Genome Sequencing Consortium and The Cold Spring Harbor, Washington University in St Louis and PE Biosystems Arabidopsis Sequencing Consortium, have completed sequencing one of the first two plant chromosomes. Significance and context Arabidopsis thaliana is the model organism of choice for modern plant biologists. Its small genome, the excellent genetic and physical maps of the genome and the lack of large amounts of repetitive DNA made it the first choice for plant genome sequencing. In 1996, a multinational organization, the Arabidopsis Genome Initiative (AGI), was formed to coordinate the worldwide effort to sequence the first higher plant. Made up of labs from the United States, Europe and Japan, AGI set a goal for the completion of the Arabidopsis genome by 2004. The members divided up the five chromosomes between them and began sequencing. Advances in sequencing and computing technology have pushed forward their initial timetable, however, and two papers report the completion of the first two plant chromosomes. Mayer et al. report the sequencing of chromosome 4, which represents about 17% of the genome. In a companion paper in the same issue of Nature, Lin et al. report the sequencing of chromosome 2, which represents approximately 15% of the Arabidopsis genome. These sequences are two of the largest pieces of DNA sequence ever assembled and together represent almost a third of the Arabidopsis genome. The complete sequences of chromosomes 2 and 4 offer unique insights into largescale genomic organization, plant heterochromatic DNA, non-coding regions, gene duplication events, and gene family organization.