Axis formation is one of the earliest patterning events in plant and animal embryogenesis. In Arabidopsis, the main axis of the embryo is evident at the asymmetric division of the zygote into a small, embryonic apical cell and a large extraembryonic basal cell. Here we show that the homeobox genes WOX2 and WOX8, which are initially coexpressed in the zygote, act as complementary cell fate regulators in the apical and basal lineage, respectively. Furthermore, WOX8 expression in the basal cell lineage is required for WOX2 expression and normal development of the proembryo, suggesting an inductive mechanism. The identified WOX cascade is required for normal expression of a reporter gene of the auxin efflux carrier PIN1 and for the formation of auxin response maxima in the proembryo. Thus, our results link the spatial separation of WOX transcription factors to localized auxin response and the formation of the main body axis in the embryo.

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.

Corresponding authorHans Kossel passed away on December 24, 1995. We mourn the loss of a fatherly friend and a great figure in contemporary science. We dedicate this paper to his memory and to his friends all over the world.Substitutional RNA editing changes single C nucleotides in higher plant chloroplast transcripts into U residues. To determine the cis-acting sequence elements involved in plastid RNA editing, we constructed a series of chloroplast transformation vectors harboring selected editing sites of the tobacco ndhB transcript in a chimeric context. The constructs were inserted into the tobacco plastid genome by biolistic transformation leading to the production of stable chimeric RNAs. Analysis of RNA editing revealed unexpected differences in the size of the essential cis elements or in their distance from the editing site. Flanking sequences of identical size direct virtually complete editing for one pair of editing sites, partial editing for a second and no editing at all for a third pair of sites. Serial 5' and 3' deletions allowed us to define the cis-acting elements more precisely and to identify a sequence element essential for editing site recognition. In addition, a single nucleotide substitution immediately upstream of an editing position was introduced. This mutation was found drastically and selectively to reduce the editing efficiency of the downstream editing site, demonstrating that position -1 is important for either site recognition or catalysis. Our results indicate that the editing of adjacent sites is likely to be mechanistically coupled. In no case did the presence in the plastome of the additional editing sites have any effect on the editing efficiency of the endogenous ndhB sites, indicating that the availability of site-specific trans-acting factors is not rate limiting.

The establishment of pluripotent stem cells is a key event during plant and animal embryogenesis, but the underlying mechanisms remain enigmatic. We show that in the flowering plant Arabidopsis thaliana, expression of the shoot meristem stem cell marker CLV3 becomes detectable in transition stage embryos. Surprisingly, the key regulator of stem cell homeostasis WUSCHEL (WUS) is expressed but dispensable for stem cell initiation. Rather, the WUS paralog WOX2, a regulator of embryo patterning initiated in the zygote, functions in this process by shielding stem cell progenitors from differentiation. WOX2 upregulates HD-ZIP III transcription factors required for shoot identity and balances cytokinin versus auxin hormone pathways, revealing that classical plantlet regeneration procedures recapitulate the natural induction mechanism. Our findings link transcriptional regulation of early embryo patterning to hormonal control of stem cell initiation and suggest that similar strategies have evolved in plant and animal stem cell formation.

Continuous hydrogen photo-production under sulfur deprivation was studied in the Chlamydomonas reinhardtii pgr5 pgrl1 double mutant and respective single mutants. Under medium light conditions, the pgr5 exhibited the highest performance and produced about eight times more hydrogen than the wild type, making pgr5 one of the most efficient hydrogen producer reported so far. The pgr5 pgrl1 double mutant showed an increased hydrogen burst at the beginning of sulfur deprivation under high light conditions, but in this case the overall amount of hydrogen produced by pgr5 pgrl1 as well as pgr5 was diminished due to photo-inhibition and increased degradation of PSI. In contrast, the pgrl1 was effective in hydrogen production in both high and low light. Blocking photosynthetic electron transfer by DCMU stopped hydrogen production almost completely in the mutant strains, indicating that the main pathway of electrons toward enhanced hydrogen production is via linear electron transport. Indeed, PSII remained more active and stable in the pgr mutant strains as compared to the wild type. Since transition to anaerobiosis was faster and could be maintained due to an increased oxygen consumption capacity, this likely preserves PSII from photo-oxidative damage in the pgr mutants. Hence, we conclude that increased hydrogen production under sulfur deprivation in the pgr5 and pgrl1 mutants is caused by an increased stability of PSII permitting sustainable light-driven hydrogen production in Chlamydomonas reinhardtii.