The cryptophyte Guillardia theta harbors a plastid surrounded by four membranes. This turns protein targeting of nucleus-encoded endosymbiont localized proteins into quite a challenge, as the respective precursors have to pass either all four membranes to reach the plastid stroma or only the outermost two membranes to enter the periplastidal compartment. Therefore two sets of nuclear-encoded proteins imported into the endosymbiont can be distinguished and their topogenic signals may serve as good indicators for studying protein targeting and subsequent transport across the outermost membranes of the cryptophyte plastid. We isolated genes encoding enzymes involved in two different biochemical pathways, both of which are predicted to be localized inside the periplastidal compartment, and compared their topogenic signals to those of precursor proteins for the plastid stroma, which are encoded on either the nucleus or the nucleomorph. By this and exemplary in vitro and in vivo analyses of the topogenic signal of one protein localized in the periplastidal compartment, we present new data implicating the mechanism of targeting and transport of proteins to and across the outermost plastid membranes. Furthermore, we demonstrate that one single, but conserved amino acid is the triggering key for the discrimination between nucleus-encoded plastid and periplastidal proteins.

The mobilization of sulfur (SUF) system is one of three systems involved in iron-sulfur cluster biosynthesis and maintenance. In eukaryotes the SUF system is specific for the plastid and therefore of symbiotic origin. Analyses in cryptophytes showed a unique genetic compartmentalization of the SUF system, which evolved by at least two different gene transfer events. We analyzed one of the components, SufD, in the cryptophyte Guillardia theta and in Arabidopsis thaliana. We demonstrated that SufD fulfils house keeping functions during embryogenesis and in adult plants in A. thaliana.

To investigate the mechanism of auxin action during pattern formation in dicot embryos, we tested the effects of the natural auxin indole-3-acetic acid (IAA), the auxin transport inhibitor N-(1-naphthyl)thalamic acid (NPA) and the antiauxin p-chlorophenoxyisobutyric acid (PCIB). In vitro treatments of isolated zygotic Brassica juncea embryos with these substances led to a wide range of morphogenetic alterations. Treatment of globular embryos with exogenous auxin (10-40 microM) either completely inhibited morphogenesis, resulting in ball-shaped embryos, or caused the development of egg- and cucumber-shaped embryos, which only consisted of a shortened hypocotyl without any apical structures. Axis duplication was observed sometimes after inhibition of auxin transport in globular embryos, and led to the development of twin embryos. During the transition from globular to heart stage, changes in auxin distribution or activity frequently caused the development of either split-collar or collar-cotyledons. Antiauxin inhibited cotyledon growth, leading to embryos with single or no cotyledons, or inhibited the development of the hypocotyl and the radicle. Inhibition of auxin transport in transition embryos sometimes led to axis broadening, which resulted in the development of two radicles. The described changes in embryo shapes represent arrests in different auxin-regulated developmental steps and phenocopy some Arabidopsis morphogenetic mutants.

A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7-9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and β-glucuronidase. Shoots are regenerated from callus-forming explants within 3-4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14-16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical β glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.

No abstract