Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Researchand Biotechnology

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25°C. In contrast, a short non-damaging heat-treatment at 38°C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25°C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. paten

Characterization and Functional Identification of a Novel Plant 4,5-Extradiol Dioxygenase Involved in Betalain Pigment Biosynthesis in Portulaca grandiflora

Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism

The CaMV 35S promoter has a weak expression activity in dark grown tissues of moss Physcomitrella patens

The constitutive Cauliflower Mosaic Virus 35S promoter (CaMV 35S) is widely used as a tool to express recombinant proteins in plants, but with different success. We previously showed that the expression of an F-actin marker, GFP-talin, in Physcomitrella patens using the CaMV 35S promoter failed to homogenously label moss tissues. Here, we show a significant diminution of the GFP fluorescence in dark grown old moss cells and complete lack of labelling in newly differentiated cells. Furthermore, we demonstrate that stable moss lines harbouring a resistance cassette driven by the CaMV 35S are unable to grow in darkness in the presence of the antibiotic. In contrast to the CaMV 35S, the heat inducible promoter, hsp17.3B showed uniform expression pattern in all cells and tissues following a mild heat shock