Jean-Pierre Zryd

Stable transformation of the moss Physcomitrella patens

SummaryWe report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens.

The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Δmcb1 strains, which grow normally, P. patens Δmcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Δmcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.

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.

Secondary metabolism in cultured red beet (Beta vulgaris L.) cells: Differential regulation of betaxanthin and betacyanin biosynthesis

Red beet cell lines exhibiting a range of cell colours were generated from secondary callus via specific induction methods. Phenotype colour ranged from white/green through yellow, orange and red to deep violet, representing all types of pigments found in red beet plant. Specific phenotypes could only be obtained through specific induction sequences and once established were stabilised by cultivation on a maintenance medium. The ratio of auxin (2,4-D) to cytokinin (6-BAP) was an important factor in the control of these processes. All coloured phenotypes were linked, but could be classified into two main groups, one yellow-red and the other orange-violet, according to their different cellular morphologies. A certain amount of instability still existed within each group. Modification of the growth regulator composition could be used to interchange specific combinations of coloured phenotypes, depending upon the initial state of cellular differentiation. Use of the DNA-methylation inhibitor 5-azacytidine demonstrated that methylation plays a key role in the repression of genes encoding enzymes involved in betacyanin biosynthesis. Furthermore, the poly(ADP-ribose) polymerase inhibitor 3-methoxybenzamide blocked the induction of the same gene set in a concentration dependent manner without affecting cell growth.

A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens

Abstract The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

Biogenesis of betalains : purification and partial characterization of dopa 4,5-dioxygenase from Amanita muscaria

Abstract 3,4-Dihydroxyphenylalanine 4,5-dioxygenase, a central enzyme in the biogenesis of betalains, has been purified from the mushroom Amanita muscaria. Like other extradiol-cleaving dioxygenases, this enzyme is an oligomer; however, DOPA 4,5-dioxygenase is composed of varying number of an identical subunit of Mr 22 000. It is inhibited by cyanide, diethylpyrocarbonate and various nitrogen-containing ion chelating agents. The enzyme does not exhibit a strict specificity for DOPA. Other p-dihydric aromatic compounds such as dopamine and catechol are also converted to α-hydroxymuconic e-semialdehyde derivatives. This is the first report of an enzyme involved in the metabolism of betalain pigments.

Characterization of a tyrosinase from Amanita muscaria involved in betalain biosynthesis

Abstract A tyrosinase was characterized from the betalain-containing pileus of the mushroom Amanita muscaria . The enzyme was located exclusively in the coloured parts of the mushroom and hydroxylated tyrosine to 3-(3,4-dihydroxyphenyl)-alanine (DOPA), suggesting that it is involved in betalain biosynthesis. The tyrosinase was not specific for tyrosine, and in addition to the monophenolase activity, the enzyme also oxidized diphenols to o -quinones (diphenolase activity). The native enzyme appeared to be a heterodimer of two subunits with M r s of 27000 and 30000, which is unusual for tyrosinases. Typical tyrosinase inhibitors such as tropolone, 2-mercaptobenzothiazole and benzoic acid strongly inhibited the enzyme.

Biochemical complementation of the betalain biosynthetic pathway in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine dioxygenase

Abstract. 3,4-Dihydroxyphenylalanine (DOPA) dioxygenase from Amanitamuscaria catalyses the key reaction of betalain biosynthesis, namely the conversion of DOPA to betalamic acid by a 4,5-ring-opening reaction. In addition, it catalyses a 2,3 opening which yields the fungal pigment muscaflavin, a compound that has never been found in plants. In this work, a cDNA clone (DodA) encoding A. muscaria DOPA-dioxygenase was expressed in white Portulacagrandiflora petals, using the particle bombardment technique. Transformation resulted in the formation of yellow and violet spots that contained betalain pigments and muscaflavin, indicating that the fungal enzyme was expressed and active in plants, and could complement the plant betalain biosynthetic pathway. The presence of muscaflavin in transformed plants indicates a difference in the specificity of the plant and A.muscaria enzymes.

The formation of betalamic acid and muscaflavin by recombinant dopa-dioxygenase from Amanita

Abstract DOPA-dioxygenase from Amanita muscaria is known to catalyse the conversion of 3-(3,4-dihydroxyphenyl)alanine (DOPA) to betalamic acid in the key reaction of betalain biosynthesis. In this work, we re-examined the reactivity of DOPA-dioxygenase using a cDNA clone encoding active DOPA-dioxygenase the kinetic parameters of which were comparable to those of the native enzyme. Using l -DOPA as a substrate, the enzyme catalysed the formation of two products. In addition to betalamic acid, the enzyme also catalysed the formation of muscaflavin, a compound that occurs naturally in A. muscaria and in mushrooms of the Hygrocybe family but not in the betalain-containing plants of the order Caryophyllales. Muscaflavin arises by a 2,3-extradiol cleavage of DOPA, whereas betalamic acid is the product of a 4,5-cleavage. Our results indicate that the recombinant enzyme has both 2,3- and 4,5-dioxygenase activity, and do not support the prevailing view that the two compounds are produced by two distinct enzymes.

The knock‐out of ARP3a gene affects F‐actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens

The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.