The plastome sequence of Bactris gasipaes and evolutionary analysis in tribe Cocoseae (Arecaceae)

The family Arecaceae is distributed throughout tropical and subtropical regions of the world. Among the five subfamilies, Arecoideae is the most species-rich and still contains some ambiguous inter-generic relationships, such as those within subtribes Attaleinae and Bactridineae. The hypervariable regions of plastid genomes (plastomes) are interesting tools to clarify unresolved phylogenetic relationships. We sequenced and characterized the plastome of Bactris gasipaes (Bactridinae) and compared it with eight species from the three Cocoseae sub-tribes (Attaleinae, Bactridinae, and Elaeidinae) to perform comparative analysis and to identify hypervariable regions. The Bactris gasipaes plastome has 156,646 bp, with 113 unique genes. Among them, four genes have an alternative start codon (cemA, rps19, rpl2, and ndhD). Plastomes are highly conserved within tribe Cocoseae: 97.3% identity, length variation of ~2 kb, and a single ~4.5 kb inversion in Astrocaryum plastomes. The LSC/IR and IR/SSC junctions vary among the subtribes: in Bactridinae and Elaeidinae the rps19 gene is completely contained in the IR region; in the subtribe Attaleinae the rps19 gene is only partially contained in the IRs. The hypervariable regions selected according to sequence variation (SV%) and frequency of parsimony informative sites (PIS%) revealed plastome regions with great potential for molecular analysis. The ten regions with greatest SV% showed higher variation than the plastid molecular markers commonly used for phylogenetic analysis in palms. The phylogenetic trees based on the plastomes and the hypervariable regions (SV%) datasets had well-resolved relationships, with consistent topologies within tribe Cocoseae, and confirm the monophyly of the subtribes Bactridinae and Attaleinae.

L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

ABSTRACTl-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous l-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of l-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess l-DOPA, both the betaxanthin and hygroaurin adducts of l-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other l-DOPA dioxygenases. The rate constants for the conversion of l-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

Expression of the aac(6′)-Ib-cr Gene in Class 1 Integrons

ABSTRACT aac(6′)-Ib-cr is a plasmid-mediated quinolone resistance gene embedded within a gene cassette, most often within an integron. It confers resistance to quinolones and aminoglycosides. We investigated the role of a 101-bp fragment frequently present upstream of the aac(6′)-Ib-cr gene cassette and found that it contributes to the expression of aac(6′)-Ib-cr and provides an alternative start codon, confirming the length of the AAC(6′)-Ib-cr protein to 199 amino acids.

Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.