Cyclotide discovery in Gentianales revisited--identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.

Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

Palynological variation in balsaminoid ericales. I. Marcgraviaceae.

BACKGROUND AND AIMS: Marcgraviaceae are a rather small family of seven genera and approx. 130 neotropical species. This study aims to present a detailed palynological survey of the family in order to comment on the intrafamily relationships and possible correlations with pollinators. METHODS: In total, 119 specimens representing 67 species and all genera are observed using light microscopy and scanning electron microscopy. Furthermore, eight species from five genera are studied with transmission electron microscopy. KEY RESULTS: Our results show that pollen grains of Marcgraviaceae are small (20-35 microm), have three equatorial apertures, granules on the colpus membrane, oblate spheroidal to prolate spheroidal shapes, mainly psilate to perforate ornamentations, and lalongate colpus-shaped thinnings at the inner layer of the exine, and show the presence of orbicules. Based on our fragmentary knowledge of the pollination biology of the family, there are no clear correlations between pollinators and pollen features. CONCLUSIONS: The genus Marcgravia has a high percentage of reticulate sexine patterns and a relatively thin nexine. Sarcopera can be defined by the presence of an oblate spheroidal to even suboblate shape, while Ruyschia and Souroubea typically show prolate spheroidal to subprolate pollen grains. The presence of a thick foot layer in the pollen wall is characteristic of the genera Norantea, Sarcopera and Schwartzia. Pollen features that are taxonomically useful within the family are the shape, sexine sculpturing, and ultrastructure of the pollen wall.