A protocol for combining fluorescent proteins with histological stains for diverse cell wall components.

Higher plant function is contingent upon the complex three-dimensional (3D) architecture of plant tissues, yet severe light scattering renders deep, 3D tissue imaging very problematic. Although efforts to 'clear' tissues have been ongoing for over a century, many innovations have been made in recent years. Among them, a protocol called ClearSee efficiently clears tissues and diminishes chlorophyll autofluorescence while maintaining fluorescent proteins - thereby allowing analysis of gene expression and protein localisation in cleared samples. To further increase the usefulness of this protocol, we have developed a ClearSee-based toolbox in which a number of classical histological stains for lignin, suberin and other cell wall components can be used in conjunction with fluorescent reporter lines. We found that a number of classical dyes are highly soluble in ClearSee solution, allowing the old staining protocols to be enormously simplified; these additionally have been unsuitable for co-visualisation with fluorescent markers due to harsh fixation and clearing. Consecutive staining with several dyes allows 3D co-visualisation of distinct cell wall modifications with fluorescent proteins - used as transcriptional reporters or protein localisation tools - deep within tissues. Moreover, the protocol is easily applied on hand sections of different organs. In combination with confocal microscopy, this improves image quality while decreasing the time and cost of embedding/sectioning. It thus provides a low-cost, efficient method for studying thick plant tissues which are usually cumbersome to visualise. Our ClearSee-adapted protocols significantly improve and speed up anatomical and developmental investigations in numerous plant species, and we hope they will contribute to new discoveries in many areas of plant research

A multi-character analysis of Struthiopteris leads to the rescue of Spicantopsis (Blechnaceae, Polypodiopsida)

The family Blechnaceae is a moderately sized leptosporangiate fern lineage, with 24 genera and around 250 species. Struthiopteris accommodates small to medium-sized, dimorphic, pinnate species. It is composed of six northern species: S. spicant is distributed in western parts of Europe and North America; S. fallax is endemic to Iceland; S. niponica, S. amabilis and S. castanea are endemic to Japan, and S. hancockii occurs in Japan and Taiwan. Due to the lack of a global review and to its highly interesting geographical distribution, this genus merits further study to clarify its taxonomy, species relationships, and distributional pattern. The present study aims to achieve the following goals: (a) identify and describe morphological characters supporting the taxonomy of Struthiopteris; (b) reconstruct a complete phylogeny for the genus; (c) study the biogeographical history of Struthiopteris at a global scale. The morphological study involved the observation of characters ranging from rhizome scales to spores over 164 individuals. Phylogenies were constructed applying ML and BI techniques over 51 newly produced sequences of three chloroplast markers (rbcL, trnL-trnF, psbA-trnH), using the species Blechnidium melanopus and Brainea insignis as closest relatives. For the molecular dating and historical biogeography analyses, we estimated and compared ancestral ranges under several models. Most of the morphological characters led us to discern two groups of species: the S. spicant group (S. spicant, S. fallax, and S. castanea) and the S. niponica group (the remaining species). In our molecular phylogeny, the supposed sister genus Blechnidium always appeared as nested within Struthiopteris, rendering this genus non-monophyletic. The two groups identified by the morphology appeared as monophyletic clades within Struthiopteris, with the clade S. spicant more closely related to Blechnidium than to the clade S. niponica. For all these reasons, we propose to rescue the now-disused genus Spicantopsis for the species belonging to the S. niponica group: indeed, this genus was created c. 100 years ago to reunite the same species S. amabilis, S. niponica, and S. hancockii. Our results suggest that all members of this group of genera (Blechnidium, Struthiopteris, Spicantopsis) emerged in East Asia about 85 mya, at a time when Japan was still part of the mainland. It appears that, for most of their history, the members of these genera have been confined to East Asia, with one dispersal to the Americas by an ancestor within Struthiopteris s.str., and additional dispersals to India and the Philippines by Blechnidium melanopus

Anatomia de estípulas e coléteres de Psychotria carthagenensis Jacq. (Rubiaceae) Stipule and colleter anatomy of Psychotria carthagenensis Jacq. (Rubiaceae)

Psychotria carthagenensis (Rubiaceae) pode ser identificada pela presença de estípulas apicais lanceoladas. Muitos gêneros da família têm estípulas com estruturas secretoras, denominadas coléteres. Sobre a estrutura de estípulas e coléteres pouco é conhecido. O objetivo do presente trabalho foi caracterizar anatomicamente estípulas e coléteres de indivíduos de P. carthagenensis ocorrentes no Estado de Santa Catarina (Brasil). Amostras de estípulas apicais dos ramos foram coletadas, fixadas e processadas para estudos em microscopias óptica e eletrônica de varredura. Testes histoquímicos foram aplicados em material in vivo. As estípulas são fundidas pela base e separadas na porção apical. Na face adaxial ocorrem tricomas multicelulares, entre os quais estão os coléteres. Estes têm base constricta, poros na superfície cuticular e epiderme em paliçada envolvendo um parênquima axial, o qual pode conter ráfides. Os coléteres secretam substâncias mucilaginosas. Esta secreção é muito importante para proteger o meristema apical caulinar e as folhas jovens.<br>Psychotria carthagenensis (Rubiaceae) is identified by the presence of lanceolate apical stipules. Several Rubiaceae genera have stipules with secretory structures called colleters. Little is known about the structures of stipules and colleters. This work aimed to characterize anatomically the stipules and the colleters of Psychotria carthagenensis from Santa Catarina state (Brazil). Apical shoot stipule samples were collected, fixed, and processed for light and scanning electron microscopy studies. Histochemical tests were made on in vivo material. The bases of stipules are connected and the apices are separate. On the adaxial surface, there are multicellular trichomes and the colleters are found among these. They have a constricted base, pores on the cuticle surface, parenchymatic middle axis surrounded by a layer of palisade-like epidermal cells, in which may have raphides. The colleters secrete a mucilaginous substance that is very important to protect the apical shoot and young leaves