Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. revealed by integrated chemical, anatomical, and RNA-Seq analyses.

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Structural Characterisation of the Leaf of Bauhinia monandra Kurz (Fabaceae - Cercidoideae)

Abstract Bauhinia monandra Kurz (Fabaceae) is an ornamental leguminous tree from Africa. Its leaf has been proven to be hypoglycaemic, and are a source of lecithin BmoLL, providing this species with great medicinal potential. Curiously, there are no reports of the anatomical description of the leaf of this species. The present work aimed at describing the leaf anatomy of B. monandra, and make histochemical inferences. To this end, anatomical procedures in light and scanning electron microscopy were performed from different parts of the leaves of plants growing in the state of Bahia, Brazil. The leaf is amphistomatous. It possesses papillate epidermis, nonglandular and navicular trichomes, and the mesophyll is dorsiventral. The central vein is distinct from the others of the first degree. The first, second, and third-degree veins possess thick fibre bundles with associated monocrystals. The other veins are a little fibrous and excessively reduced. The petiole vascularisation consists of circularly arranged collateral bundles, plus two accessory bundles. The pulvinus have a wide parenchymatic cortex with abundant druses and vascularisation restricted to the central region, surrounded by a crystalline sheath and by a starch sheath. The motile cushion has the vascular bundles arranged in series. Associated with the phloem from the pulvinus vascular bundles and the motile apparatus, occur non-lignified septate fibres that confer flexibility to these organs. The absence of lateral projections and the arrangement of vascular bundles in the petiole was the most distinctive anatomical features and of the greatest taxonomic potential observed in the leaf of B. monandra.

Utilisation of Toluidine blue O pH 4.0 and histochemical inferences in plant sections obtained by free-hand.

For the observation of plant material under a light microscope, free-hand sectioning is usually the fastest and most practical procedure. The staining of the obtained cuts is a crucial step and the action mechanism of the dye is variable. Toluidine blue O is a cationic dye whose metachromatic potential for free-hand sections has long been known. Thus, this work aimed at analysing the performance of Toluidine blue O for this type of sectioning with a systematic and comparative approach. The action of Toluidine blue O pH 4.0 was tested for both fresh and previously clarified free-hand sections from leaves of different species of plant. These sections were compared with sections embedded in glycol-methacrylate resin stained with the same dye or submitted to different histochemical tests. The fresh sections obtained by free-hand and stained with Toluidine blue O permitted the accomplishment of histochemical inferences, with the advantage of maintaining the natural colours from the tissues. The previously clarified sections presented the advantage of having a clearer image, allowing a better cellular delimitation. We concluded that Toluidine blue O is very informative for sections of plant material obtained by free-hand, being, therefore, a procedure that is fast, inexpensive and with excellent results.

Working Optimally with Serial Sections in Glycol Methacrylate Resin

ABSTRACT The present work presents an optimized form for distributing serial sections in a glycol methacrylate slide set. This consists of filling the first row with sections from each slide, thus proceeding in the same way with the other rows. Therefore each slide will contain sections of three distinct sample segments.

An inexpensive alternative equipment for the plant material embedding in the paraffin under the vacuum

The present work describes an equipment constructed using inexpensive material for embedding the plant material in the paraffin under the vacuum, using an oven and a vacuum pump. The equipment was tested using the samples of Rodriguezia venusta (Orchidaceae) buds embedded in paraffin, where half of the samples were submitted to the vacuum by the equipment during the embedding. The material was sectioned with a rotary microtome, obtaining full series of quality sections. The control was hard to section with the microtome, obtaining damaged sections due the air bubbles, making the ribbon formation difficult. These results proved the effectiveness of the equipment, making it a practical, inexpensive and more portable solution for newly established laboratories.

O presente trabalho apresenta um equipamento feito com material barato, destinado à inclusão de material botânico em parafina sob vácuo, utilizando-se uma estufa e uma bomba de vácuo. O equipamento foi testado utilizando-se amostras de botão floral de Rodriguezia venusta (Orchidaceae) incluídas em parafina, das quais metade foi submetida ao vácuo pelo equipamento durante a infiltração. O material foi seccionado em micrótomo rotativo, obtendo-se séries completas de cortes de boa qualidade das amostras submetidas ao vácuo. O controle foi de difícil microtomia, obtendo-se cortes danificados pela presença de bolhas de ar, dificultando assim a formação de fitas. Estes resultados comprovam a eficácia do equipamento proposto, sendo este uma solução prática, barata e portátil para laboratórios em início de estruturação.