Autophagy and vacuolar biogenesis during the nectary development.

MAIN CONCLUSION: Different autophagy pathways are a driver of vacuolar biogenesis and are development stage specific during the extrafloral nectary development in Citharexylum myrianthum. Plant autophagy plays an important role in various developmental processes such as seed germination, pollen maturation and leaf senescence. However, studies that address the evidence of autophagy and its role in the development of plant glands are scarce and largely restricted to laticifers. Regarding nectary, studies have repeatedly pointed to signs of degradation associated with the end of the secretory cycle, without exploring autophagy. Likewise, the relationship between autophagy and biogenesis of vacuoles remains an unexplored issue. In this study, using conventional and microwave fixation in association with ultracytochemical methods for transmission electron microscopy, we investigated the occurrence of autophagy and its implication in the differentiation of extrafloral nectary in Citharexylum myrianthum (Verbenaceae) under natural conditions, focusing on the vacuole biogenesis. We described a variety of vacuole types associated with the stage of nectary epidermis development, which differs with respect to origin, function and nature of the products to be stored. Three distinct autophagy pathways were detected: macroautophagy, microautophagy (both restricted to the undifferentiated epidermal cells, at the presecretory stage) and megaautophagy (circumscribed to the differentiated epidermal cells, at the postsecretory stage). Our study clearly demonstrated that the vacuole variety and autophagy processes in the nectary epidermal cells are development specific. This study highlights the role of autophagy in vacuole biogenesis and its implications for the development of nectary and opens new venues for future studies on regulation mechanisms for autophagy in plant secretory structures under normal conditions.

Nectar Replaced by Volatile Secretion: A Potential New Role for Nectarless Flowers in a Bee-Pollinated Plant Species.

The presence of nectarless flowers in nectariferous plants is a widespread phenomenon in angiosperms. However, the frequency and distribution of nectarless flowers in natural populations, and the transition from nectariferous to nectarless flowers are poorly known. Variation in nectar production may affect mutualism stability, since energetic resource availability influences pollinators' foraging behavior. Here, we described the spatial and temporal nectar production patterns of Jacaranda oxyphylla, a bee-pollinated species that naturally presents nectarless flowers. Additionally, we compared nectariferous and nectarless floral disks in order to identify histological, subcellular and chemical changes that accompanied the loss of nectar production ability. For that we used standard methods for light and transmission electron microscopy, and gas chromatography coupled to mass spectrometry for chemical analyses. We verified that 47% of flowers did not produce nectar during the whole flower lifespan (nectarless flowers). We also observed remarkable inter-plant variation, with individuals having only nectarless flowers, others only nectariferous ones and most of them showing different proportions of both flower types, with variable nectar volumes (3-21 µl). Additionally, among nectariferous flowers, we registered two distinct rhythms of nectar production. 'Early' flowers produced nectar from 0 to 24 h, and 'late' flowers produced nectar from 24 to 48 h of anthesis. Although disks from nectariferous and nectarless flowers displayed similar histological organization, they differed strongly at subcellular level. Nectariferous ('early' and 'late') flowers exhibited a cellular apparatus typical of nectar secretion, while nectarless flowers exhibited osmophoric features. We found three aliphatic and one aromatic compound(s) that were detected in both the headspace of flowers and the disks of nectarless flowers, but not the disks of nectariferous flowers Although the remarkable variation in nectar availability may discourage pollinator visits, nectarless flowers might compensate it by producing volatile compounds that can be part of floral scent, acting as chemical attractants. Thus, nectarless flowers may be helping to maintain pollination in this scenario of trophic resource supply scarcity. We suggest that J. oxyphylla can be transitioning from a nectar-based pollination system to another resource-based or even to a deceit mechanism of pollination.

Preparation and characterization of D, L-PLA loaded 17-ß-Estradiol valerate by emulsion/evaporation methods.

PLA microparticles containing 17-ß-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert micro-particles) and 3-4 µm (drug loaded microparticles). The encapsulation efficiency was ∼80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-ß-estradiol.

Anatomy and ontogeny of the pericarp of Pterodon emarginatus Vogel (Fabaceae, Faboideae), with emphasis on secretory ducts.

Discrepant and incomplete interpretations of fruits of Pterodon have been published, especially on the structural interpretation of the pericarp portion that remain attached to the seed upon dispersal. The present work clarified these doubts and analyzed ultrastructural aspects of the Pterodon emarginatus diaspores using light and transmission electron microscopes. Cell divisions are prevalent among the initial phases of development, and the subadaxial and adaxial meristems form the fibrous inner mesocarp and the endocarp composed of multi-seriate epidermis, respectively. At the median mesocarp, numerous secretory ducts differentiate between the lateral bundles, by lytic process. After lysis of the central cells and the formation of the lumen, the ducts show unistratified secretory epithelium with dense cells; oil droplets are observed on the secretory epithelium and the subadjacent tissues. At maturity, the uniseriate exocarp and the outer mesocarp slough off in an irregular fashion, leaving the diaspore composed of a papery and brittle wing linked to a seed chamber that includes the median mesocarp composed of lignified cells, bordering vascular bundles and many secretory ducts whose epithelial cells develop large vacuoles that accumulate oleoresins. The Pterodon emarginatus fruit is a cryptosamara.

Anatomy and ontogeny of the pericarp of Pterodon emarginatus Vogel (Fabaceae, Faboideae), with emphasis on secretory ducts

Discrepant and incomplete interpretations of fruits of Pterodon have been published, especially on the structural interpretation of the pericarp portion that remain attached to the seed upon dispersal. The present work clarified these doubts and analyzed ultrastructural aspects of the Pterodon emarginatus diaspores using light and transmission electron microscopes. Cell divisions are prevalent among the initial phases of development, and the subadaxial and adaxial meristems form the fibrous inner mesocarp and the endocarp composed of multi-seriate epidermis, respectively. At the median mesocarp, numerous secretory ducts differentiate between the lateral bundles, by lytic process. After lysis of the central cells and the formation of the lumen, the ducts show unistratified secretory epithelium with dense cells; oil droplets are observed on the secretory epithelium and the subadjacent tissues. At maturity, the uniseriate exocarp and the outer mesocarp slough off in an irregular fashion, leaving the diaspore composed of a papery and brittle wing linked to a seed chamber that includes the median mesocarp composed of lignified cells, bordering vascular bundles and many secretory ducts whose epithelial cells develop large vacuoles that accumulate oleoresins. The Pterodon emarginatus fruit is a cryptosamara.

Interpretações discrepantes e incompletas têm sido conferidas ao fruto de Pterodon, especialmente no que tange à determinação estrutural da porção pericárpica que acompanha a semente na dispersão. Assim, com o objetivo de dirimir tais dúvidas e analisar a organização ultra-estrutural das estruturas secretoras presentes no diásporo de Pterodon emarginatus, realizaram-se estudos convencionais aos microscópios de luz e eletrônico de transmissão. Nas fases iniciais de desenvolvimento do fruto, prevalecem divisões celulares; pela ação do meristema subadaxial e do adaxial, formam-se, respectivamente, o mesocarpo interno fibroso e o endocarpo composto por epiderme multisseriada. No mesocarpo mediano, entre os feixes vasculares laterais, diferenciam-se numerosos ductos secretores lisígenos. Após a lise das células centrais e formação do lume, os ductos apresentam epitélio secretor uniestratificado, com células densas; gotas de óleo são observadas no epitélio secretor e tecido subjacente. Na maturidade, o exocarpo unisseriado e o mesocarpo externo, ambos fenólicos, descamam irregularmente, sendo o diásporo constituído pela ala papirácea e quebradiça, ligada ao núcleo seminífero que abrange o mesocarpo mediano de células lignificadas, margeando feixes vasculares e muitos ductos secretores, que apresentam acúmulo de oleorresina e cujas células epiteliais tornam-se vacuoladas. O fruto de Pterodon emarginatus é, portanto, uma criptossâmara.

Leaf emergences in Microlepis oleaefolia (DC.) Triana (Melastomataceae) and their probable function: an anatomical and ultrastructural study.

Microlepis oleaefolia (DC.) Triana, an endemic species of Melastomataceae from the Brazilian cerrado, presents very complex leaf structures called as "hairs with root-like base" embedded in the mesophyll. This paper describes the ontogenesis, histochemical and structural aspects of these leaf structures as a framework for further functional studies. Samples of leaves in different developmental stages were processed according to common light and electron microscopy techniques. Fresh material was subjected to histochemical tests to examine the chemical composition of cell walls. The apoplastic transport between leaf emergences and the vascular system was verified by staining with 1% aqueous safranin. The structures are emergences of mixed protoderm and ground meristem origin. They are persistent, predominantly short-stalked and dendritic. Their arms have thick non-lignified cellulosic walls with a loose and heterogeneous aspect; on the inner face, the wall, which appears labyrinthine, presents small irregularly shaped projections directed towards the protoplast. The base of the emergences is composed of sclereids embedded in the mesophyll that reach the vascular system. Assays with aqueous safranin solution revealed it penetrates the cell walls of the arms and showed a connection between the emergence and xylem. Anatomical, chemical and ultrastructural features of leaf emergences of M. oleaefolia indicate that these structures are able to transport substances via apoplast and can absorb or exude solutions.

Pulvinus functional traits in relation to leaf movements: a light and transmission electron microscopy study of the vascular system.

Previous studies on legume pulvini suggest that the vascular system plays an important role in the redistribution of ions and transmission of stimuli during leaf's movements. However, the number of anatomical and ultrastructural studies is limited to few species. The aim of this paper is to investigate the structure and cellular features of the pulvinus vascular system of nine legume species from Brazilian cerrado, looking for structural traits pointing to its participation in the leaf's movements. Samples were excised from the medial region of opened pulvinus of Bauhinia rufa, Copaifera langsdorffii, Senna rugosa (Caesalpinioideae), Andira humilis, Dalbergia miscolobium, Zornia diphylla (Faboideae), Mimosa rixosa, Mimosa flexuosa and Stryphnodendron polyphyllum (Mimosoideae), and were prepared following light microscopy, transmission electron microscopy and histochemical standard techniques. The vascular system occupies a central position, comprises phloem and xylem and is delimited by a living sheath of septate fibers in all the species studied. This living cells sheath connects the cortex to the vascular tissues via numerous plasmodesmata. The absence of fibers and sclereids, the presence of phenolic idioblasts and the abundance and diversity of protein inclusions in the sieve tube members are remarkable features of the phloem. Pitted vessel elements, parenchyma cells with abundant cytoplasm and living fibriform elements characterize the xylem. The lack of lignified tissues and extensive symplastic continuity by plasmodesmata are remarkable features of the vascular system of pulvini of the all studied species.