Unraveling the secretion mechanism of the curious nectaries in Gentianaceae.

Unusual nectaries were anatomically described as being usual traits for Gentianaceae. They are small, avascularized, and formed by 3 to 5 rosette cells with labyrinthine walls around one central cell. Such as nectaries have been reported for stems, petals, and sepals of different species of the family, however, there is no information on the mechanisms involved with the synthesis and release of secretion. Thus, this work aimed to unravel the mechanism of secretion and exudation of nectar for these curious nectaries using Calolisianthus speciosus as a model. Samples were processed according to standard methods for light and electron microscopy. Leaf and sepal nectaries were described, as were those of the apex of petals where ants were observed patrolling a darkened region. The enzymatic method was used for the detection of sugars, proteins, and amino acids in leaf and sepal exudates. The nectaries of petals of C. speciosus are similar to those of its leaves, sepals, and stem, although their activities are asynchronous. Polysaccharides were detected on the labyrinthine walls of rosette cells and protein in the opposite region of the cytoplasm. Labyrinthine walls increase the contact surface between rosette cells and the central cell, allowing for the transfer of secretion. After accumulation, the secretion is released to the subcuticular space of the central cell through disruption of the cuticle. The secretion and exudation of nectar were elucidated and involve distinct organelles.

Anatomy, Flow Cytometry, and X-Ray Tomography Reveal Tissue Organization and Ploidy Distribution in Long-Term In Vitro Cultures of Melocactus Species.

Cacti have a highly specialized stem that enables survival during extended dry periods. Despite the ornamental value of cacti and the fact that stems represent the main source of explants in tissue culture, there are no studies on their morpho-anatomical and cytological characteristics in Melocactus. The present study seeks to address the occurrence of cells with mixed ploidy level in cacti tissues. Specifically, we aim to understand how Melocactus stem tissue is organized, how mixoploidy is distributed when present, and whether detected patterns of ploidy change after long periods of in vitro culture. To analyze tissue organization, Melocactus glaucescens and Melocactus paucispinus plants that had been germinated and cultivated in vitro were analyzed for stem structure using toluidine blue, Xylidine Ponceau, Periodic Acid Schiff, ruthenium red, and acid floroglucin. To investigate patterns of ploidy, apical, medial, and basal zones of the stem, as well as, periphery, cortex, and stele (vascular tissue and pith) regions of the stem and root apexes from four- and ten-year old cultured in vitro were analyzed by flow cytometry. X-ray micro-computed tomography (XRµCT) was performed with fragments of stems from both species. The scarcity of support elements (i.e., sclereids and fibers) indicates that epidermis, hypodermis, and wide-band tracheids present in cortical vascular bundles and stele, as well as water stored in aquifer parenchyma cells along the cortex, provide mechanical support to the stem. Parenchyma cells increase in volume with a four-fold increase in ploidy. M. glaucescens and M. paucispinus exhibit the same pattern of cell ploidy irrespective of topophysical region or age, but there is a marked difference in ploidy between the stem periphery (epidermis and hypodermis), cortex, stele, and roots. Mixoploidy in Melocactus is not related to the age of the culture, but is a developmental trait, whereby endocycles promote cell differentiation to accumulate valuable water.

Rare earth elements induce cytoskeleton-dependent and PI4P-associated rearrangement of SYT1/SYT5 endoplasmic reticulum-plasma membrane contact site complexes in Arabidopsis.

In plant cells, environmental stressors promote changes in connectivity between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM). Although this process is tightly regulated in space and time, the molecular signals and structural components mediating these changes in interorganelle communication are only starting to be characterized. In this report, we confirm the presence of a putative tethering complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+- and lipid-binding protein 1 (CLB1/SYT7). This complex is enriched at ER-PM contact sites (EPCSs), has slow responses to changes in extracellular Ca2+, and displays severe cytoskeleton-dependent rearrangements in response to the trivalent lanthanum (La3+) and gadolinium (Gd3+) rare earth elements (REEs). Although REEs are generally used as non-selective cation channel blockers at the PM, here we show that the slow internalization of REEs into the cytosol underlies the activation of the Ca2+/calmodulin intracellular signaling, the accumulation of phosphatidylinositol-4-phosphate (PI4P) at the PM, and the cytoskeleton-dependent rearrangement of the SYT1/SYT5 EPCS complexes. We propose that the observed EPCS rearrangements act as a slow adaptive response to sustained stress conditions, and that this process involves the accumulation of stress-specific phosphoinositide species at the PM.

Calycinal secretory structures in Calolisianthus pedunculatus (Cham. & Schltdl) Gilg (Gentianaceae): anatomy, histochemistry, and functional aspects.

Secretory structures were little studied in Gentianaceae. Glandular areas on the calyx dorsal region are commonly reported for Helieae species, the main tribe of Gentianaceae. So, the elucidation of nature of glandular areas is particularly relevant. Trichomes secreting mucilage, interpreted as colleters, are reported only for the sepals of Gentianinae species. We aimed to anatomically characterize and identify the nature of the calycinal secretory structures in Calolisianthus pedunculatus. Samples from floral buds, flowers, and fruits were collected, fixed, and processed following usual procedures for light and scanning electron microscopies. Histochemical tests were performed to determine the nature of the secretion. Glucose, fructose, and sucrose were measured with an ELISA reader. Colleters occur on the sepal ventral region and are composed of a multicellular secretory head and a stalk. These structures secrete polysaccharides and proteins, and the secretion is probably released through cuticle microchannels. Nectaries, on the other hand, occur on the sepal dorsal region. They are formed by 3-5 cells arranged in rosettes circling a central cell or pore. These structures also secrete polysaccharides (mainly fructose), lipids, and proteins. The identification of the secretory structures in the sepals of Calolisianthus pedunculatus highlights the importance of anatomical studies in this family. The interpretation of the glandular areas on the calyx of the Helieae species as nectaries has been proven, as well as the confirmation of colleters as common structures in the sepals of Gentianaceae. Besides the taxonomic and phylogenetic importance of nectars and colleters, we highlight the importance of the secretion for the protection of floral buds against dehydration.

Arsenic hyperaccumulation in Pityrogramma calomelanos L. (Link): adaptive traits to deal with high metalloid concentrations.

Pityrogramma calomelanos is interestingly the single non-Pteris arsenic (As)-hyperaccumulating fern. It has been pointed as a potential species for phytoremediation and a model plant to study the As toxicity and its mechanisms of action. In order to investigate the morphoanatomical traits associated to As tolerance, P. calomelanos plants were exposed to different As concentrations in hydroponic solution. At low As dose (1 mM As), 90% of the As accumulated in plants was allocated in shoots, and no symptoms of As stress were observed in fronds and roots. Under higher As exposure (10 and 30 mM As), 81-74% of the total As in plants was present in shoots, and apical and marginal necroses on pinnae were observed. Anatomical observations showed that As induces damages mainly in the secondary veins and adjacent cells. High amounts of phenols were observed in pinna tissues of control and treated plants. In the roots, As promoted slight alterations as detachment of border-like cells and accumulation of granular substances in cortical cells. The high root-to-shoot As translocation and the constitutive presence of phenols and border-like cells protecting the root tips showed to be adaptive traits that allow P. calomelanos to survive in contaminated sites.

Clusia hilariana and Eugenia uniflora as bioindicators of atmospheric pollutants emitted by an iron pelletizing factory in Brazil.

The objectives of this work were to evaluate if the pollution emitted by the pelletizing factory causes visual symptoms and/or anatomical changes in exposed Eugenia uniflora and Clusia hilariana, in active biomonitoring, at different distances from a pelletizing factory. We characterize the symptomatology, anatomical, and histochemistry alterations induced in the two species. There was no difference in the symptomatology in relation to the different distances of the emitting source. The foliar symptoms found in C. hilariana were chlorosis, necrosis, and foliar abscission and, in E. uniflora, were observed necrosis punctuais, purple spots in the leaves, and increase in the emission of new leaves completely purplish. The two species presented formation of a cicatrization tissue. E. uniflora presented reduction in the thickness of leaf. In C. hilariana, it was visualized hyperplasia of the cells and the adaxial epidermis did not appear collapsed due to thick cuticle and cuticular flanges. Leaves of C. hilariana showed positive staining for iron, protein, starch, and phenolic compounds. E. uniflora showed positive staining for total phenolic compounds and starch. Micromorphologically, there was accumulation of particulate matter on the leaf surface, obstruction of the stomata, and scaling of the epicuticular wax in both species. It was concluded that the visual and anatomical symptoms were efficient in the diagnosis of the stress factor. C. hilariana and E. uniflora showed to be good bioindicators of the atmospheric pollutants emitted by the pelletizing factory.

Differential responses of C3 and CAM native Brazilian plant species to a SO2- and SPMFe-contaminated Restinga.

Aiming to evaluate responses in terms of growth rates, physiological parameters, and degree of sensitivity to SO2 and SPMFe in Eugenia uniflora L. (Myrtaceae, a C3 species) and Clusia hilariana Schlecht (Clusiaceae, a CAM species); saplings were exposed to emissions from a pelletizing factory for 7 months. The species were distributed along a transect (200, 500, 800, 1400, and 1700 m away from the emission source), and analyses were performed after 71, 118, and 211 days of exposure to the pollutants. E. uniflora received higher superficial deposition of particulate iron. The highest total iron foliar contents were observed 200 m away from the emission source in both plant species, while the highest total sulfur foliar contents were observed 200 m away in C. hilariana and 800 m away in E. uniflora. E. uniflora presented decreased values of height growth rate, number of necrotic leaves, chlorophyll analysis (SPAD index) and transpiration, in relation to the distances from the emission source. C. hilariana showed decreased values of height growth rate, number of leaves, number of necrotic leaves, total ionic permeability, stomatal conductance, transpiration, net CO2 assimilation, and total dry matter, in relation to distances from the emission source. In relation to the days of exposure, both species presented increased number of necrotic leaves and foliar phytotoxicity index, and decreased values in the chlorophyll analysis. The two native plant species, both of which occur in the Brazilian Restinga, showed damage when exposed to emissions from an iron ore pelletizing factory. C. hilariana was considered the most sensitive species due to the decreased values in a higher number of variables after exposition.

Morpho-anatomical and growth alterations induced by arsenic in Cajanus cajan (L.) DC (Fabaceae).

Arsenic (As) is a toxic element to most organisms. Studies investigating anatomic alterations due to As exposure in plants are scarce but of utmost importance to the establishment of environmental biomonitoring techniques. So, this study aimed to investigate the effects of As on the development and initial root growth in Cajanus cajan (Fabaceae), characterize and quantify the possible damages, evaluate genotoxic effects, and identify structural markers to be used in environmental bioindication. Plants were exposed hydroponically to 0.5, 1.0, 1.5, and 2.0 mg As L(-1), as sodium arsenate. Growth parameters were measured, and in the end of the exposure, root samples were analyzed for qualitative and quantitative anatomical alterations. Arsenic genotoxicity was evaluated through analysis of the mitotic index in the root apex. Compared to the control, As-treated seedlings showed an altered architecture, with significantly decreased root length (due to the lower mitotic index in the apical meristem and reduced elongation of parenchyma cells) with darkened color, and abnormal development of the root cap. A significant increase in vascular cylinder/root diameter ratio was also detected, due to the reduction of the cellular spaces in the cortex. The secondary xylem vessel elements were reduced in diameter and had sinuous walls. The severest damage was visible in the ramification zone, where uncommon division planes of phellogen and cambium cells and disintegration of the parenchyma cells adjacent to lateral roots were observed. The high sensibility of C. cajan to As was confirmed, since it caused severe damages in root growth and anatomy. The main structural markers for As toxicity were the altered root architecture, with the reduction of the elongation zone and increase of ramification zone length, and the root primordia retained within the cortex. Our results show a new approach about As toxicity and indicate that C. cajan is a promising species to be used for bioindication of environmental contamination by As.

Morphoanatomical responses induced by excess iron in roots of two tolerant grass species.

We aimed to verify whether morphoanatomic alterations occur in response to excess iron, in roots of Setaria parviflora and Paspallum urvillei (Poaceae), and to localize the presence of the sites of iron accumulation. Plants were subjected to 0.009, 1, 2, 4, and 7 mM Fe-EDTA in nutrient solution. Both species presented iron contents in the roots above the critical toxicity level. The presence of iron plaque on roots of the two species was confirmed, and it may have reduced iron absorption by the plants. Roots from the two species showed typical visual symptoms of stress by excess iron: change in color and mucilaginous and flaccid appearance. Anatomical damage was observed in both species: aerenchyma disruption, alterations in endodermal cells, and irregular shape of both vessel and sieve tube elements. The metal was histolocalized in the cortex and in protoxylem and metaxylem cell walls in both species, which suggests a detoxification strategy for the excess iron. Phenolic compounds were not histolocalized in roots. Microscopic analyses were therefore effective in evaluating the real damage caused by excess iron.

Leaf morphoanatomy of species tolerant to excess iron and evaluation of their phytoextraction potential.

Setaria parviflora (Poir.) Kerguélen and Paspalum urvillei Steudel are grasses that grow naturally in a soil with high iron contents. This study aimed to characterize morphoanatomically and histochemically the iron phytotoxicity on leaves and evaluate the phytoextraction potential of these grasses. Saplings were cultivated in hydroponic solution with and without excess Fe-EDTA. Regarding measurements taken on leaves, reduction was observed among treatments of Fe-EDTA on height values of abaxial epidermis and bundle sheath in both species. As for iron histolocalization, stronger reaction was observed in leaves of S. parviflora, in comparison with P. urvillei. Anatomical damage, such as protoplast retraction, irregular xylem, changes in cell volume, and cell collapse, and visual symptoms, like leaf bronzing, chlorosis, and necrosis, were similar in both species when exposed to excess iron; however, P. urvillei showed more severe damage. This species accumulated more iron in shoots than S. parviflora and therefore is more favorable for use in phytoextraction. The root system of both species accumulated higher iron concentrations in relation to shoots.

Tolerance to iron accumulation and its effects on mineral composition and growth of two grass species.

This study aimed to assess the influence of excess iron on the capacity of accumulation of this heavy metal, mineral composition, and growth of Setaria parviflora and Paspalum urvillei. Seedlings were submitted to 0.009; 1; 2; 4; and 7 mM of Fe-EDTA. In both species there was an increase in the concentration of Fe, Zn, P, and Ca and a decrease in Mn, K, and Mg in the iron plaque. Both species accumulated more iron in roots. In the shoots, S. parviflora showed higher iron content, except at 7 mM. Iron altered the contents of Fe, Cu, K, and Mg in roots, and of Fe, Mn, Zn, N, P, K, Ca, and Mg in shoots. The two species tolerated high iron concentrations and accumulated high content of this element in both shoots and roots. The iron did not reduce their growth. Both species are indicated for studies aiming restoration of iron-contaminated areas.

Extrafloral nectaries in neotropical Gentianaceae: occurrence, distribution patterns, and anatomical characterization.

PREMISE OF THE STUDY: Extrafloral nectaries (EFNs) are structures that secrete nectar and protect plants against herbivores and pathogens. In Gentianaceae, these structures have been described in species of Calolisianthus, Fagraea, and Anthocleista and are important morphological markers for taxonomic and phylogenetic studies. To establish a foundation for further studies, we investigated the occurrence, distribution patterns, and anatomy of EFNs on leaves of 27 species belonging to 13 genera and three tribes of neotropical Gentianaceae. • METHODS: Leaf samples were diaphanized, stained with basic fuchsin, and mounted in glycerinated gelatin. Cross sections were obtained from material embedded in methacrylate or paraffin, stained, and mounted in Permount. Polysaccharides were histochemically stained with periodic acid-Schiff stain. Samples were also examined with scanning electron microscopy. • KEY RESULTS: Unusual EFNs, visible only with light microscopy, were formed of modified epidermal cells. Each EFN consisted of 2-5 secretory cells encircling a central cell. The EFNs varied in size and in the shape and arrangement of the adjacent cells surrounding the secretory cells. EFNs occurred in all analyzed species as isolated units distributed throughout the leaf blade or as aggregates; aggregates were generally visible to the naked eye. Based on their occurrence as aggregates or isolated units and on their location on the leaf blade, six distribution patterns were identified. • CONCLUSIONS: This is the first comprehensive study of EFNs on the leaves of neotropical Gentianaceae. The data suggested that NEFs evolved from isolated units for NEFs in aggregates. The results represent a new source of data for future ecological, systematic, and phylogenetic studies in Gentianaceae.

Iron plaque formation and morphoanatomy of roots from species of restinga subjected to excess iron.

The restingas, a sandy coastal plain ecosystem of Brazil, have received an additional amount of iron due to the activity of mining industries. The present study aims to characterize morphoanatomically and histochemically the iron plaque formation on roots of Ipomoea pes-caprae L. and Canavalia rosea DC, cultivated in hydroponic solution with and without excess iron. The iron plaque formation as well as changes in the external morphology of the lateral roots of both species were observed after the subjection to excess iron. Changes in the nutrient uptake, and in the organization and form of the pericycle and cortex cells were observed for both species. Scanning electron microscopy showed evident iron plaques on the whole surface of the root. The iron was histolocalized in all root tissues of both species. The species of restinga studied here formed iron plaque in their roots when exposed to excess of this element, which may compromise their development in environments polluted by particulated iron.

Effects of simulated acid rain on leaf anatomy and micromorphology of Genipa americana L. ( Rubiaceae )

Experimentos foram conduzidos para avaliar o grau de susceptibilidade e determinar as injúrias causadas pela chuva ácida simulada na anatomia e micromorfologia foliar de Genipa americana. Plantas foram expostas à chuva com pH 3,0 durante 10 dias consecutivos. No tratamento controle utilizou-se apenas água destilada (pH 6,0). Amostras foliares foram coletadas e fixadas para microscopia de luz e eletrônica de varredura. Foram observados nas folhas expostas à chuva ácida: necroses pontuais intervenais, colapso das células do mesofilo e da epiderme; hipertrofia do parênquima lacunoso e acúmulo de compostos fenólicos e grãos de amido. A análise micromorfológica evidenciou, nas áreas necrosadas, plasmólise das células-guarda e ruptura da cutícula e da crista estomática. Alterações anatômicas ocorreram antes que sintomas visuais fossem observados nas folhas. Estes resultados comprovam a importância de dados anatômicos na diagnose precoce da injúria e na determinação da sensibilidade de G. americana à chuva ácida.