Updating the Knowledge on the Secretory Machinery of Hops (Humulus lupulus L., Cannabaceae).

Cannabaceae species garner attention in plant research due to their diverse secretory structures and pharmacological potential associated with the production of secondary metabolites. This study aims to update our understanding of the secretory system in Hops (Humulus lupulus L.), an economically important species especially known for its usage in beer production. For that, stems, leaves, roots, and inflorescences were collected and processed for external morphology, anatomical, histochemical, ultrastructural and cytochemical analyses of the secretory sites. Our findings reveal three types of secretory structures comprising the secretory machinery of Hops: laticifer, phenolic idioblasts and glandular trichomes. The laticifer system is articulated, anastomosing and unbranched, traversing all plant organs, except the roots. Phenolic idioblasts are widely dispersed throughout the leaves, roots and floral parts of the species. Glandular trichomes appear as two distinct morphological types: capitate (spherical head) and peltate (radial head) and are found mainly in foliar and floral parts. The often-mixed chemical composition in the secretory sites serves to shield the plant from excessive UVB radiation, elevated temperatures, and damage inflicted by herbivorous animals or pathogenic microorganisms. Besides the exudate from peltate glandular trichomes (lupulin glands), latex and idioblast content are also likely contributors to the pharmacological properties of different Hop varieties, given their extensive presence in the plant body.

The putative cannabinoid-secreting trichome of Trema micrantha (L.) Blume (Cannabaceae).

Trema, a genus of the popularly known Cannabaceae, has recently been the subject of cannabinoid bioprospection. T. micrantha is a tree with pharmacological potential widely used in folk medicine. It has two types of glandular trichomes, bulbous and filiform, spread throughout the plant body. Considering the proximity of this species to Cannabis sativa and Trema orientalis, species containing cannabinoids, the glandular trichomes of T. micrantha are also expected to be related to the secretion of these compounds. Thus, this study aims to detail the morphology of secretory trichomes during the synthesis, storing and release of metabolites in T. micrantha. We tested the proposition that they could be a putative type of cannabinoid-secreting gland. Pistillate and staminate flowers and leaves were collected and processed for ontogenic, histochemical, and ultrastructural analyses. Both types of glandular trichomes originate from a protodermal cell. They are putative cannabinoid-secreting sites because: (1) terpene-phenols and, more specifically, cannabinoids were detected in situ; (2) their secretory subcellular apparatus is consistent with that found in C. sativa: modified plastids, polyribosomes, an extensive rough endoplasmic reticulum, and a moniliform smooth endoplasmic reticulum. Plastids and smooth endoplasmic reticulum are involved in the synthesis of terpenes, while the rough endoplasmic reticulum acts in the phenolic synthesis. These substances cross the plasma membrane by exocytosis and are released outside the trichome through cuticle pores. The study of the cell biology of the putative cannabinoid glands can promote the advancement of prospecting for natural products in plants.

Diversity of Leaf Glands and Their Putative Functions in Rhamnaceae Species.

Leaf glands are found in many Rhamnaceae species, the buckthorn family, and are frequently used in taxonomic studies of the group, especially because they are easily visible to the naked eye. Despite the many records and extensive use in the taxonomy of the family, few studies deal with the classification of these glands and their roles for the plant. Thus, this study aimed to unravel the type, functioning, and putative functions of the leaf glands of three Brazilian forest species: Colubrina glandulosa Perkins, Gouania polygama (Jacq.) Urb., and Rhamnidium elaeocarpum Reissek. Leaves were collected and processed for surface, anatomical, histochemical, and ultrastructural analyses. In addition, the presence of visitor animals was registered in the field. The leaf glands of C. glandulosa and G. polygama are defined as extrafloral structured nectaries due to their anatomical structure, interaction with ants, and the presence of reduced sugars and of a set of organelles in the secretory cells. The unusual mechanism of nectar release and exposure in an apical pore stands out in G. polygama. The glands of R. elaeocarpum are ducts or cavities that secrete phenolic oil resin. Their presence is an atypical condition in the family, although they are often confused with mucilage reservoirs, much more common in Rhamnaceae. The extrafloral nectary, secretory cavity, and duct are associated with plant protection against phytophages, either by attracting patrol ants or by making the organs deterrent. Our data, combined with other previously obtained data, attest to the great diversity of gland types found in Rhamnaceae species.

Floral Development of Rhamnaceae and Origin of Its Unique Floral Features.

Rhamnaceae flowers have a peculiar morphology, including keeled sepals, one stamen whorl closely related to the petals, and a broad perigynous hypanthium that supports a voluminous nectary. In the present investigation, we detailed the flower development of five Rhamnaceae species to understand the origin of such specific floral characteristics. Floral buds and flowers were processed for surface and histological analyses. The sepals emerge in sequential order and the other organs in simultaneous order. The development of the perigynous hypanthium renders the floral apex broad and concave. The sepals undergo abaxial thickening early on, forming a keel and strongly influencing the floral merosity. Petals and stamens appear close to each other on the same radius in a very short plastochron. The carpels unite soon after their emergence, forming a syncarpous ovary and free style branches. Differences in intercalary carpel growth promote the formation of inferior (Gouania virgata) and semi-inferior ovaries (Colubrina glandulosa, Hovenia dulcis, and Sarcomphalus joazeiro). Rhamnidium elaeocarpum does not undergo such growth, and the resulting ovary is superior. The keeled sepals promote the isolation of the petal-stamen pair inside the flower bud. The possibility of a common primordium that the originates petal and stamen is refuted. Comparisons with other Rosales families provide insights into the floral origin and diversification of Rhamnaceae.

Mucilage cells in the flower of Rosales species: reflections on morphological diversity, classification, and functions.

The presence of mucilage cells in plants, studied mainly in vegetative organs, is a condition shared by several taxonomic groups and aspects related to their diversity have been discussed with systematic purposes. This study explores the flower distribution and classification of mucilage cells in Rosales species, with inferences about flower functions. Floral buds from fifty-seven species representing seven of nine families recognized in the Rosales were sampled and processed for light and transmission electron microscopy. Mucilage cells were found in about 40% of the studied species of Cannabaceae, Rhamnaceae, Ulmaceae, and Urticaceae families, whereas no floral mucilage cells were found in species of Elaeagnaceae, Moraceae, and Rosaceae. Mucilage cells were found in the epidermis and internal tissues of many organs of different floral morph types. There is a great diversity of forms of presentation of mucilage in cells, from smaller individualized single cells to very bulky cells and to completely filled mucilage reservoirs. In some cases, cells with mucilage apparently in the cell wall and others with mucilage in the vacuole seem to occur side by side. This diversity challenges the existing classifications of mucilage cells and reinforces the importance of ontogenetic and ultrastructural studies following the path of mucilage in cells in order to propose a more natural classification and to elucidate the evolution of mucilage cells in plants.

Connective modifications and origin of stamen diversity in Melastomataceae.

The androecium of Melastomataceae presents notable modifications in its merosity, morphology between whorls and in prolonged connectives and appendages. We carried out a comparative study of six Melastomataceae species to shed light on the developmental processes that originate such stamen diversity. The development of stamens was studied using scanning electron microscopy and histological observations. The stamens of all species studied have a curved shape because they emerge on a plane displaced by the perigynous hypanthium. They are the last flower organs to initiate and therefore their growth is inwards and towards the floral center. Despite the temporal inversion between carpels and stamens in Melastomataceae, the androecium maintains the centripetal pattern of development, the antepetalous stamens emerging after antesepalous stamens. The isomerous androecium can be the result of abortion of the antepetalous stamens, whereas heterostemony seems to be caused by differences in position and the stamen development time. Pedoconnectives and ventral appendages originate from the basal expansion of the anther late in floral development. The delay in stamen development may be a consequence of their dependence on the formation of a previous space so that they can grow. Most of the stamen diversity is explained by the formation of the connectives and their appendages. The formation of a basal-ventral anther prolongation, which culminates in the development of the pedoconnective, does not differ from other types of sectorial growth of the connective, which form shorter structures.

Evolution of the Anther Gland in Early-Branching Papilionoids (ADA Clade, Papilionoideae, Leguminosae).

Papilionoideae is the most diverse subfamily of Leguminosae, especially in terms of floral morphology. The ADA clade shows some exciting floral features among papilionoids, such as anther glands. However, the evolution of the anther glands in such early-branching papilionoids remains unknown. Thus, we compared the occurrence, distribution, morphology, and evolutionary history of the anther glands in species of the ADA clade. Floral buds and/or flowers in 50 species were collected from herbarium specimens and investigated using scanning electron and light microscopy and reconstruction of ancestral character states. The anther apex has a secretory cavity, secretory duct, and phenolic idioblast. The lumen shape of the cavity and duct is closely related to the shape of the anther apex. The oval lumen is located between two thecae, the spherical lumen in the prominent anther apex and the elongated lumen in anthers with a long apex. The occurrence of cavities/ducts in the anther in only two phylogenetically closely related subclades is a unifying character -state. The floral architecture is not correlated with cavity/ducts in the anther but is possibly related to the type of pollinator. Future research needs to combine floral morphology and pollination systems to understand the evolution of floral designs and their diversification.

Resolving the non-papilionaceous flower of Camoensia scandens, a papilionoid legume of the core genistoid clade: development, glands and insights into the pollination and systematics of the group.

Camoensia scandens is a papilionoid legume inserted in the core genistoid clade. It has large, crepuscular, scented flowers but the corolla is non-papilionaceous, which deviates from the pattern found in the subfamily. The vexillum has a folded claw, forming a tube, which is opposed to the androecium opening; all petals have yellow-gold crinkled margins. In addition, there is a long hypanthium, which stores a translucid liquid. The goal of this study is to elucidate the ontogenetic pathways that result in such a peculiar flower and the glands responsible for the sweet fragrance of the petals. Floral buds and flowers were processed for SEM, TEM and light microscopy analyses. Five sepals arise unidirectionally followed by five petals that initiate simultaneously. After the petals, 11 stamens emerge unidirectionally; a pair of adaxial stamens is opposite to the vexillum. In the intermediate developmental stages the sepals unite basally; the two adaxial sepals unite with each other to a greater extent than with the other sepals. The filaments are basally connate, forming a tube with an adaxial opening at the base. The carpel emerges concomitantly with the two abaxial antepetalous stamens. The long hypanthium forms from the outer floral organs (base of the sepals, petals, filaments) and is attached to the base of the stipe. The corolla is noticeable in the intermediate stages of development. The crinkled golden margins house scent glands formed of a secretory epidermis with secretory trichomes and secretory subepidermal cells. The odor is composed of neutral polysaccharides, nitrogenous substances and essential oils. An extensive nectariferous region is found on the inner surface of the hypanthial tube. The nectar is translucent, viscous and released through large pores. The comparison of our data with that of other genistoid flowers enabled discussions about the pollination and systematics of the group.

Bloodwood: the composition and secreting-site of the characteristic red exudate that gives the name to the Swartzia species (Fabaceae).

The Swartzia species are commonly known as bloodwood due to the red exudate released from the stem after injury. This exudate has aroused great interest, and an integrative study is essential to describe it in detail. Thus, this work aimed to identify the red exudate's secreting-site in S. flaemingii and S. langsdorffii, and determine if it is a latex or a resin. Samples of the stem bark and the secondary xylem were prepared for histological analysis. Fresh exudates were dissolved in deuterated methanol and analyzed by 1H-NMR; other samples were resuspended in MeOH:H2O (9:1), partitioned with organic solvents and analyzed by direct infusion mass spectrometry. Total phenolic and total flavonoid contents were determined spectrophotometrically, and antioxidant capacity was determined using ferric reducing antioxidant power assay. The results showed that the exudate is a red latex produced by articulated laticifers located among the phloem cells. The latex is composed of sucrose, catechin glucosides, chlorophyll derivatives, and hederagenin-type saponins. Both samples of S. flaemingii and S. langsdorffii presented high amounts of phenolics and flavonoids, as well as a strong antioxidant capacity. The anatomical study showed that the secreting-site of the Swartzia red exudates were laticifers. This finding allows us to exclude other substances such as resin or oleoresin, generally produced by secretory cavities or ducts. Furthermore, since laticifers are rare in Fabaceae, this finding is significant, and represents an essential taxonomic feature. The showy red color is due to the large amounts of flavonoids. This latex probably has a protective role against microorganisms and photodamage. The bioactive potential of this exudate inspires further studies, which may boost the economic importance of Swartzia.

What makes a fig: insights from a comparative analysis of inflorescence morphogenesis in Moraceae.

BACKGROUND AND AIMS: Moraceae, the family of mulberry and fig trees, displays small homogeneous flowers but extremely diverse inflorescences ranging from simple and branched to complex and condensed. Inflorescences also vary in flower organization in the receptacle, in the degree of flower condensation and in receptacle shape. Thus, the objective of the present study was to compare the inflorescence morphogenesis of Moraceae species, to investigate whether clades with a similar pollination mode share the same patterns of inflorescence development and the developmental stages at which we observe the key changes resulting in the diversified inflorescence architecture that culminates in the Ficus syconium. METHODS: Inflorescences at different developmental stages were sampled from Brosimum gaudichaudii, Castilla elastica, Clarisia ilicifolia, Ficus pertusa, Maclura tinctoria and Morus nigra and processed for surface and anatomical analyses. KEY RESULTS: The inflorescence morphogenesis of the studied species is highly variable. The shape of the inflorescence meristem (bulging, hemispheric or elongated), the initiation order and arrangement of flowers along the receptacle and the occurrence of bracts vary between related species. This diversity originates early during inflorescence development. Brosimum gaudichaudii, C. elastica and F. pertusa have flowers enclosed or immersed within the receptacle, although inflorescences begin their development as flat and open structures, as occurs in the other three study species. CONCLUSION: Comparison of the inflorescence morphogenesis in Moraceae species allows us to infer that evolutionary ontogenetic changes driven by pollinators culminate in the enclosure of flowers inside the receptacle, as occurs in the Ficus syconium.

Expanding the laticifer knowledge in Cannabaceae: distribution, morphology, origin, and latex composition.

Cannabaceae is a known family because of the production of cannabinoids in laticifers and glandular trichomes of Cannabis sativa. Laticifers are latex-secreting structures, which in Cannabaceae were identified only in C. sativa and Humulus lupulus. This study aimed to expand the knowledge of laticifers in Cannabaceae by checking their structural type and distribution, and the main classes of substances in the latex of Celtis pubescens, Pteroceltis tatarinowii, and Trema micrantha. Such information is also updated for C. sativa. Samples of shoot apices, stems, leaves, and flowers were processed for anatomical, histochemical, ultrastructural, and cytochemical analyses. Laticifers are articulated unbranched in all species instead of non-articulated as previously described for the family. They occur in all sampled organs. They are thick-walled, multinucleate, with a large vacuole and a peripheral cytoplasm. The cytoplasm is rich in mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, and plastids containing starch grains and oil drops. Pectinase and cellulase activities were detected in the laticifer wall and vacuole, confirming its articulated origin, described by first time in the family. These enzymes promote the complete dissolution of the laticifer terminal walls. The latex contains proteins, lipids, and polysaccharides in addition to phenolics (C. sativa) and terpenes (C. pubescens, T. micrantha). The presence of laticifers with similar distribution and morphology supports the recent insertion of Celtis, Pteroceltis, and Trema in Cannabaceae. The articulated type of laticifer found in Cannabaceae, Moraceae, and Urticaceae indicates that the separation of these families by having distinct laticifer types should be reviewed.

Cellulases and pectinases act together on the development of articulated laticifers in Ficus montana and Maclura tinctoria (Moraceae).

The presence of articulated laticifers in the Moraceae family was recently discovered, which means that the location of pectinase and cellulase activities must be of great importance for their growth. Thus, the present study aimed to determine the role of these enzymes in the laticifer growth in Ficus montana and Maclura tinctoria. Reproductive meristems were collected and fixed in Karnovsky. Pectinase and cellulase labeling was performed in part of the samples, while another part was processed for usual TEM analyses. Pectinase and cellulase activities were detected in the vacuole and close to the middle lamella in both species. The presence of cellulases in the laticifers supports their articulated origin. Therefore, the occurrence of pectinase and cellulase activity in the laticifers points out that these enzymes could act in the dissolution of the transverse walls and in the processes of intrusive growth (through the dissolution of the middle lamella) and cell elongation (through the partial disassembly of components of the wall making it more plastic). Both enzymes are synthesized in the endoplasmic reticulum and transported to the cell wall by exocytosis or stored in the vacuole. The species studied showed a diverse subcellular composition, which is probably related to the species and not to the laticifer type (they present the same type) and to the composition of the latex (they show similar latex composition). We conclude that the presence of pectinases and cellulases can be used as a diagnostic condition for the laticifer types (articulated vs. non-articulated).

Aluminium detoxification in facultative (Passovia ovata (Pohl ex DC.) Kuijt and Struthanthus polyanthus Mart. - Loranthaceae) and dependent (Psittacanthus robustus (Mart.) Marloth - Loranthaceae) Al-accumulating mistletoe species from the Brazilian savanna.

Mechanisms to detoxify aluminium (Al) is a hot topic for cultivated plants. However, little information is known about the mechanisms used by native plants to deal with Al-toxicity. In Cerrado, some generalist mistletoe species, such as Passovia ovata (Pohl ex DC.) Kuijt and Struthanthus polyanthus Mart. can parasitize Al-accumulating and Al-excluding plant species without any clear symptoms of toxicity and mineral deficiency, while Psittacanthus robustus (Mart.) Marloth, a more specialist mistletoe, seems to be an Al-dependent species, parasitizing only Al-accumulating hosts. Here we (i) characterized the forms and compartmentalization of Al in leaves of P. robustus; (ii) compared Ca and Al leaf concentration, and leaf concentration of organic acids and polyphenols between facultative Al-accumulating (P. ovata and S. polyanthus) and Al-dependent (P. robustus) mistletoe species infecting Miconia albicans (Sw.) Steud. (Al-accumulating species). P. robustus chelated Al3+ with oxalate and stored it in the phloematic and epidermic leaf tissues. Leaf Ca and Al concentration did not differ among species. Leaf oxalate concentration was higher in the Al-dependent species. Concentrations of citrate and phenolic compounds were higher in the leaves of the facultative Al-accumulating species. These results show that facultative Al-accumulating and Al-dependent species use different mechanisms to detoxify Al. Moreover, this is the first report on a mistletoes species (P. robustus) with a potential calcifuge behaviour in Cerrado.

The ontogenetic bases for variation in ovary position in Melastomataceae.

PREMISE OF THE STUDY: Although the ovary position is considered a stable character in angiosperms, Melastomataceae species have perigynous flowers in which the ovary varies from superior to inferior. Thus, we investigated the ontogenetic process involved in variation of the ovary position in Melastomataceae. We focused on histogenesis of the floral apex in search of developmental patterns for each type of ovary position. METHODS: Six species in which the ovary varies from superior to inferior were chosen: Henriettea saldanhae, Leandra melastomoides, Miconia dodecandra, Microlicia euphorbioides, Rhynchanthera grandiflora, and Tibouchina clinopodifolia. Buds and flowers were processed for surface and histological examinations. KEY RESULTS: The floral apex changes from convex to concave, resulting in a perigynous hypanthium. Cell divisions in the margins of the floral apex form an annular intercalary meristem that elevates the base of the primordia of almost all whorls. The joint growth of the carpel base with the gynoecial hypanthium originates semi-inferior ovaries in Leandra melastomoides, Miconia dodecandra, and Tibouchina clinopodifolia and inferior ovaries in Henriettea saldanhae. In Microlicia euphorbioides and Rhynchanthera grandiflora, the carpels are not affected by this hypanthial growth; flowers have a superior ovary. CONCLUSIONS: Changes in ovary position of Melastomataceae are due to intercalary meristematic activity, which is one of the main mechanisms for the origin of morphological innovations among plants. Our data illustrate the importance of the intercalary meristems in floral development, and we discuss the implications of this ontogenetic model for understanding the evolution of ovary position in Melastomataceae.

Comparative development of rare cases of a polycarpellate gynoecium in an otherwise monocarpellate family, Leguminosae.

PREMISE OF THE STUDY: Apocarpy (i.e., free carpels) is considered to be the basal condition for ovary development in angiosperms. Yet it only occurs in 10% of angiosperm species, of which another 10% are monocarpellate. Most legume flowers are monocarpellate. Species with polycarpellate gynoecia occur in about 15 genera with most representatives in Mimosoideae. In the present study, we analyze legumes with polycarpellate flowers with the aim of improving our understanding of gynoecium evolution. METHODS: Flowers of nine legume species from five genera were analyzed using a scanning electron microscope (SEM). KEY RESULTS: In Leguminosae, carpels usually form as individual primordia or protuberances. Inga congesta differs slightly from this pattern in that the central apex bulges outward before the formation of individual carpel primordia. While legumes usually develop entirely plicate carpels, flowers of Acacia celastrifolia and Inga bella show an intermediate type of carpel morphology with a distal plicate zone and a small proximal ascidiate zone. Carpels in Inga congesta and Archidendron glabrum are sometimes slightly fused at the ovary base. The orientation of carpel clefts seems to reflect the floral symmetry. They are directed to the floral center in mimosoids and caesalpinioids, whereas in Swartzia dipetala carpel clefts are oriented to the adaxial side. CONCLUSIONS: Polycarpelly arose at least seven times independently in Leguminosae. The polycarpellate condition appears to be correlated with polyandry, and in most instances, it is accompanied by a profound change in floral organization from a closed to an open system.

Mass spectrometry of flavonoid vicenin-2, based sunlight barriers in Lychnophora species.

Lychnophora salicifolia plants collected from four different places in Brazil (three states: Goias, Minas Gerais and Bahia) revealed a conserved accumulation of vicenin-2, a di-C-glycosyl flavonoid. Quantitative studies by UPLC-MS/MS showed high concentration of vicenin-2 in leaves from sixty specimens of six Lychnophora species. So the tissue distributions of vicenin-2 were evaluated in wild Lychnophora leaves (Asteraceae) by laser based imaging mass spectrometry (IMS) to propose its distributions and possible functions for the species analyzed. Mass spectrometric imaging revealed that vicenin-2, unlike other flavonoids, was produced at the top of the leaves. The combination of localization and UV absorption properties of vicenin-2 suggests that it could act as a UV light barrier to protect the plants, since plants are sessile organisms that have to protect themselves from harsh external conditions such as intense sunlight.