General morphology
The inflorescences of Vigna adenantha have 9 or more nodes in the main axis; a globose secondary axis originates in each node, which bears 2 flowers and 5 to 7 EFNs (Figure 1). In the first node of this secondary axis three buds originate, two of them develop into flowers, the third one becomes in an EFN, in the following nodes, only EFNs develop (Figure 2A, B). The nectaries are sessile, tightly disposed (Figure 3A). Each one has a structure that consists of a group of cells located in the center, surrounded by a complete or middle-moon shape ring (Figure 3A). The formation of a middle moon or complete ring depends on the relative position of each EFN in the secondary axis and the consequent available space.
Ontogeny
Four stages of development were distinguished in the EFNs. In the following description the development of the first EFN is assessed and it is correlated with the microsporogenesis, microgametogenesis, megasporogenesis and megagametogenesis of the flowers of the same node. The following EFNs of the secondary axis have the same ontogeny.
Stage 1. The bud of the EFN is placed in the axil of a bract of the secondary axis and is constituted by a protodermis which protects the underlaying meristematic tissue (Figure 2C). The bract has simple and glandular pluricellular trichomes in its abaxial surface (Figure 3B). Around or beside the bud, a ring begins to develop; it is constituted by a protodermis which protects the underlaying parenchyma. Beneath the bud as well as beneath the developing ring, procambial bands can be observed which originate phloem and xylem (Figure 2C).
In the same node of the secondary axis, two floral buds are developing as floral primordia, the microspore mother cells in their first meiotic division are found in the anthers, but the megaspore mother cell has not differentiated yet in the ovules.
Stage 2. Beneath the bract, two bracteoles are formed (Figure 2D), which are pilose in their abaxial face, with simple and glandular pluricellular trichomes (Figure 3C). In the meristematic apex of the bud, sepals primordia can be observed (Figures 2D and 3D). At the base of the bud, a band of cells more vacuolated than the surrounding ones are distinguished, which would correspond to a future abscission zone (Figure 2D). Below this zone, there are longitudinally enlarged cells, with thin primary walls and large nuclei. The ring of the future EFN is already totally differentiated: it is constituted by an epidermis and a parenchyma without intercellular spaces, with highly vacuolated cells and with conspicuous nuclei (Figure 2D). At the base of the EFN in formation, vascular bands are observed (Figure 2E, F).
In the corresponding floral buds of the same node, microspore tetrads are present in the anthers and the megaspore mother cell begins the meiotic division in the ovule.
Stage 3. The bud of the EFN does not continue its development and detaches from the abscission zone, leaving exposed the longitudinally enlarged cells in the center of the nectary (Figures 2G and 3E, F). These central cells elongate longitudinally even more, remaining as papillae, whose walls partially degrade exhibiting perforations (Figure 3G). Bellow these central cells there is a parenchymatic tissue. The secretion, which stains strongly with safranin-fast green (Figure 2G) and toluidine blue (Figure 2H), accumulates among the central cells. The parenchyma cells of the ring have conspiscuous vacuoles with content that also stain intensely with toluidine blue (Figure 2H).
In the corresponding floral buds of the same node, the anthers have pollen grains already formed and some tapetum rests, while in the ovules the megaspore is in the mitotic process which ends in the megagametophyte formation.
Stage 4. Nectar secretion ceases and the central cells desintegrate (Figure 3H).
In the corresponding floral buds of the same node anthers are dehiscent and the tapetum is completely dissolved while in the ovules, the megagametophyte is totally formed. At this moment, anthesis begins.
Ultrastructure of the secretory stage (stage 3)
Ring
The epidermal cells of the ring exhibit a very thick outer tangential wall, crossed by conspicuous ectodesmata (Figure 4A), some of them filled with a highly electrondense content or with vesicles inside (Figure 4B). An equivalent electrodense content accumulates outside the invaginated plasmalemma (Figure 4B, arrows) and outside the outer tangential wall (Figure 4B, arrowheads). The middle lamella of the radial walls appears distended and cavities can be seen along it (Figure 4A). The parenchyma cells have very dense cytoplasm, evident vacuoles with fibrilar content of varying electrondensity, rough endoplasmic reticulum, numerous free ribosomes, dictyosomes, abundant amyloplasts with one to several starch granules, mitochondria and lipidic globules (Figure 4, C-F). Plasmodesmata can be observed between parenchymatic cells (Figure 4F).
Central cells
The cytoplasm of the elongated and papillose cells has numerous free ribosomes, abundant mitochondria and dictyosomes (Figure 5A, B). The underlying parenchymatic cells exhibit vacuoles with medium-electrondense content, amyloplasts, plastids that contain both starch granules and lipidic globules, free lipidic globules and mitochondria (Figure 5 C, D). Plasmodesmata connect these cells (Figure 5D).
Nectary activity period
During the inflorescence development, once the first EFN of a secondary axis arrives to stage 3, the second EFN begins to differentiate in the next node and so on, in the acropetal sense of the secondary axis (Figures 2B and 3A).
When the fruits have seeds with embryos in the globular stage, at least one EFN in stage 4, one EFN in stage 3 and one EFN in stage 1 coexist in the same secondary axis of the inflorescence, so the EFNs activity is continuous during maturation of the fruits (Figure 2A).
Nectar concentration
Nectar removed from nectaries in stage 3 was highly viscose, the concentration ranged from 0.02 to 12.5 (3.01 ± 4.78, n = 5) percent of total sugars. During sunny days with low ambient humidity, crystallization of the nectar was observed on the EFN surface.