Comparative floral spur anatomy and nectar secretion in four representatives of Ranunculaceae

Abstract

Nectaries are common in Ranunculaceae. These secretory structures, however, have not been studied in detail despite their importance in plant-animal interactions, and data relating to the structure of nectary spurs, which are so characteristic of several genera of this family, remain scarce. In order to redress this imbalance, we sought, in the present paper, to analyze the anatomical and ultrastructural organization of the nectary spurs of four representatives of Ranunculaceae, i.e., Aconitum lycoctonum L., Aquilegia vulgaris L., Consolida regalis Gray, and Delphinium elatum L. Nectary spurs were examined using light, fluorescence, scanning electron, and transmission electron microscopy. The floral nectaries of A. lycoctonum and A. vulgaris are situated at the apices of the spurs, whereas in C. regalis and D. elatum, the nectary is located along the floor surface of the spurs. Nectar in C. regalis and D. elatum is exuded through micro-channels in the cuticle, whereas in A. lycoctonum and A. vulgaris, it is released by means of cell wall disruption, indicating that the method of nectar secretion here is holocrine. Structurally, the nectary of all four investigated species is quite similar, and its cells are typical of nectar-producing cells described in the literature. It is proposed that in A. lycoctonum and A. vulgaris, disruption of the cell wall and the release of the entire cell contents into the spur cavity contribute to the composition of the nectar that the latter contains, enriching it with cytoplasmic components. We conclude that the manner of nectar exudation may vary considerably between closely related plant species, regardless of their geographical origin and phylogeny.

Keywords: Cell ultrastructure; Cuticle micro-channels; Holocrine secretion; Morphology and anatomy; Nectary structure; Secretory structures.

Fig. 1

Habit of the flower and floral histology of Aconitum lycoctonum: d–h light micrographs. a Inflorescence with zygomorphic, pale yellow flowers. Scale bar = 10 mm. b Lateral view of a dissected flower with two curled nectary spurs (arrows) visible. Scale bar = 5 mm. c Longitudinal section through the nectary spur, note secretory area at the apex of the spur (arrow). Scale bar = 2 mm. d Section of nectary showing internal epidermis, underlying secretory parenchyma, and non-glandular parenchyma. Scale bar = 50 μm. e The secretory cells after PAS staining, note ruptured outer cell walls of the internal epidermis (arrows). Scale bar = 20 μm. f Section of nectary showing plastids with starch grains (arrows) and these are more abundant toward the external epidermis. Scale bar = 50 μm. g Cuticle covering internal epidermal cells that stains only slightly with auramine O, note secretory material extruding from ruptured epidermis (arrow), which is also positive with auramine O (asterisk). Scale bar = 20 μm. h Section showing lack of autofluorescence of chlorophyll in the secretory cells. Scale bar = 100 μm

Fig. 2

Scanning electron micrographs and ultrastructure of nectary spur of Aconitum lycoctonum: a–d scanning electron micrographs, e–h transmission electron micrographs. a Spur cut longitudinally showing spur cavity. Scale bar = 200 μm. b Glabrous surface of the internal epidermis of the nectary spur with several secreting cells (arrows). Scale bar = 20 μm. c Details of internal epidermis with secretory material extruded by disrupted cells (asterisks). Scale bar = 10 μm. d Internal epidermis with cells at different stages of secretion: Secreting cell forms blister containing cytoplasm (arrow), whereas another (asterisk) releases its secretion via the ruptured cell wall. Scale bar = 10 μm. e Internal epidermis with relatively thick outer cell wall and thin cuticle, note pit field in the radial cell wall (arrow). Scale bar = 2 μm. f Secretory material (asterisk) with nucleus, mitochondria, cytoplasm, and other disorganized organelles being visible in the spur cavity. Scale bar = 5 μm. The inset shows broken outer cell wall of the internal epidermis with a secretory material (asterisk) dispersing into the spur cavity. Scale bar = 2.5 μm. g Subepidermal nectariferous parenchyma cells, each containing a large nucleus, mitochondria, secretory vesicles, and plastids, note numerous plasmodesmata (arrows) between the radial cell walls. Scale bar = 2 μm. h Cells of the nectar-producing parenchyma with nucleus, several small vacuoles, and arrays of rough endoplasmic reticulum. Scale bar = 5 μm

Fig. 3

Habit of the flower and floral histology of Aquilegia vulgaris: c–g light micrographs. a Purple flower with long nectary spurs (arrows). Scale bar = 10 mm. b Longitudinal section through the spur showing green nectariferous tissue located at its apex (arrow). Scale bar = 1 mm. c Transverse section of the spur with nectariferous tissue and underlying, non-glandular parenchyma with vascular bundles. Scale bar = 200 μm. d Details of nectariferous cells with intensely staining cytoplasm and numerous vacuoles, note several internal epidermal cells with broken outer cell walls (arrows). Scale bar = 20 μm. e Section of nectary tissue showing nectar-producing and non-glandular parenchyma cells, both accumulating starch grains in plastids, note phloem elements penetrating nectar-producing parenchyma. Scale bar = 50 μm. f Internal secretory epidermis with a thin cuticle layer that stains only slightly with auramine O. Scale bar = 20 μm. g Strong autofluorescence of chlorophyll occurs both in nectar-producing and ground parenchyma cells. Scale bar = 100 μm

Fig. 4

Scanning electron micrographs and ultrastructure of nectary spur of Aquilegia vulgaris: a–d scanning electron micrographs, e–j transmission electron micrographs. a Micrograph showing spur cavity. Scale bar = 200 μm. b Interface between nectariferous (n) and non-glandular surfaces. Scale bar = 100 μm. c Surface of nectariferous tissue showing several secreting cells; secreted material is present (asterisks). Scale bar = 20 μm. d Details of internal epidermis with extruded secretory material (asterisk); following the release of secretory material, depressions are evident on some epidermal cells (arrows). Scale bar = 10 μm. e Internal epidermal cells with large, centrally positioned nucleus and dense cytoplasm; numerous pit fields occur in radial cell walls (arrows). Scale bar = 5 μm. f The outer cell wall of the internal epidermis becomes ruptured, and the cytoplasm and disorganized organelles can be seen in the secreted material. Scale bar = 5 μm. g General view of nectar-secreting parenchyma showing large central vacuole and dense parietal cytoplasm with mitochondria and plastids. Scale bar = 5 μm. h Secretory parenchyma cells with parietal cytoplasm, large central vacuole, and profiles of rough endoplasmic reticulum. Scale bar = 500 nm. i Cytoplasm with mitochondria and plastids containing partly hydrolyzed starch grains, note plasmodesmata connecting adjacent cells (arrow) and intravacuolar myelin-like figure (ml). Scale bar = 1 μm. j Nectariferous parenchyma cells with numerous mitochondria, dictyosomes, and plastids with eroded starch grains, note the formation of electron-translucent profile within plastids (asterisks). Scale bar = 500 nm

Fig. 5

Habit of the flower and floral histology of Consolida regalis: c–g light micrographs. a Inflorescence with dark purple flowers and spurred organs (arrows). Scale bar = 5 mm. b Longitudinal section through floral spur showing light green nectary located at the floor of the spur (arrows). Scale bar = 0.5 mm. c Transverse section of the spur showing nectariferous tissue (n) and spur cavity (sc). Scale bar = 200 μm. d Section showing secretory cells with parietal cytoplasm, relatively large nuclei, and vascular bundles containing both phloem and xylem. Scale bar = 50 μm. e Parenchyma cells adjacent to sieve tubes contain numerous plastids with large starch grains (arrows). Scale bar = 50 μm. f Cuticle overlying internal epidermis is thick and stains intensely with auramine O. Scale bar = 20 μm. g Section showing chlorophyll autofluorescence both in secretory and ground parenchyma cells. Scale bar = 100 μm

Fig. 6

Scanning electron micrographs and ultrastructure of nectary spur of Consolida regalis: a–b scanning electron micrographs, c–g transmission electron micrographs. a Glabrous surface of inner epidermis. Scale bar = 100 μm. b Details of internal epidermis with highly striate cuticle and nectar residues (arrows) between rows of adjacent epidermal cells. Scale bar = 20 μm. c Details of outer tangential wall of internal epidermis with thick cuticle layer, containing numerous micro-channels. Scale bar = 2 μm. d Internal epidermal cells containing long profiles of rough endoplasmic reticulum, vesicles, large nuclei, and central vacuole, note the numerous plasmodesmata connecting the epidermal cells (arrows). Scale bar = 2 μm. e Section showing cytoplasm of internal epidermal cell containing mitochondria and numerous small vacuoles, note relatively large intercellular space (is) between these cells and adjoining nectary parenchyma. Scale bar = 2 μm. f Cytoplasm of secretory parenchyma cells, note numerous plasmodesmata connecting adjacent cells (arrows). Scale bar = 2 μm. g Secretory parenchyma with rough ER profiles and plastids containing partly hydrolyzed starch grains, note the formation of electron-translucent profile in plastids (asterisk). Scale bar = 2 μm

Fig. 7

Habit of the flower and floral histology of Delphinium elatum: d–g light micrographs. a Inflorescence with dark blue flowers. Scale bar = 20 mm. b Lateral view of a dissected flower showing two nectary spurs (arrows) (dorsal sepal removed). Scale bar = 10 mm. c Longitudinal section through the spur showing nectariferous tissue at the ventral surface (arrows). Scale bar = 2 mm. d Section through the nectary spur showing spur cavity (sc) and nectariferous tissue at the spur floor (n). Scale bar = 200 μm. e Section of nectary showing internal epidermis, underlying nectariferous parenchyma cells, and vascular bundles containing phloem and xylem, located in ground parenchyma, note large starch grains (arrows) in parenchyma cells, especially around sieve tubes. Scale bar = 50 μm. f Red chlorophyll autofluorescence in the ground parenchyma cells. Scale bar = 100 μm. g Thick cuticle on outer walls of the internal epidermis stains intensely with auramine O, note pit fields between secretory parenchyma cells (arrows). Scale bar = 20 μm

Fig. 8

Scanning electron micrographs and ultrastructure of the nectary spur of Delphinium elatum: a, b scanning electron micrographs, c–h transmission electron micrographs. a Glabrous and striate cuticle of the inner epidermis. Scale bar = 50 μm. b Details of internal epidermis with nectary residues (arrows) between rows of adjacent cells. Scale bar = 20 μm. c Details of outer tangential wall of internal epidermis with cuticle containing numerous micro-channels. Scale bar = 500 nm. d Internal epidermal cell with large nucleus, mitochondria, and plastids, note phenolic comounds (phe). Scale bar = 2 μm. e Epidermal and subepidermal cells connected by plasmodesmata (arrows). Scale bar = 1 μm. f–g Nectariferous parenchyma cells containing long profiles of rough ER, note numerous secretory vesicles that coalesce and fuse with the plasmalemma. Scale bar = 1 μm. h Details of secretory parenchyma cell showing central vacuole and numerous mitochondria closely associated with starch-containing plastids. Scale bar = 2 μm. Abbreviations used on the figures: C Cuticle, CW Cell wall;, ER Endoplasmic reticulum, ie Internal epidermis, is Intercellular space, m Mitochondrion, ml Myelin-like figure, N Nucleus, n Nectary, ne Nectar, np Nectary parenchyma, P Plastid, ph Phloem elements, phe Phenolic compounds, sc Spur cavity, st Starch, sv Secretory vesicle, V Vacuole, vb Vascular bundles, x Xylem