The species from Bulbophyllinae are generally regarded as fly-pollinated: myophilous or sapromyophilous. The section Cirrhopetalum is characterized by umbellate inflorescence and elongated lateral sepals. The aim of the floral anatomical investigation (micromorphology, histochemistry, ultrastructure) in Bulbophyllum wendlandianum (section Cirrhopetalum) was the detection of secretory activity. The appendages of dorsal sepal and petals function as osmophores. The exudation is transported inside vesicles via granulocrine secretion. The cuticle stretches and forms swellings on the entire cell surface. Such swellings of cuticle on the appendages of dorsal sepal and petals were not previously described in Bulbophyllum species. The nectary is located in the central groove on the adaxial lip surface. It comprises epidermal epithelial cells and few subepidermal layers.
Floral spurs are regarded as features affecting pollinator behaviour. Anacamptis pyramidalis is regarded as a deceitful, non-rewarding orchid species. In the form fumeauxiana, additional spurs occur on the lateral sepals. In this study we analyse micromorphological and ultrastructural floral features and suggest the mechanism of deception in A. pyramidalis and A. pyramidalis f. fumeauxiana. In f. fumeauxiana, the adaxial surface of the lip, the lip calli, the tips of the lateral sepals, the abaxial and adaxial epidermises of the lip spur, and the lateral sepal spur have a secretory function. Numerous stomata were observed on the abaxial surfaces of spurs and sepals. The characteristic features of the ultrastructure of osmophore cells were noted: dense cytoplasm with numerous profiles of ER, mitochondria, plastids with plastoglobuli and tubular structures, a large nucleus, lipid droplets, and vesicles fusing with the plasmalemma. The similarity of the floral morphology and anatomy, the flowering period, and pollinators of A. pyramidalis, A. pyramidalis f. fumeauxiana and Gymnadeniaconopsea suggest a possible food-deceptive mechanism—imitation of nectar presence produced in the spurs of Gymnadenia.
Isolated thylakoids from halophytic Eutrema salsugineum (Thellungiella salsuginea) produces more H2O2 in comparison to glycophytic Arabidopsis thaliana. The first objective of this study was to verify whether this feature is relevant also to the intact chloroplasts and leaves. Enhanced H2O2 levels in chloroplasts and leaves of E. salsugineum were positively verified with several methods (electron microscopy, staining with Amplex Red and with diaminobenzidine). This effect was associated with a decreased ratio of O2.-/H2O2 in E.s in comparison to A.thaliana as detected by electron paramagnetic resonance (EPR) method. As a next step, we tested how this specific ROS signature of halophytic species affect the antioxidant status and down-stream components of ROS signaling. Comparison of enzymatic antioxidants revealed a decreased activity of ascorbate peroxidase (APX), enhanced activity of glutathione peroxidase (GPX), and the presence of thylakoid-bound forms of iron superoxide dismutase (FeSOD) and ascorbate peroxidase (APX) in E.s.. These cues were, however, independent from application of salt stress. The typical H2O2-dependent cellular responses, namely the levels of glucosinolates and stress-related hormones were determined. The total glucosinolate content in E.s water-treated leaves was higher than in A.t. and increased after salinity treatment. Treatment with salinity up-regulated all of tested stress hormones, their precursors and catabolites (abscisic acid, dihydrophaseic acid, phaseic acid, 1-aminocyclopropane-1-carboxylic acid, salicylic acid, jasmonic acid, cis-(+)-12-oxo-phytodienoic acid and jasmonoyl-L-isoleucine) in A.t., whereas in E.s. only a stimulation in ethylene synthesis and abscisic acid catabolism was noted. Obtained results suggest that constitutively enhanced H2O2 generation in chloroplasts of E.s. might be a crucial component of stress-prepardeness of this halophytic species. It shapes a very efficient antioxidant protection (in which glucosinolates might play a specific role) and a fine tuning of hormonal signaling to suppress the cell death program directed by jasmonate pathway.
Digestive structures of carnivorous plants produce external digestive enzymes, and play the main role in absorption. In Lentibulariaceae, the ultrastructure of digestive hairs has been examined in some detail in Pinguicula and Utricularia, but the sessile digestive hairs of Genlisea have received very little attention so far. The aim of this study was to fill this gap by expanding their morphological, anatomical and histochemical characterization. Several imaging techniques were used, including light, confocal and electron microscopy, to reveal the structure and function of the secretory hairs of Genlisea traps. This report demonstrates the application of cryo-SEM for fast imaging of whole, physically fixed plant secretory structures. The concentration of digestive hairs along vascular bundles in subgenus Genlisea is a primitive feature, indicating its basal position within the genus. Digestive hairs of Genlisea consist of three compartments with different ultrastructure and function. In subgenus Tayloria the terminal hair cells are transfer cells, but not in species of subgenus Genlisea. A digestive pool of viscous fluid occurs in Genlisea traps. In spite of their similar architecture, the digestive-absorptive hairs of Lentibulariaceae feature differences in morphology and ultrastructure.
Flowers of Bulbophyllum weberi and B. cumingii are characterized by fly-pollinated features. The secretory activity was described in dorsal sepals in both species (putative osmophores), petals in B. weberi (possible osmophores) and adaxial surface of lips in both species. In the cells of dorsal sepals and petals of B. weberi proteins, dihydroxyphenols, lipids and starch grains were detected, in lateral sepals—lipids. Whereas in dorsal sepal of B. cumingii only lipids and starch grains were noted, in lateral sepals—proteins and dihydroxyphenols and in petals—proteins and starch grains. The lips in both species differed histochemically and ultrastructurally. The epidermal cells of lip groove in B. weberi contained lipids, proteins, starch grains in cytoplasm, dihydroxyphenols in vacuoles and pectic acids/mucilage on surface. Whereas in B. cumingii—few lipids, starch grains, no proteins, no dihydroxyphenols and no mucilage were noted. Ultrastructurally, in B. weberi, the secretory material was present on surface and vesicles building into plasmalemma, while in B. cumingii—cell wall ingrowths and microchannels in cuticle. The osmiophilic irregular materials and globular, osmiophilic globules in B. weberi are probably tannin-like materials. For the first time, we described the cell wall ingrowths in Bulbophyllum species: in lip of B. cumingii and petals of B. weberi.
This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2•−) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3′-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2•− was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2•− is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth.