Evidence for the Dual Role of Floral Secretory Cells in Bulbophyllum

Abstract

Floral epidermal cells of most species of Bulbophyllum Thouars studied to date produce both lipid-rich foodrewards and fragrance. Since fragrances largely consist of terpenoids and have an affinity for lipophilic stains, the simultaneous presence of lipid-rich food-rewards frustrates identification of fragrance-secreting cells by conventional histochemistry. Furthermore, since both lipid-rich food-rewards and fragrances are probably synthesized by a similar complement of organelles, interpretation of TEM images can prove difficult. All members of section Racemosae Benth. & Hook. f. investigated to date, however, are unusual in their secretion of a predominantly proteinaceous food-reward, and lipids are seemingly absent. This might enable their use as models for the identification and characterization of fragrance-secreting tissues and organelles. Three members of sect. Racemosae were chosen, namely Bulbophyllum dissitiflorum Seidenf., B. lilacinum Ridl. and B. tricorne Seidenf. & Smitinand. All produced food-rewards. Of these, one (B. dissitiflorum) lacked fragrance and was used as a control, whereas the remaining two species produced fragrance. Having established that the food-reward was mainly proteinaceous in each case, and did not test positively for lipid, we undertook further histochemical investigations, as well as light microscopy, SEM and TEM. Specialized palisade-like epidermal cells of all species contained protein bodies and rough endoplasmic reticulum consistent with the production and secretion of a protein-rich food-reward. Cuticular pores were also present. In fragrant species, these cells also contained abundant smooth endoplasmic reticulum, oil droplets and many, well-developed, spherical plastids with numerous plastoglobuli, similar to those found in the osmophores (fragrance-producing structures) of other orchids. Indeterminate, osmiophilic cytoplasmic inclusions were also present. By contrast, the non-fragrant species lacked oil droplets and other osmiophilic inclusions and the plastids were scant, poorly developed, often elongate or irregular in shape and contained few plastoglobuli. Smooth endoplasmic reticulum was also less frequent. Since food-rewards tested negatively for lipid, it is probable that any oil droplets present were involved in fragrance production, especially since they were absent from the non-fragrant species. Thus, the unusual absence of lipids from the food-rewards of sect. Racemosae provided a rare opportunity, permitting, for the first time, the unraveling of these two secretory processes (food-reward and fragrance) in Bulbophyllum and clearly demonstrating the plasticity of these cells and their dual role in secretion.

Nectar Replaced by Volatile Secretion: A Potential New Role for Nectarless Flowers in a Bee-Pollinated Plant Species

“…Small droplets of lipophilic material in the disk epidermal cells that stained with Sudan IV and Nadi reagent, and also observed in TEM analysis, were reported in osmophore epidermal cells of several Orchidaceae species ( Davies and Stpiczyńska, 2014 ; Stpiczyńska and Davies, 2016 ) and indicate the possible role of the epidermal cells in scent production. In addition, a layer of osmiophilic material lined the tonoplast inner surface, as occurs in the epidermal and parenchyma vacuole of J. oxyphylla nectarless flowers is also evidence of scent production.…”

“…Some authors, studying Orchidaceae species ( de Melo et al, 2010 ; Kowalkowska et al, 2012 , 2015 ; Wiśniewska et al, 2018 ), found that floral nectaries and osmophores are somewhat similar in structure and ultrastructure features, except that in the latter there is predominance of SER and low frequency of Golgi bodies. Therefore, abundance of globular mitochondria, SER with peripheral distribution and oil droplets in the cytosol and in amyloplasts, together with the scarce Golgi bodies here observed may be associated with fragrance production ( Curry et al, 1991 ; Stpiczyńska and Davies, 2016 ). The occurrence of polymorphic plastids containing many lipophilic droplets and numerous large oil inclusions in the cytoplasm of parenchyma cells both in nectariferous flowers at 48 h of anthesis and in nectarless flowers is a strong evidence of the involvement of the floral disk in scent production, as similar plastids also occur in osmophores ( Pridgeon and Stern, 1983 , 1985 ; Curry et al, 1991 ; de Melo et al, 2010 ; Antoń et al, 2012 ).…”

“…This data is compatible with the predominance of elaioplasts in the nectary parenchyma cells at 48 h of anthesis, when most of amyloplasts are degenerated and nectar secretion has already stopped. Plastid change occurred simultaneously with the increase in the amount of oil drops inside vacuoles or dispersed in the cytosol, and SER proliferation, which are ultrastructural evidences of lipophilic secretion ( Gleizes et al, 1980 ; Figueiredo and Pais, 1992 ; Turner et al, 1999 ; Machado et al, 2005 ; Stpiczyńska et al, 2005 ; Stpiczyńska and Davies, 2016 ; Possobom and Machado, 2018 ). Although these features are unusual in nectary tissues, the ability of nectary cells to produce both nectar and lipids ( Baker and Baker, 1975 ; Durkee et al, 1984 ; Subramanian et al, 1990 ; Possobom et al, 2010 ; Tölke et al, 2015 ; Guimarães et al, 2016 ; Stpiczyńska and Davies, 2016 ; Machado et al, 2017b ) and transition from a true nectary to a lipophilic secretory gland ( Durkee, 1982 ; Durkee et al, 1984 ) has been reported in some angiosperm species.…”

“…In fact, prominent periplasmic space containing lipid droplets in regions underlying the outer tangential walls indicate secretory activity of epidermal cells associated with the accumulation and release of the volatile compounds. Likewise, the occurrence of large mitochondria and abundant SER located in the cortical cytoplasm, besides plastids featured by a reduced electron density and lipid inclusions, together with numerous lipid droplets in the cytosol and vacuoles of the subepidermal layers, are subcellular features commonly associated with synthesis of volatile compounds ( Cheniclet and Carde, 1985 ; Figueiredo and Pais, 1994 ; Ascensão et al, 1997 ; Turner et al, 1999 ; Possobom et al, 2015 ; Stpiczyńska and Davies, 2016 ).…”

“…The increase in the amount of different materials (lipid–carbohydrate–phenolic) within the plastids throughout anthesis, as here observed, has been demonstrated in Orchidaceae osmophores ( Wiśniewska et al, 2018 and references therein). The involvement of plastids in the synthesis of scent components, mainly terpenoids and phenolic compounds, has been broadly discussed ( Pridgeon and Stern, 1985 ; Stern et al, 1987 ; Pais and Figueiredo, 1994 ; Kowalkowska et al, 2012 ; Stpiczyńska and Davies, 2016 ; Wiśniewska et al, 2018 ). Besides protection against herbivores, pathogens, and UV radiation ( Brillouet et al, 2013 ), phenolic compounds are known to occur in floral scent ( Jürgens and Dötterl, 2004 ; Jürgens et al, 2006 ; Steiner et al, 2011 ; Wiśniewska et al, 2018 ).…”