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Annals of Botany 105: 349 –354, 2010 doi:10.1093/aob/mcp302, available online at www.aob.oxfordjournals.org VIEWPOINT On the mechanisms of nectar secretion: revisited A. E. Vassilyev* Department of Anatomy and Morphology, V.L. Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg 194376, Russia * E-mail [email protected] Received: 20 May 2009 Returned for revision: 21 July 2009 Accepted: 26 November 2009 Published electronically: 5 January 2010 † Background and Scope Models of nectar formation and exudation in multilayered nectaries with modified stomata or permeable cuticle are evaluated. In the current symplasmic model the pre-nectar moves from terminal phloem through the symplasm into the apoplasm (cell walls and intercellular spaces) with nectar formation by either granulocrine or eccrine secretion and its diffusion outwards. It is concluded, however, that no secretory granules are actually produced by the endoplasmic reticulum, and that secretory Golgi vesicles are not involved in the transport of nectar sugar. Therefore, the concept of granulocrine secretion of nectar should be discarded. The specific function of the endomembrane system in nectary cells remains unknown. According to the apoplasmic model, the pre-nectar moves from the terminal phloem in the apoplasm and, on the way, is transformed from phloem sap into nectar. However, viewed ultrastructurally, the unloading (terminal) phloem of nectaries appears to be less active than that of the leaf minor veins, and is therefore not actively involved in the secretion of pre-nectar components into the apoplasm. This invalidates the apoplasmic model. Neither model provides an explanation for the origin of the driving force for nectar discharge. † Proposal A new model is proposed in which nectar moves by a pressure-driven mass flow in the nectary apoplasm while pre-nectar sugars diffuse from the sieve tubes through the symplasm to the secretory cells, where nectar is formed and sugars cross the plasma membrane by active transport (‘eccrine secretion’). The pressure originates as the result of water influx in the apoplasm from the symplasm along the sugar concentration gradient. It follows from this model that there can be no combinations of apoplasmic and symplasmic pre-nectar movements. The mass-flow mechanism of nectar exudation appears to be universal and applicable to all nectaries irrespective of their type, morphology and anatomy, presence or absence of modified stomata, and their own vascular system. Key words: Apoplasm, nectar secretion mechanism, nectary, phloem unloading. Nectaries and nectar are an area of intensive botanical research, as demonstrated by the extensive listings in the table of contents of recent volumes of the Annals of Botany. However, one of the most important problems of nectarology, i.e. the structural mechanism(s) of nectar secretion, has not been unambiguously resolved. It is well known that nectar is the phloem sap that is modified by nectary cells (Durkee, 1983; Fahn, 1988), and that sugars, the dominant component of nectar (together with water), enter the nectary from the phloem. In most species studied (about 48 %), the floral nectaries are vascularized by phloem only, the minority (about 12 %) receive direct vascular supply comprising both xylem and phloem, and the remainder ( perhaps surprisingly, an important 40 %) are not vascularized at all (Frei, 1955; Kartashova, 1965; Davis et al., 1986, 1988). Extrafloral nectaries often receive no direct vascular supply either (Elias, 1983). In the latter case photosynthates are unloaded from the phloem of the adjacent vascular bands into neighbouring parenchyma cells and diffuse further symplasmically into the secretory cells of the nectary. Most authors (for reviews see Lüttge and Schnepf, 1976; Fahn, 1988; Nepi, 2007) adhere to the so-called symplasmic pathway of post-phloem sugar transport. In this hypothesis, sugars are unloaded from the sieve tubes and companion cells into the cytoplasm of adjoining secretory (nectariferous) or subglandular cells (when the vascular bundles are terminated in the subglandular tissue). These sugars then move in the cytoplasm of the secretory cells through the plasmodesmata as a pre-nectar. Nectar, produced by the cytoplasm of the secretory cells from pre-nectar as a modified phloem sap, is released into the apoplasm (i.e. the cell walls and intercellular spaces). The nectar that is prepared for exudation moves in the apoplasm to be released on the nectary surface through modified stomata or a permeable cuticle. This hypothesis does not explain why the nectar is discharged, because it was assumed (O’Brien et al., 1996) that its components move in the apoplasm by way of diffusion. In the above (symplasmic) hypothesis, two contrasting mechanisms have been proposed to explain the nectar secretion from the symplasm to the apoplasm of secretory cells, namely eccrine and granulocrine secretion (Fahn and Rachmilevitz, 1970; Lüttge, 1971; Durkee, 1983; Fahn, 1988; O’Brien et al., 1996; Nepi, 2007). These mechanisms appear to be taxono-specific (Nepi, 2007). Eccrine secretion involves an active molecular transport of nectar sugars across the plasma membrane. In granulocrine secretion nectar sugar molecules and other components of the nectar are accumulated beforehand in the cisternae of the # The Author 2010. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 350 Vassilyev — Mechanisms of nectar secretion endoplasmic reticulum or Golgi bodies ( previously dictyosomes). They then supposedly produce vesicles with nectar that are released through fusion with the plasma membrane. The presence of a well-developed endoplasmic reticulum, numerous Golgi bodies and vesicles is considered to be the distinctive feature of granulocrine secretion of nectar. However, the hypothesis of granulocrine secretion of nectar contradicts significant advances that have been made in the study of the secretory system of plant cells, the endoplasmic reticulum (reviewed by Quader and Zachariadis, 2006) and Golgi apparatus (reviewed by Matheson et al., 2006) and should be discarded. No secretory granules are produced by the endoplasmic reticulum and secretory Golgi vesicles are involved in the production of polysaccharides rather than the sugar components of the nectar. Study of the specific structure and function of the endoplasmic reticulum and Golgi apparatus is a promising area of nectary research. As an alternative to the symplasmic hypothesis, 40 years ago I offered an apoplasmic theory of pre-nectar transport (Vassilyev, 1969). This theory was further described in detail in Vassilyev (1977) in which I wrote: ‘From the vascular bundle endings, a sap that is transformed into the nectar moves mainly by-passing cell protoplasts. Its main path is a so-called free space of cells, i.e. their cell walls and most of intercellular spaces. The “pump” ensuring nectar transport is localized in the parenchyma cells of the vascular bundles. These cells absorb sugars from the sieve elements and secrete them into the free space (now apoplasm, A.V.) in the form of pre-nectar . . .’ ( p. 116; translated from Russian). Pre-nectar moves through the apoplasm and is transformed into nectar by the activity of secretory cells. In the 1970s the idea of the apoplasmic (‘apoplastic’ at that time) phloem loading of photosynthates in leaves was a popular concept among plant physiologists. Unfortunately, my theory regarding the central role of phloem cell unloading as a moving force of pre-nectar transport that occurs in the apoplasm rather than in symplasm was published in Russian, although the essence of the theory was described in English by other authors (for example Lüttge and Schnepf, 1976; Durkee, 1983; Fahn, 1988). However, those authors who became familiar with the apoplasmic hypothesis (Fahn, 1988; Nichol and Hall, 1988) criticized it because it was not applicable to trichomatous nectaries with barrier cells whose fully cutinized cell walls blocked the apoplasmic movement of pre-nectar. Nevertheless, the hypothesis was not abandoned completely. Subsequent articles have appeared (Davis et al., 1988; Razem and Davis, 1999; Gaffal and El-Gammal, 2003; Wist and Davis, 2006; Gaffal et al., 2007) from authors unfamiliar with my theory, and who assumed the direct unloading of sugars from the nectary phloem into the apoplasm. Their conclusion about the active role of the nectary phloem in sugar unloading was vulnerable for criticism since it was based on ultrastructural studies of the phloem of nectaries only. They did not consider it in light of the ultrastructure of the phloem in leaf minor veins of the same species. Meanwhile, I made comparative ultrastructural studies of terminal phloem in leaves (from minor veins) and nectaries 4 1 1 2 1 4 A 2 3 2 3 4 1 5 4 B F I G . 1. Cirsium arvense. (A) Leaf minor vein. Note that the diameter of all parenchyma cells is much larger than that of sieve tubes, and they bear highly developed wall ingrowths characteristic of transfer cells. (B) Basal (transporting) phloem of nectary. Wall ingrowths are formed only in a few cells; they are short and infrequent (rudimentary). (1) Sieve tube, (2) companion cell, (3) phloem parenchyma cell, (4) wall ingrowths. Scale bars ¼ 1.5 mm. F I G . 2. Strand of terminal (unloading) phloem of Cirsium arvense floral nectary. Note that companion and phloem parenchyma cells are of similar (small) diameters to sieve tubes. All lack wall ingrowths. The number of phloem parenchyma cells is greatly reduced. (1) Sieve tube, (2) companion cell, (3) phloem parenchyma cell, (4) secretory cell, (5) intercellular space. Scale bar ¼ 5 mm. (Adapted, with permission, from Vassilyev and Koteyeva, 2004.) Vassilyev — Mechanisms of nectar secretion 351 MASS FLOW NECTAR CUTICLE SUCROSE NECTAR CELL WALLS + INTERCELLULAR SPACES (APOPLASM) SECRETORY CELLS PLASMODESMATA (SYMPLASM) ACTIVE TRANSPORT DIFFUSION PLASMA MEMBRANE COMPANION CELL SIEVE TUBE F I G . 3. Mechanism of pressure-driven mass flow of nectar in nectaries with stomata and terminal phloem. Five blue-tone gradations mirror relative sugar concentrations: (1) in the apoplasm and nectar (maximal); and (2) in the sieve tube contents ( phloem sap) and the cytoplasm of companion cells. The other three tones reflect a gradient in pre-nectar sugar concentrations in the symplasm of secretory cells, namely: (3) in the cytoplasm of the cells near companion cells; (4) in the cytoplasm of cells away from companion cells; and (5) in the epidermal cytoplasm (minimal). This gradient is derived as a result of nectar sugar discharge from the symplasm by secretory cells and provides for pre-nectar sugar diffusion across plasmodesmata (red arrows). Arrows with a circle indicate active transport (secretion) of sugars from symplasm into apoplasm across the plasma membrane. Nectar moves in the apoplasm by pressure-driven mass flow into substomatal cavities, from where it enters the nectary surface through modified stomata. The cell walls are shown exaggeratedly thickened. (Modified from Vassilyev, 2003.) 352 Vassilyev — Mechanisms of nectar secretion of six species with apoplasmic loading of photosynthates (Vassilyev and Koteyeva, 2004) and five species with symplasmic loading (Vassilyev, 2005). These two papers were again in Russian, but with lengthy English abstracts. The data obtained showed that in nectaries, bundle sheath parenchyma was not differentiated, and that phloem elements including sieve tubes were in direct contact with secretory cells. Sometimes a common wall with plasmodesmata between sieve tube and secretory cell was observed. Wall ingrowths characteristic of companion cells and phloem parenchyma cells of Cirsium arvense (Fig. 1A) and Impatiens parviflora leaf minor veins were found in reduced form only in a few cells of nectary basal phloem (Fig. 1B). The ratio of companion cells to sieve elements was also reduced. Unlike leaf companion cells, those of nectary terminal phloem were of the same (small) diameter as the sieve tubes (Fig. 2). A similar ultrastructure is apparently characteristic of the nectary phloem of Echinacea purpurea (another member of the Asteraceae) (Wist and Davis, 2006, fig. 6). In nectaries of Barbarea vulgaris, Alliaria petiolata and Rorippa sylvestris, phloem parenchyma is not differentiated in terminal phloem and the sieve tubes have very lengthy contacts with the secretory cells. In Cirsium arvense, Impatiens parviflora and Convolvulus arvensis, ultrastructural differences between companion cells and phloem parenchyma cells characteristic of leaf minor veins were indistinguishable in nectary phloem. The data obtained from these studies indicated that the companion cells in nectary terminal phloem were not actively involved in the secretion of pre-nectar components into the apoplasm. Instead, the nectary vascular parenchyma are thought to function in the symplasmic (via plasmodesmata) transport of photosynthates from the sieve tubes into secretory cells. Consequently, I assigned companion cells of the nectary terminal phloem only a passive role in nectar secretion, i.e. sugar unloading into secretory cells by diffusion. Given the above, the ultrastructural evidence indicates that an apoplasmic pathway of the pre-nectar is unlikely. In addition, analysis of recent information has shown (see Vassilyev and Koteyeva, 2004) that companion cells of terminal phloem do not have an important role in photosynthate unloading in non-nectary tissues. In the unloading zones, the size of companion cells diminishes to the size of the sieve elements, with concomitant disappearance of wall ingrowths, or companion cells simply do not differentiate. In the latter case, sieve elements are connected by plasmodesmata with the ground parenchyma cells. The structural data (mainly high plasmodesmatal frequency) as well as the results of physiological and biochemical studies (Dejong et al., 1996) indicate passive (i.e. along a concentration gradient) PLASMODESMA ATP H+ ADP PLASMA MEMBRANE H+-ATPase H+ + H -SUCROSE ANTIPORTER SUCROSE H2O SYMPLASM APOPLASM (CELL WALLS + INTERCELLULAR SPACES) F I G . 4. Molecular mechanisms of discharge of nectar components (sugars and water) from secretory (nectariferous) cells. Sugars of nectar are transferred from the symplasm into apoplasm against the concentration gradient by way of antiporters. Simultaneously, in the opposite direction, protons enter the symplasm along the concentration gradient. However, they are actively (i.e. against a concentration gradient) pumped out from the symplasm by Hþ-ATPase ( plasma membrane proton pump shown in red) using ATP energy, providing a moving force for sugar transport. The difference between sugar concentration in the apoplasm versus that in the symplasm is shown by the intensity of the blue colour. Water is exuded into the apoplasm via aquapores in the plasma membrane along an osmotic gradient. Thus, hydrostatic pressure in the apoplasm is produced that is necessary for ensuring mass flow of nectar on the surface. (Modified from Vassilyev, 2003.) Vassilyev — Mechanisms of nectar secretion photosynthate unloading and their further diffusion in the symplasm without entering the apoplasm. The situation in these systems appears to be similar to that occurring in nectaries devoid of their own phloem. Based on recent information regarding the cell ultrastructure and physiology of nectaries, as well as the mechanisms of sugar transport across membranes, I have proposed a new mechanism of nectar secretion, the pressure-driven mass flow of nectar in the nectary apoplasm (Vassilyev, 2003). Pre-nectar sugars, like other substances (Roberts and Oparka, 2003), are transported from the phloem into nectary secretory cells in the symplasm (in the cytoplasm and through plasmodesmata) by diffusion. The driving force for nectar flow in the apoplasm is the active transmembrane transfer of sugars into this compartment (‘eccrine secretion’) from the cytoplasm of all secretory (¼ nectariferous) cells rather than sugar unloading from phloem cells alone (Fig. 3). The pressure responsible for the mass flow of nectar originates as the result of water influx in the apoplasm of the nectary cells along the sugar concentration gradient produced by the active transport of sugars from the symplasm by way of antiporters (Fig. 4). Nectar sugars, together with the minor components of nectar, then flow in the apoplasm of a nectary towards the outside. Models of nectar flow in nectaries with stomata (see Fig. 3) and trichomatous nectaries with barrier cells, as well as in the leaf of an ‘apoplasmic’ plant, have been formulated (Vassilyev, 2003). It follows from these models that there can be no combinations of apoplasmic and symplasmic prenectar movements (see Nepi, 2007). In fact, nectar moves by mass flow only in the apoplasm, whereas in the symplasm simple diffusion of pre-nectar sugar molecules along the concentration gradient occurs. It is pertinent to compare this process with the mass flow of photosynthates in the sieve tubes, where all solutes are moving unidirectionally and with the same speed. The idea of the active nectar sugar transport by the secretory cells of the nectaries into the apoplasm has been supported by Peng et al. (2004). Using cytochemical techniques, they detected plasma membrane Hþ-ATPase activity in the secretory cells of Cucumis sativus nectaries that was indicative of active transport of nectar. Preliminary histochemical investigation using a fluorescent dye, sulphorodamine G, as an indicator of proton extrusion from the cells also showed that the active transmembrane transport of nectar sugar molecules was characteristic of nectary secretory cells of the other three species studied, namely Barbarea vulgaris, Helleborus foetidus and Artrostema ciliatum (Koteyeva et al., 2005). The proposed mechanism of nectar exudation appears to be universal and applicable to all nectaries irrespective of their types, morphology and anatomy, presence or absence of modified stomata, and their own vascular system. This conclusion is based on three foundations: (1) nectar moves in the apoplasm (cell walls and intercellular spaces) to the surface by pressuredriven mass flow; (2) the flow is generated by the active (i.e. against concentration gradient) transport of sugars from the cytoplasm (symplasm) of secretory cells and concomitant water efflux from their cytoplasm; and (3) pre-nectar sugars and water are transported in the symplasm from the phloem by diffusion. 353 ACK NOW LED GE MENTS I am indebted to Richard Crang, Art Davis and Martin Steer for valuable advice. L I T E R AT U R E CI T E D Davis AR, Peterson RL, Shuel RW. 1986. Anatomy and vasculature of floral nectaries of Brassica napus (Brassicaceae). Canadian Journal of Botany 64: 2508–2516. 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