Download On the mechanisms of nectar secretion: revisited

Annals of Botany 105: 349 –354, 2010
doi:10.1093/aob/mcp302, available online at www.aob.oxfordjournals.org
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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
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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.
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