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Phenolic Vacuoles and Auxin Transport |
Much interest has attended the
discovery by Paris et al. (1995)
that two different species of
"vacuoles" can be clearly distinguished within the same barley
(Hordeum vulgare) root tip cell by labeling with
antibodies to two different tonoplast intrinsic proteins, 
-TIP and
TIP-Ma27. Moreover, because barley lectin is exclusively contained
within the -TIP compartment, and the protease aleurain within the
TIP-Ma27 compartment, it appears that these two compartments are also
functionally distinct. The general conclusion that plants have a
diversity of vacuole types has been borne out by subsequent research
(e.g., Fleurat-Lessard et al., 1997; Swanson et al., 1998; Jauh et al.,
1999). Scarcely mentioned in the rush and tumble associated with these
molecular biological discoveries is the fact that the idea that plant
cells have a multiplicity of vacuolar types is quite old. In fact, it
was the dominant paradigm in the first half of the 20th century.
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Distinct Vacuolar Types: An Old Concept |
Cytologists of the classic era of light microscopy often
observed two types of vacuoles coexisting within the same protoplast of
higher plant cells (Bailey, 1930; Zirkle, 1937; Guilliermond, 1941; Buvat, 1969). Of these two types of vacuoles, the types variously referred to as "specialized vacuoles" or "vacuoles with `full' saps" (Härtel, 1951) or "A-type vacuoles" (Bailey,
1930) are the cytological entities of concern here. Although molecular biology will undoubtedly soon serve as the basis of a new vacuolar nomenclature, let us for the time being refer to these structures simply as phenolic vacuoles. The distinguishing features of phenolic vacuoles include their high phenolic content, their avidity for certain
basic dyes (e.g. neutral red), their unusually acidic interior, their
greater sap viscosity, and their greater refractivity. The avidity of
phenolic vacuoles for basic dyes would seem to be due to the
precipitation of the dyes by endogenous phenols.
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1970-1990: The Dark Ages of Phenolic Vacuole Research |
In the second half of the 20th century, research concerning
phenolic vacuoles became increasingly rare. This was largely because the technologies used by plant cell biologists of that era were not
nearly as suitable for the study of phenolic vacuoles as the bygone,
mostly observational techniques of previous generations. Indeed, as
late as 1969, Buvat was able to state fairly accurately that most of
what was then known about plant vacuoles had been "obtained by use of
the light microscope, and from looking at living cells." Soon
thereafter, the situation changed dramatically, and most of the classic
light microscopy studies relating to phenolic vacuoles or, indeed,
vacuoles in general, faded from view. Instead, the new conception of
vacuole was instilled largely from experiments involving two methods:
electron microscopy and in vitro studies upon either "isolated
vacuoles" or "vacuole-enriched" fractions. Both approaches,
unfortunately, are severely limited insofar as probing the structure
and function of phenolic vacuoles.
A problem inherent in all electron microscopy studies is that the
technique offers only a static view of the cell. This problem is
especially acute in the case of phenolic vacuoles, the extremely dynamic nature of which has been stressed by nearly all who have studied them (e.g. Bailey, 1930). Additional problems arise during the
fixation of phenolic-storing cells because of the facility with which
these compounds leach from the vacuoles and "auto-fix" the
surrounding cytoplasm, although this problem can be overcome to some
extent by precipitation of the phenolic compounds with caffeine during
fixation (Mueller and Greenwood, 1978).
The biochemical utility of the techniques of cell fractionation and
density gradient ultracentrifugation rests on the assumption that the
densities of membrane vesicles of interest are homogenous: a condition
not achievable in the case of phenolic vacuoles. Milovidov (1930) found
that the phenolic vacuoles within the youngest cells of the teeth of
rose leaves were heavier than the cytoplasm, while the phenolic
vacuoles of older cells were lighter than the cytoplasm. He obtained
similar results with barley root cells. Åkerman (1917) also found that
the density of phenolic vacuoles in Drosera rotundifolia tentacles changed dramatically upon stimulation. Centrifugation revealed that that the cytoplasm is denser than the vacuole in non-stimulated cells, but the situation is reversed in stimulated cells. In fractionating plant tissues, one typically takes organs composed of heterogenous cells and tissues of different age and subjects them to intense shearing and wounding. One can well imagine, therefore, that the vesicles derived from phenolic vacuoles following fractionation would, upon centrifugation, be smeared through many fractions.
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Phenolic Vacuoles and Auxin Secretion: A Link? |
Many researchers have noted the strong correspondence between
the prevalence of phenolic vacuoles in a given plant tissue and the
motor and/or secretory properties of those tissues. For example,
phenolic vacuoles are unusually prevalent in pulvinar cells (e.g.
Fleurat-Lessard et al., 1997), guard cells (e.g. Guyot and
Humbert, 1970), and glandular cells (e.g. Dufrénoy, 1927). Here,
I call attention to another collection of tissues that appear, based on
their basophilic staining properties, to be rich in phenolic vacuoles:
auxin-secreting tissues.
Neither auxin secretion nor phenolic vacuoles occur in a neatly defined
set of tissues. If, however, one considers only those tissues regarded
to be most active in auxin secretion, one finds a close correspondence
between auxin-secreting tissues and the presence of phenolic vacuoles.
For example, the auxin-secreting vascular cambium of woody plants is
rich in phenolic vacuoles (Bailey, 1930). In the case of herbaceous
dicots, Jacobs and Gilbert (1983) demonstrated that naphthylphthalamic
acid receptors, the proteins thought to be involved in the polar efflux
of auxin from plant cells, are localized in the perivascular parenchyma
of pea (Pisum sativum) stems. Sorokin (1956) found that
phenolic vacuoles were most prevalent in the starch sheath and adjacent
cells of pea stems as well (see also Dufrénoy, 1930; Van
Fleet, 1950). The current model of auxin transport in plant roots is
that auxin, continuing its downward polar movement from the stem, moves
basipetally through the stele of the root. Upon reaching the root
tip, this stream of auxin is distributed back upward along the
epidermis and subtending cortical cells (Jones, 1998). Soran and Lazar
(1965) found that the central cortex of maize (Zea mays)
roots stained weakly with neutral red compared with endodermal and
epidermal cells.
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What Do Phenolic Vacuoles Do in Auxin Secreting
Tissues? |
Jacobs and Rubery (1988) found that certain phenolics, including
flavonoids such as quercetin, apigenein, and kaempferol, can
specifically compete with naphthylphthalamic acid for binding to its
receptor. They proposed that these widely distributed compounds act as
natural regulators of polar auxin transport in plants. Consistent with
this idea, Peer et al. (2001) have recently demonstrated that the
flavonoid quercetin is present at higher concentrations at the basal
end of auxin-secreting cells. Perhaps the efficient secretion of auxin
requires that these flavonoids be tightly sequestered in phenolic
vacuoles, or perhaps that these phenolic vacuoles may act as reservoirs
that release flavonoids at certain points in development.
Alternatively, the prevalence of phenolic vacuoles in auxin-secreting
tissues may be related to the moto-secretory properties of the tissue.
Tronchet (1961), for example, noted a close correspondence between
flavonoid (quercetin and kaempherol) levels in the stem cortex of
plants and the extent to which they twined or circumnutated.
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