Download New aspects of plant aquaporin regulation and

Journal of Experimental Botany, Vol. 50, No. 339, pp. 1541–1545, October 1999
REVIEW ARTICLE
New aspects of plant aquaporin regulation and specificity
Martin Eckert, Alexander Biela, Franka Siefritz and Ralf Kaldenhoff1
Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
Received 15 April 1999; Accepted 29 June 1999
Abstract
Recent findings concerning the sensitivity of aquaporins to heavy metal ions and their possible consequences to plant physiology are discussed.
Furthermore, results obtained from heterologous
expression of plant aquaporin cRNAs in Xenopus
oocytes are related to the situation in the plant. An
assessment of the significance of water flux regulation
by modification of the aquaporin protein or change
of gene activity is given and new data concerning
the selectivity of plasma membrane aquaporins are
presented.
Key words: Aquaporin, regulation, specificity, sensitivity to
heavy metal ions.
Introduction
The idea of a molecular water transport mechanism has
been substituted by a revised model since evidence for
the existence of water-conducting protein components in
membranes of living cells were found (Agre et al., 1995;
Maurel, 1997; Schäffner, 1998). In mammals, the significance of these so-called aquaporins was rapidly conceived and also rather obvious for specialized tissues such
as the renal descending limb of Henle’s loop ( Knepper et
al., 1996). Due to water reabsorption rates of up to 200
l d−1 and the fact that in renal collecting tubes the cellular
water permeability could be increased by vasopressin,
physiologists had long been suggesting that specialized
water transport molecules must exist. A subsequent
molecular search for the corresponding gene succeeded
in the cloning of the first aquaporin CHIP28 or AQP1
(Preston and Agre, 1991). The functional and molecular
characterization of this and similar proteins was achieved
by heterologous expression in Xenopus oocytes and subsequent physiological experiments, confirming the per-
meability of these channel proteins for water (Preston
et al., 1992).
For a long time, plant physiologists, on the other hand,
have not accepted the requirement of these molecules for
the simple reason that the majority of plants do not
possess organs or sites where comparable tremendous
amounts of water are transported through living cells. It
was assumed that the water permeability of a biomembrane itself is sufficient for the requirements of plant
cellular water movement (Fettiplace and Haydon, 1980;
Cullis et al., 1996). Since the counterparts of AQP1 were
identified in the plant plasma membrane and tonoplast,
this opinion is questioned. To date the existence of
aquaporins in virtually all living organisms, including
plants, is widely accepted, although their function is
sometimes still obscure. As outlined below, certain aspects
about the molecular mechanisms of aquaporin function
give rise to controversies and, consequently, the role of
these proteins in plant physiology is still not well understood and a matter of debate. Here, the controversial
aspects of plant aquaporins are outlined. Recent findings
concerning sensitivity to heavy metals and aspects of
selectivity for water as well as the important issue of
membrane water permeability regulation will be discussed.
Sensitivity of aquaporins to heavy metal ions
There is no doubt that the cellular biochemistry and
physiology of a living organism is seriously affected by
heavy metal ions. As early as the last century, mercury
compounds were frequently used to study different aspects
of plant metabolism and transport processes. In 1884,
Haberlandt was probably one of the first who could
inhibit water transport by the addition of a mercury
chloride solution (Haberlandt, 1884). When a detached
twig was incubated at the cut end with a heavy metal
solution, guttation of the attached leaves was inhibited.
This indicates that the substance somehow interacts with
1 To whom correspondence should be addressed. Fax: +49 931 8886158. E-mail: [email protected]
© Oxford University Press 1999
1542 Eckert et al.
a mechanism leading to the active sequestering of water.
Since then, techniques for studying the effects of heavy
metal solutions on plant physiology have been greatly
improved and some of them were concerned with the
mechanism of water transport. The notion that heavy
metals interact with a component of the water transport
process was strongly supported by the findings of aquaporin functional analysis assays in Xenopus oocytes. The
system has been successfully used for animal membrane
proteins and the technique was adapted to the requirements of water permeability measurement studies (Zhang
and Verkmann, 1991). After injection of cRNA corresponding to a putative aquaporin, the oocytes were incubated for protein synthesis. It is assumed that the proteins
were correctly integrated into the plasma membrane and
the capacity for increasing the membrane water permeability was calculated from the velocity of cell expansion in
hypo-osmotic conditions compared to a water injected
control. Following this procedure a couple of plant tonoplast ( TIP) and plasma membrane intrinsic proteins (PIP)
could be identified as aquaporins (Maurel et al., 1993;
Daniels et al., 1994; Kammerloher et al., 1994; Chaumont
et al., 1998; Biela et al., 1999). An argument in favour of
the assumption that the addition of a heavy metal solution
specifically blocks a water transport component was the
fact that the swelling of aquaporin expressing oocytes
could be dramatically reduced by mercury chloride solutions with a concentration of up to 1 mM. To account
for these results, many plant physiologists concluded that
heavy metal ions specifically block aquaporins and, consequently, could indicate the significance of these proteins
in whole plant or cellular water transport.
Although these conclusions appeared to be reasonable,
it turned out that the pharmacology of mercurials includes
numerous secondary effects (Schütz and Tyerman, 1997).
Since it is likely that some of these effects are still
unknown, the possibility of mercury interaction with the
transport components for osmolytes in plants cannot be
ruled out. The conditions used for the heterologous frog
oocyte system are rather artificial because water is forced
into the cell by a steep osmotic gradient accomplished by
the dilution of the incubation medium with water. This
treatment is far from what happens in the whole plant,
so that observations obtained in the oocyte system have
to be confirmed in planta, where the driving force for
transmembrane water flow is originated by changes in
osmotic gradients through solute or ion transporting
components or metabolism. Some of the ion channels are
known to be sensitive to heavy metals (Becker and Hoth,
personal communication). The water flux is, in cases
where high flow rates are required, facilitated by aquaporins. Consequently, and regardless of the metabolic
poisoning of the cell, a living plant might be inhibited in
at least two processes, i.e. the block of mercury-sensitive
aquaporins and the block of proteins transporting osmot-
ically active substances. These would be difficult to dissect
by physiological methods. In addition, even the results
obtained by the Xenopus oocyte expression system do not
always indicate a sensitivity of aquaporin-induced water
permeability to heavy metal ions. For the tonoplast
aquaporins investigated so far, a sensitivity to mercurials
seems to be requisite. In contrast, some plant aquaporins
of the plasma membrane, when expressed in Xenopus
oocytes, facilitate the passage of water no matter if
mercurials were added or not. The first aquaporin of this
type was described in Arabidopsis thaliana ( YamaguchiShinozaki et al., 1992; Daniels et al., 1994) and Biela et
al. reported a second type which is expressed in tobacco
roots (Biela et al., 1999). The functional implications of
sensitivity or resistance to heavy metals are still unknown.
The possibility that these aquaporins are rather common
in the plant plasma membrane should be considered in
the interpretation of experiments where heavy metals
were applied to plants. In this case, another component,
i.e. mercurial-resistant water channels, as well as sensitive
aquaporins and ion channels, have to be considered. In
our opinion, this prevents a precise conclusion about
aquaporin function in the living plant. The investigation
of organisms lacking one of the aquaporins either due to
a mutation (Shiels and Bassnett 1996; Preston et al., 1994;
Schnermann et al., 1998; Deen et al., 1994; Chou et al.,
1998) or by expression of an antisense RNA targeted
against an aquaporin mRNA (Agre, 1998; Kaldenhoff et
al., 1995, 1998) are possible means of getting information
about the physiological function of aquaporins in the
living organism.
Regulation of aquaporin water permeability
Further information about the implication of aquaporins
for plant water transport can be drawn from the developmental or tissue specific expression of aquaporins. High
levels of promoter activity, mRNA or AQP-protein were
detected during processes of cell elongation (Ludevid et
al., 1992; Kaldenhoff et al., 1995, 1996), in and adjacent
to stomatal guard cells (Sarda et al., 1997; Kaldenhoff et
al., 1995) or vessels of roots, stems or leaves ( Kaldenhoff
et al., 1995; Grote et al., 1998; Ludevid et al., 1992;
Yamada et al., 1997). A single aquaporin gene seems to
be expressed in a specific tissue and the respective gene
activity is probably additionally regulated by a factor
related to the developmental stage. Adult Arabidopsis
thaliana plants, for example, do not express each of the
plasma membrane aquaporins in every organ to the same
extent (Grote et al., 1998). From the physiological point
of view, a continuous high cellular water permeability
does not make sense under all circumstances. In instances
where a reduced water transport occurs, for example, if
transpiration is low during water stress or when cell
elongation is terminated, a high water permeability of
Plant aquaporin
certain tissues would not be necessary or could possibly
lead to excess loss of water and, in extreme situations,
to death. The most sensible conclusion is that the aquaporins are regulated, either by a block or a shift
from an activated to an inactivated, less permeable state.
Phosphorylation and dephosphorylation seems to suggest
itself as a molecular mechanism for this transition. An
argument to support these assumption is the existence of
multiple phosphorylation sites present in all aquaporins
(Reizer et al., 1993). Furthermore, there are reports
about increased water permeability of Xenopus oocytes
expressing aTIP after the addition of cAMP, adenylate
cyclase activator forskolin and phosphatase inhibitor
(Maurel et al., 1995). It is tempting to suppose that this
is a common regulation mechanism for water permeability. On the other hand, aTIP seems to be an exception in
this respect. Experiments under the same conditions confirm the results obtained by Maurel et al. (1995), but
undoubtedly show no activation of plasma membranelocated aquaporins (Fig. 1).
In experiments starting from the hypothesis that the
plasma membrane-located PM28 from spinach is phosphorylated and the aquaporin water permeability in
oocytes upregulated (Johansson et al., 1998), the addition
of a phosphatase inhibitor maintains and increases the
water permeability, a protein kinase inhibitor has the
opposite effect. The two classes of plant aquaporins,
located in tonoplast and plasma membrane, respectively,
are not only divergent in the positions of phosphorylation
sites which affect the protein function (amino acid 99 in
aTIP, respectively, 274 in PM28), but possibly differ also
in the type of modifying kinase which originates from
frog oocytes. At this point it should be mentioned that
the increase or decrease in water permeability is just a
variation of the flux rate of aquaporin expressing oocytes.
Fig. 1. Xenopus oocytes expressing cRNAs of aTIP or NtAQP1,
respectively, were either incubated with cAMP, forskolin and proteinase
inhibitor (cAMP) or not treated and subjected to hypo-osmotic
conditions (n>10). Water permeability (P ) was calculated as described
f
previously (Zhang and Verkmann, 1991).
1543
Although, it was demonstrated that the serine in PM28
at position 274 is phosphorylated in plants, the effect and
significance of the possible regulation at the protein level
on plant water transport remains to be demonstrated.
Regarding the dramatic changes in gene activity obtained
in various physiological and developmental situations,
the more important and potent regulation mechanism
seems to be at the expression level. A strong regulation
at the transcriptional level was shown by promoter
reporter gene expression for tonoplast as well as plasma
membrane aquaporins (Ludevid et al., 1992; Yamada et
al., 1997; Kaldenhoff et al., 1995), those by RNA- or
protein-stability have not been demonstrated yet. The
emerging picture about the modulation mechanism of
plant membrane water permeability, therefore, is a strong
regulation at the level of transcription and a possible finemodulation by phosphorylation, where its physiological
consequences remain obscure.
Selectivity of plant aquaporins
Besides the sensitivity to heavy metal ions and regulation,
a third point of conflict between data obtained in the
heterologous frog oocyte system and the situation in the
plant concerns the selectivity of plant water channels.
Aquaporins are membrane-located proteins which facilitate the osmotically driven transport of water from one
site of the membrane to the other. They are selective for
water, other molecules do not permeate. The latter statement relies on experiments where various solutions
containing diverse molecules were added to aquaporinexpressing oocytes and no uptake was measured. This is
an assertion which is believed to be true as long as
nothing but water is transported by the aquaporins. In
the instance of the human AQP3 and AQP1, those
substances could be detected. The former was described
to be permeable also for urea or glycerol (Ishibashi et
al., 1994) and the latter for CO (Cooper et al., 1998;
2
Nakhoul et al., 1998). Because plant plasma membrane
aquaporins are quite similar to human AQPs it is possible
that some of the PIP-like water channels are permeable
for these substances too. Permeability for glycerol in
addition to water has been shown for the soybean symbiosome membrane located NOD26 (Rivers et al., 1997;
Dean et al., 1999) and for the plasma membrane located
NtAQP1, which is highly expressed in tobacco roots
(Biela et al., 1999). This aquaporin is also permeable for
urea as illustrated by Fig. 2.
It remains to be demonstrated whether the membrane
transport of other substances like CO , PO or boron,
2
4
which are possibly more important for plant metabolism
than glycerol or urea, is facilitated by this type of aquaporin too. Again, it is questionable whether or not these
protein features are simply due to the artificial gradient
applied to the Xenopus oocyte expressing the particular
1544 Eckert et al.
Fig. 2. Xenopus oocytes injected with water instead of cRNA (control )
or cRNA corresponding to NtAQP1 or PIP2b and incubated for 10 min
with radioactive labelled urea (n>10). Uptake of urea is shown relative
to that of controls.
aquaporin. It is also not clear and hard to predict which
consequences for the physiology of the plant arise from
these protein features in a specific tissue or the whole
plant. The situation becomes even more puzzling if the
fact is taken into consideration that a membrane is
permeable for water or CO per se and that passage of
2
the one or other substance through aquaporins could
only increase the flux. On the other hand, if the exact
molecular features of plant aquaporins are known, plant
sites and cells that require a high influx or efflux of the
particular substance could be recognized. By resolving
these cellular bottlenecks and the connected physiological
events, the analysis of aquaporins, no matter if they are
solely water channels or permeable to other substances
as well, would give an opportunity for a better understanding of plant physiology and responses to environmental changes or developmental stages. A manipulation
of these sites either by influencing the regulation or
expression of aquaporins, could provide a possible means
for an adaptation of plants to certain stress conditions.
Acknowledgements
We thank Drs C Maurel (Institut des Sciences Végétales,
CNRS, Gif-sur-Yvettes Cedex, France) and AR Schäffner
(Institut
für
Biochemische
Pflanzenpathologie,
GSF
Forschungszentrum Oberschleissheim, Germany) for providing
cloned cDNAs of aTIP or PIP2b, respectively. We also thank
DFG (SFB251) for providing financial support.
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