 |
NATRIURETIC PEPTIDES IN PLANTS? |
Although molecular biology has
helped to blur many subdisciplines in biology, the same is less true
for physiology. Traditional animal physiologists still read almost
exclusively animal physiology journals, and plant physiologists still
read predominantly plant biology journals. Regrettably, the work of
researchers who try to bridge this gap often goes unappreciated. A case
in point is a provocative paper published in the American
Journal of Physiology by Vesely, Gower, and Giordano
(1993)
that addresses the possibility that plants, like
animals, may have natriuretic peptides (NPs). NPs are a family of
peptide hormones that have been strongly implicated in the regulation
of salt and water homeostasis in vertebrates. Vesely et al.
(1993) provided evidence that atrial natriuretic-like peptides are present throughout the plant kingdom and that these peptides increase the flow of solute and water upward to leaves and
flowers of plants. Although the contributions of Vesely et al.
(1993) have gained the advocacy of one laboratory
(Gehring, 1999) and the skepticism of one reviewer
(Takei, 2001), on the whole, the provocative hypothesis
of Vesely et al. (1993), now a decade old, remains
regrettably underexplored. It is hoped that this short review will draw
more attention to the ideas of Vesely et al.
(1993).
| |
ANPs in Plants |
NPs and their receptors have been identified in vertebrate
species ranging from elasmobranchs to mammals. NPs are hormones important for volume regulation in mammals, while they act more specifically for Na+ regulation in fishes. Atrial NPs
(ANPs) are stored in animals as a 126-amino acid prohormone that
circulates as a 98-amino acid NH2 terminus and as a COOH
terminus consisting of amino acids 99 through 126.
Atrial natriuretic factor (ANF) consists of the 28-amino acid
COOH-terminal end of the prohormone and three peptides from the
NH2 terminus (Gehring, 1999).
Vesely, Gower, and Giordano (1993) found that atrial
natriuretic-like peptides are present in the plant kingdom and that
these peptides increase the flow of solute and/or water upward to
leaves and flowers of plants. They determined that the 126-amino acid prohormone of atrial natriuretic factor (proANF)-(1-30),
proANF-(31-67), and ANF-like peptides were present in the roots,
stems, and leaves of a wide variety of Embryophyta. proANF-(1-30),
proANF-(31-67), and proANF-(79-98), but not ANF, significantly
increased the flow of colored water up stems, coloring their
flowers 15 to 35 min earlier than the other half of the same flowers
without exogenous peptide addition. These same peptides increased the
rate of transpiration (i.e. loss of water from the leaves) and
the absorption of solutions. Moreover, high-performance gel permeation
chromatography revealed that proANF-(1-30), proANF-(31-67), and
ANF extracted from plants are very similar to their pure synthetic
human sequences.
| |
Binding Studies of ANP in Plants |
Building upon earlier pharmacological binding studies by
Gehring et al. (1996), Suwastika et al.
(2000) demonstrated that 125I-rat ANP (rANP)
binds to plasma membranes isolated from the leaves and stem
tissue of Tradescantia multiflora and, importantly, that both unlabeled rANP and immunoaffinity-purified plant NP (PNP) from
English ivy (Hedera helix) can competitively
displace each other. In addition, autoradiography measurements revealed
the specific binding of 125I-rANP to leaf and stem tissue
in situ.
| |
Effects on Stomata and Other Leaf Cells |
Consistent with the original findings of Vesely et al.
(1993), Gehring et al. (1996) found that
rANP induced stomatal opening in Tradescantia
spp. in a concentration-dependent manner. Pharmawati, Billington, and Gehring (1998) demonstrated that the
effect of rANP is critically dependent on the secondary structure of
the peptide hormone. The native circular molecule is active,
whereas the linearized molecule shows no biological activity.
Furthermore, they found that rANP-induced stomatal opening is
reversibly inhibited by two inhibitors of guanylate cyclase, LY 83583 and methylene blue. Stomatal opening was also induced in a
concentration-dependent manner by the cell-permeant cyclic
guanosine-3',5'-monophosphate (cGMP) analog 8-Br-cGMP, and this effect
was prevented by abscisic acid. PNP also induced stomatal
opening in a concentration-dependent manner
(Billington, Pharmawati, and Gehring, 1997).
Pharmawati et al. (2001) found that rANP and PNP
significantly elevated cGMP in guard cell protoplasts. Stomata opened
by PNP could be induced to close by LY 83583. Evidence was
provided that this effect of cGMP on stomatal opening may be linked to Ca2+ levels. rANP, PNP, and 8-Br-cGMP all induced stomatal
opening and this was inhibited by compounds that
lower intracellular Ca2+ levels such as ethylene glycol
bis(3-aminoethyl ether)
N,N,N',N'-tetraacetic acid (EGTA), ruthenium red, and procaine. The connection between Ca2+ and cGMP was further supported by the fact that
PNP-induced increases in cGMP levels do not occur in the presence of
EGTA. Since the plasma membrane H+-ATPase is a key enzyme
driving stomatal opening, Pharmawati et al. (2001)
examined whether a causal relationship exists between cGMP, rANP, or
PNP and H+ transport across the guard cell plasma membrane.
Their results showed that the activity of the H+-ATPase was
reduced by 8-Br-cGMP and increased by rANP and PNP. However,
ATP-dependent transmembrane H+ gradients were found to be
enhanced only by rANP and not by PNP. Conceivably, this difference in
the response of plants to rANP and PNP may be due to specific effects
of PNP on H+-coupled symporters.
More recently, evidence has been presented indicating that rANP may
have physiological effects on mesophyll cells too. Maryani et al.
(2001) found that a synthetic peptide identical to the C
terminus (amino acids 99-126) of rANP modulates the osmotically induced swelling of potato (Solanum tuberosum) mesophyll
cell protoplasts (MCPs) in a concentration- and time-dependent manner. Osmotically induced volume changes in MCPs were enhanced by plant extracts with NP immunoreactivity. In contrast, pretreatment of the
plant extracts with rabbit anti-human ANP (99-126) antiserum suppressed enhanced osmoticum-induced swelling. PNP also enhanced osmotically induced swelling. A curious finding was that while rANP and
PNP caused increases in cGMP levels in MCPs, elevated cGMP in itself
did not induce osmotic swelling but rather exerted an inhibitory effect.
| |
Effects on Water Uptake by Roots |
Pharmawati et al. (1998) found that PNP
fractions rapidly and specifically increased cGMP levels in stele
tissue isolated from maize (Zea mays) roots within
30 s. Unlike the case with stomata, LY 83583, an inhibitor of
guanylate cyclase, was without effect. The authors did not consider
this finding to be problematic since LY 83583 does not always inhibit
particulate guanylate cyclase. Rather, the authors suggested that
their results support the existence in the stele of plant roots of a
membrane-bound PNP receptor containing intrinsic guanylate cyclase
activity analogous to animal NP receptors.
Suwastika and Gehring (1998) found that both rANP
and PNP significantly increased radial water movements out of the xylem of shoots of T. multiflora. Enhanced radial water
movements were also observed in response to 8-Br-cGMP. LY 83583, an
inhibitor of soluble guanylate cyclase, reduced radial water movements, but had no effect on cGMP levels, suggesting that LY 93583 may have
more than one site of action in plants. Suwastika and Gehring (1998) speculated that as in vertebrates, NP effects
might, at least in part, be mediated via the regulation of
membrane-bound guanylate cyclases and aquaporin water channels.
| |
Molecular Biology |
By means of Southern blot hybridization, Vesely et al.
(2001) discovered the presence of an ANP-encoding gene
within plants. Northern blots indicated that this gene expressed ANP
prohormone mRNA. Southern blots of English ivy genomic DNA revealed
that the ANP gene sequence was present in its roots, stems, and leaves. Northern blot analysis of total plant RNA isolated from leaves, roots,
and stems of English ivy revealed a single 0.85-kb prohormone ANP
transcript in stems similar to that detected in rat
(Rattus norvegicus) heart.
TOP
NATRIURETIC PEPTIDES IN PLANTS?
ANPs in Plants
a new class of plant hormone?
Ann Bot
83: 329-334