384
Plants embrace a stepchild: the discovery of peptide growth
regulators
Henk J Franssen
Over the past decade, peptides have been added to the
collection of signalling molecules in plants. As the impact of
peptide hormones in non-plants is enormous, a comparison of
plant and non-plant peptide signal molecules at this stage
deserves our attention—not only to reveal common and unique
features, but also to point to new avenues of future research
on plant hormones.
Addresses
Department of Molecular Biology, Agricultural University, Dreijenlaan 3,
6703HA Wageningen, The Netherlands; e-mail:
[email protected]
Current Opinion in Plant Biology 1998, 1:384–387
http://biomednet.com/elecref/1369526600100384
signalling cascades in plants do exist; avirulence genes of
several pathogens encode peptides and the corresponding
plant resistence genes encode putative receptor proteins,
containing leucine-rich repeats (LRRs) [4•]. Indeed, in
animals several LRR receptors recognise peptide hormones [5]. Strikingly, LRR encoding genes have also been
shown to be crucial in controlling shoot and flower meristem size [6] and embryogenesis [7]. Although the
(peptide) ligands for these LRR containing receptors have
not been identified as yet, the above data support the role
of peptides in plant development.
This review will describe the state-of-art of plant peptides
within the context of the broader knowledge of non-plant
peptide signals.
© Current Biology Ltd ISSN 1369-5266
Abbreviations
GFP
green fluorescent protein
LRR
leucine rich repeat
ORF
open reading frame
Introduction
Intrinsic to multicellular organisms is the continuous flow
of information between cells to co-ordinate the role and
functioning of individual cells within the organism.
Intercellular communication in plants has long been
thought to be the exclusive playground of the classic plant
hormones [1]. In recent years, however, this group of signal
molecules has been enriched with oligosaccharides, brassinosteroids, polyamines and jasmonates [2]. Although at first
glance these signal molecules seem to be specific to plants,
several structurally related molecules are operational in nonplants as well; for example, serotonin resembles auxin, nitric
oxide ethylene, steroids brassinosteroids and prostaglandins
jasmonic acid. Apparently, the nature of signal molecules
has been conserved throughout plant and animal kingdoms.
It is amazing, therefore, that a prominent group of signal
molecules in non-plant eukaryotes, peptide hormones, had
to await its discovery in plants for so long.
The imperium of non-plant signalling peptides
Research on non-plant peptides has resolved the successive steps in peptide signalling: peptide production,
transport and perception and, finally, integration at the cellular level [3]. The generation of active peptides is
controlled at the transcriptional- and post-translational
level. At the latter, this is regulated by proteolytic processing, usually by proteases belonging to the family of serine
proteases which recognise a dibasic motif [3,8] and/or by
post-translational modification of the primary translation
products [3]. Translocation out of the cell involves either
the secretory pathway or a membrane targeting secretory
mechanism [3,9,10]. The presence of specific amino acids
within the prohormones determines by which of these two
secretory pathways the active hormone is secreted, in
numerous cases only after external stimuli. Perception of
the peptide signals requires the presence of specific cell
surface receptors on receiving cells that provide the first
link in the signal perception- transduction cascade. Four
major classes of peptide hormone receptors can be distinguished; G-protein linked receptors, ligand gated
ion-channel receptors, receptor kinases and receptors with
intrinsic enzymatic activity [3].
Stepchild spotted
In non-plant eukaryotes, hundreds of peptide hormones
have been discovered that play a key role in embryogenesis, cell division and proliferation and neural transmission
[3]. Indeed, several of these processes are also intrinsic to
plant development but, strikingly, non-peptide signal molecules have traditionally been ascribed the major roles in
most of these processes [1]. In other words, there was felt
to be no need to look for a potential role for peptide signals in these processes.
The unraveling of the genetic basis of plant–pathogen
interactions, however, has revealed that peptide-activated
To date, four peptide signal molecules have been discovered in plants; systemin, enod40, cyi1a and sulfokins
(Table 1).
Systemin was identified as the active factor that is transported out of the wounds of wounded tomato plants to
distal tissues to induce the expression of two well characterised wound-inducible proteinase inhibitor encoding
genes [11,12]. These genes are systemically induced in
leaves of pathogen-attacked tomato and potato plants and,
therefore, are part of the inducible defense repertoire of
the plant. In the systemin perceiving cells the induction of
Plants embrace a stepchild Franssen
Table 1
Amino acid sequences of the four plant signalling peptides
Systemin [11,37]
Tomato
A V Q
Potato-1
A V H
Potato-2
A A H
Nightshade A V R
Pepper
A V H
S
S
S
S
S
K
T
T
T
T
P
P
P
P
P
P
P
P
P
P
S
S
S
P
S
K
K
K
K
K
R
R
R
R
R
D
D
D
D
P
P
P
P
P
P
P
P
P
P
P
K
K
K
K
K
M
M
M
M
M
Q
Q
Q
Q
Q
T
T
T
T
T
D
D
D
D
D
enod40 [16,21]
Tobacco
M
Q W D E A I H G S
Soybean
M E
L C W Q T S I H G S
Pea
M K L L C W Q K S I H G S
cyi1a [19·]
Tobacco
M A S S R H Q M Q C T K Y N K S L H T HG T
Sulfokin [20,22·]
Asparagus/rice
Y I Y T Q
Y I Y T
the proteinase inhibitor encoding genes is accomplished
through the induction of jasmonic acid biosynthesis [13].
385
assay, the mutant plant cyi shares several phenotypes consistent with an altered cytokinin metabolism or response.
Furthermore, it is striking that in this T-DNA tagging, a
gene was tagged that is transcribed into an mRNA of 1000
bases and that in this mRNA the first ATG is the start
codon of the longest ORF, yet it codes for a protein of just
22 amino acids.
Sulfokins were recognised as a group of mitogenic factors
that induce proliferation of plant cells in low-density suspension cell cultures. The activity of sulfokins are
indispensable by auxin or cytokinin [20].
Strikingly, for three out of the four peptides isolated so far
— enod40, sulfokins and cyi1a — a role in cell division and
proliferation is plausible. Herein, enod40 and cyi1a seem
to interplay with the activity of the classical hormones
auxin and cytokinin, whereas sulfokins are working independently of these hormones. Thus, a picture emerges
indicating that plant peptides are involved in fine tuning
the activity of other (hormone) signals in plants.
Are we relatives?
Enod40 was originally isolated as a gene that becomes activated during root nodule formation on legumes as a result
of the interaction of these plants with soil-borne Rhizobium
bacteria [14,15].
From transient expression studies of a transgene construct
consisting of a translational fusion of the enod40 open reading frame (ORF) and green fluorescent protein (GFP) in
protoplasts, it can be inferred that the enod40 ORF is translated in plant cells and the existence of the resulting
peptide in nodules has been shown by ELISA [16].
Ballistic targeting into Medicago truncatula (Mt) of a
Mtenod40 cDNA clone comprising the peptide encoding
region leads to division of root cortical cells [17•]. Alfalfa
transformed with antisense enod40 leads to impaired regeneration of calli, while overexpression of enod40 gives rise to
embryogenic tumors [18], both indicating that the function
of enod40 is not restricted to nodule formation. The identification of an enod40 homolog in tobacco is in line with this
hypothesis [16]. Furthermore, from these observations it
can be inferred that enod40 plays a role in cell proliferation, which in most cases is controled by an
auxin–cytokinin balance. Strikingly, both synthetic tobacco and soybean enod40 peptides allow tobacco protoplasts
to divide at supra optimal auxin concentrations [16].
Although doubts now exist about the value of the tobacco
protoplast assay used, tomato cell suspension cultures
exposed to tomato enod40 are altered in their response to
auxin as well, reflecting the significance of enod40 like
peptides in non legumes (H Franssen, unpublished data).
The gene encoding cyi1a was identified through activation
T-DNA tagging of tobacco and screening for tagged lines of
which protoplasts division occurs independent of cytokinin
and auxin [19•]. Again, despite the current dispute over this
As the research field covering plant peptide signalling
comes of age, it is impelling to address the question as to
what extent plant peptides and non-plant peptides share
properties. This is not only useful for the sake of comparison but also important to shed light on the gaps in our
knowledge.
Plant peptides are active in the nanomolar to picomolar
range [11,16,20].
Clones have been isolated for systemin, enod40 and cyi1a
[11,14,15,19•]. Systemin is synthesised as part of a propeptide of 200 amino acids from which the active systemin,
consisting of 18 amino acids (Table 1), is released — most
likely through proteolyses [12]. In contrast, both enod40
and cyi1a are encoded by a short open reading frame comprising 10–13 [16] and 22 amino acids [19•] (Table 1),
respectively. These peptides seem to be synthesised
directly as active entities. Thus, the question arises as to
what are the mechanisms underlying the control of activity of these peptides.
It has been put forward that a second region that is highly
conserved among all enod40 mRNAs and is located in the
3′UTR and non-coding [16,17•,18,21] might have a regulatory role in the translation of the peptide encoding part.
This hypothesis is formed on the observation that the
responses after transient expression of DNA constructs
containing either the peptide spanning region or the
3′UTR are identical [16,17•]. This can be explained by
assuming that the endogenous enod40 is expressed at low
levels, but not translatable as a result of the binding of a
protein in the 3′UTR. As a consequence of transfection of
cells with 3′UTR containing constructs the inhibitory protein is diluted and thus released from the endogenous
386
Cell signalling and gene regulation
enod40 mRNA, which is now translatable. Such a mechanism is reminiscent of, for instance, the translational
control of bicoid mRNA by the caudal gene product in
Drosophila [16]. Sulfokins have been isolated from the
dicot Asparagus officinalis and the monocot rice [20,22•].
Sulfokins consist of 4–5 amino acids: among them two
tyrosines, the sulfation of which is required for biological
activity (Table 1) [20]. The plant sulfokins share this posttranslational modification with cholecystokinin and
leucosulfakinin, two sulfated peptides isolated from animals and insects [23,24], respectively. The non-plant
sulfokins are released from larger primary translation products. Genes encoding the plant peptides, however, have
not yet been isolated.
Only for systemin has translocation out of the cell been
demonstrated [11], but as prosystemin lacks all the characteristic features of a secretory polypeptide, the mechanism
involved is unknown. Sulfokins have been purified from
conditioned medium implying they have been translocated.
There is no evidence as yet for secretion of enod40 or cyi1a.
In the search for receptors, sytemin- and sulfokin binding
proteins have been identified. Systemin binds to a 50kD
protein that has kex2p-protease like activity (i.e. belongs
to the family of serine proteases recognising a dibasic
motif) [25], and the nature of the sulfokin binding proteins
awaits elucidation [22•].
Despite the patchy knowledge of peptide signalling, it
seems plausible to conclude that plant peptides are part of
signal transduction cascades that resemble those resolved
for peptide hormones in non-plants. At the same time,
many questions concerning the constitution of these cascades arise that await further research to be resolved.
Perspectives: a bright future for Cinderella
Now the paradigm of no peptide signals in plants has been
abandoned, it can be anticipated that more signalling peptides will be discovered. The progress made in plant
genome- and expressed sequence tag (EST) sequencing
projects will certainly contribute to this. Furthermore, the
number of genes encoding (putative) receptors is increasing, among them LRR containing proteins [4•,6,7,26,27],
crinkly4, a tumor necrosis factor-like receptor [28] and a
putative G-protein-coupled receptor [29]. It can be expected that, like in non-plants, peptides serve as ligands for
these receptors in plants as well.
In addition, so-called non-translated RNAs should be reinvestigated for the presence of short ORFs[30,31,32]. A
surprising discovery in the course of identifying plant signalling peptides, is the identification of mRNAs encoding
short peptides. Studies on transgenes consisting of a translational fusion of the putative ORF and a marker gene, like
GFP, either transiently expressed or after stable integration, will be valuable in determining whether small
peptides are made in planta.
Purposeful research can now be initiated to unravel existing peptide hormone involving cascades. Genetic
approaches will be crucial in identifying the participants
involved, and physiological and biochemical approaches
will be needed to answer questions related to the mechanisms by which they work.
To get an impression of how long it might take to fill in the
gaps in our knowledge, the history of insulin, the first peptide hormone discovered in mammals, provides an
illustrious and also somewhat frightening example.
In 1922 Banting and Best [33] identified and partially purified insulin. Forty five years later it was found that insulin
is proteolytically cleaved from a larger precursor, proinsulin
[34]. In the mid 80’s the receptor for insulin was isolated
[35] and even 75 years after the discovery of insulin it
turned out that its story is not completed — recently, it was
found that also the C-peptide that is released from the prohormone by proteolytical events displays biological
activity [36].
It is clear that we are standing at the beginning of the
exploration of a new field in plant biology and the excitement about plant peptide signal molecules will certainly
last for a great part of the 21st century.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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• of special interest
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