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OCR for page 65
Colloquium
Systemins: A functionally defined family of pepticle
signals that regulate defensive genes in
Solanaceae species
Clarence A. Ryan* and Gregory Pearce
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340
Numerous plant species have been known for decades that re-
spond to herbivore attacks by systemically synthesizing defensive
chemicals to protect themselves from predators. The nature of
systemic wound signals remained obscure until 1991, when an
18-aa peptide called systemin was isolated from tomato leaves and
shown to be a primary signal for systemic defense. More recently,
two new hydroxyproline-rich, glycosylated peptide defense sig-
nals have been isolated from tobacco leaves, and three from
tomato leaves. Because of their origins in plants, small sizes,
hydroxyproline contents (tomato systemin is proline-rich), and
defense-signaling activities, the new peptides are included in a
functionally defined family of signals collectively called systemins.
Here, we review structural and biological properties of the syste-
min family, and discuss their possible roles in systemic wound
. .
slgna Ing.
prosystemin I systemin receptor I plant defense
Systemin, the initial peptide signal found in plants, is an
intracellular signaling molecule that is synthesized within the
amino acid sequence of a 200-aa precursor, called prosystemin
(1, 2~. Systemin induces proteinase inhibitor protein synthesis in
leaves of young tomato plants when supplied for a few minutes
through their cut stems at nanomolar concentrations (14. Ra-
dioactively labeled systemin, when placed on wound sites on
leaves, is found in the phloem (1, 3~. A key role for systemin in
systemic signaling was established by showing that tomato plants
expressing an antisense prosystemin gene become deficient in
long-distance wound signaling and are more susceptible to insect
attacks than wild-type plants (44.
In contrast to animal peptide hormones, the systemin precur-
sor protein lacks a leader or signal sequence that is required for
synthesis and processing through the secretory pathway (2~.
Immunolocalization techniques revealed that prosystemin is
localized in parenchyma cells of vascular bundles (5~. This
localization in the vicinity of the sieve tubes of the phloem may
facilitate transport of systemin and the oxylipins it induces in
response to wounding to distal cells. Systemin activates defensive
genes by interacting with a cell-surface receptor, called SR160,
a 160-kDa transmembrane protein with an extracellular leucine-
rich domain, and an intracellular receptor kinase domain (6, 7~.
The interaction of systemin with the receptor is the first step of
a complex intracellular signaling pathway that involves the
activation of a mitogen-activated protein kinase (MAPK) (8),
the- rapid alkalinization of the extracellular medium (7, 9), the
activation of a phospholipase (10, 11), and the release of
linolenic acid that is converted into oxylipins such as phytodi-
enoic acid and jasmonic acid that are powerful signals for
defense genes (Fig. 1) (12, 13~. The pathway exhibits analogies
to the inflammatory response in animals (14) in which wounding
activates MAPKs, phospholipases, the release of arachidonic
www.pnas.org/cgi/doi/ 10.1 073/pnas. 1934788100
acid from membranes, and its conversion to prostaglandins,
which are analogs of phytodienoic acid and jasmonic acid
The early alkalinization in response to systemin in tomato
suspension cultures was the basis for the development of an
assay system that led to the identification and characterization
of the systemin receptor, SR160 (7~. SR160 is homologous to
the BRI1 receptor from Arabidopsis (15), with a high percent-
age of amino acid identity. This was the first indication that the
systemin receptor may be a close relative of the BRI1 receptor.
This possibility was confirmed by the identification and clon-
ing of the tomato brassinolide receptor, BRI1 (16), which was
found to be identical to the tomato SR160 receptor. The
identity of a receptor with two functions, i.e., defense and
development, was unique in plants, but examples are known in
the animal kingdom. The dual function of the SR160/BRI1
receptor was supported by experiments in which the tomato
SR160/BRI1 receptor cDNA was expressed in tobacco, which
does not express a prosystemin gene and therefore does not
produce systemin as a defense signal (17~. Transformed to-
bacco suspension-cultured cells synthesized the receptor and
targeted it to the cell surface membranes of tobacco, where it
displayed the identical binding characteristics with systemin as
SR160 in tomato cells. The systemin-receptor interaction in
tobacco cells induced the alkalinization response, indicating
that signaling components for the early steps in the systemin
signaling pathway were present in tobacco and could be
activated by the tomato SR160 receptor when it interacted with
systemin. Additionally, a tomato mutant cu-3, which was
caused by a mutation in the BRI1 receptor and led to the
isolation of the BRI1 gene (16), is severely impaired in
systemin signaling (17~.
Because tobacco does not produce systemin, the presence of
components in tobacco cells that react to the systemin-receptor
interaction indicated that the BRI1 receptor may have, or may
have had in the past, a defensive role in plants that was co-opted
by systemin as the prosystemin gene evolved in species of the
Solaneae subtribe of the Solanaceae family. Tobacco does
exhibit a fairly strong systemic defense response to wounding in
young plants, but it is much weaker in older plants. Wounded
tobacco olants sYnthesize a trypsin inhibitor (TTI) that is a
paralog of tomato inhibitor II (18), which is induced in tomato
leaves in response to wounding. The induction of TTI in tobacco
This paper results from the Arthur M. Sackier Colioquium of the Nationai Academy of
Sciences, "Chemical Communication in a Post-GenomicWoricl," helcl January 17-19, 2003,
at the Arnolcl anc! Mabel Beckman Center of the National Academies of Science anc!
Engineering in Irvine, CA.
Abbreviations: MAPK, mitogen-activatecl protein kinase; TobHypSys, tobacco hyciroxypro-
line-rich systemin; TomHypSys, tomato hydroxyproline-rich systemin.
*To whom corresponcJence shoulcl be aciciressecl. E-mail: cabuciryan~hotmail.com.
2003 by The National Acaclemy of Sciences of the USA
PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2 1 14577-14580
OCR for page 66
PROSYSTEMIN
| herbivore attacks,
~ wounding
SYSTEMIN
~pHI _
~ a_,_
it' ~
Proton ATPase ~ \
Defense
Genes
_ lUAPKs ~ PL ~ membranes
~ VIA
LA
Ioctadecanoid
pathway
Fig. 1. A simplified diagram of the systemin signaling pathway. The pathway
shows several key steps of the signaling pathway, and in particular the steps
leading to the blockage of a proton ATPase that leads to the alkalinization of
the extracellular medium, which is the basis of the assay developed to identify
signaling peptides.
leaves in response to wounding indicates a genetic link between
the wound-signaling systems of tomato and tobacco, despite the
absence of systemin in tobacco. The synthesis of TTI in young
tobacco plants is strongly induced by jasmonic acid (18), indic-
ative of the early steps of signaling that result in the release of
linolenic acid from membranes, similar to tomato plants. The
roles of both systemin and jasmonate in systemic signaling have
been the subject of considerable speculation (19, 20~. We
hypothesize here that the evolution of the prosystemin gene in
species of the Solaneae subtribe resulted in the production of
systemin, a strong systemic signal that is not found in other
plants, that amplifies the jasmonate signaling pathway. Prosys-
temin released from cells at the wound site is likely processed to
systemin by proteinases also released from damaged cells. This
would allow diffusion of systemin to the apoplast of nearby
unwounded vascular cells to interact with its receptor and induce
the synthesis of jasmonates. As jasmonates move through the
plant, it would induce more prosystemin along with proteolytic
enzymes that are known to be induced by jasmonate (14) that
could process the nascent prosystemin to systemin to continue to
amplify the jasmonate signal in nearby cells.
A major source of jasmonates at wound sites is from
linolenic acid that is produced by the degradation of mem-
brane lipids within the cellular debris. This source of jas-
monates would likely provide an important "kick-start" for
defense signaling, as the oxylipins diffuse into the vascular
system and are transported to parenchyma cells (5) to up-
regulate signaling pathway genes, including the prosystemin
gene (in Solaneae species). This hypothetical scenario led us
to suspect that other peptide signals that were not systemic may
be present in tobacco and tomato plants that might help
amplify wound signaling. Such peptides in tobacco, which lacks
a systemic peptide signal, might contribute to a localized
amplification of the synthesis of jasmonates in response to
wounding, and to amplification of jasmonate synthesis in the
absence of systemin.
TDbacco Systemins I and 11
The search for peptide signals in tobacco was facilitated by the
development of a biological assay that is based on the alkalin-
ization of the medium of tomato suspension-cultured cells in
response to systemin, which is characterized by an increase in pH
(up to 1 pH unit per 10 min) in the culture medium (9~.
Suspension cultured tobacco cells do not exhibit an alkaliniza-
14578 1 www.pnas.org/cgi/doi/ 10.1 073/pnas. 1934788100
Peptide Amino Acid Sequence Pentose Units
1 18
TobEypSys I RGANLPOOSOASSOOSRE
TobEypSys I I NRRP~SOOSOKPADGQRP
1 18
Syst~min AVQSKPPSKRDPPRMQTD
9
6
o
Fig. 2. The amino acid sequences of tobacco hydroxyproline-rich systemins,
TobHypSys I and TobHypSys II, are shown compared with the sequence of
tomato systemin. Hydroxyproline (O), proline (P), threonine (T), and serine (S)
residues are in red, charged amino acids are in black, and neutral amino acids
are in blue. The ranges of pentose units attached to each peptide, determined
by mass spectrometry, are shown at the right.
tion response when supplied with tomato systemin, but do so in
response to a crude peptide fraction obtained from tobacco
leaves, suggesting that a peptide-receptor interaction may be
occurring that is coupled to an intracellular response. The
alkalinization of 1 ml of suspension cultured tobacco cells in
response to 1-,ul aliquots from fractions eluting from HPLC or
other columns revealed the presence of two peptides that, when
purified and characterized, were found to be 18-aa glycopeptides
that contained multiple hydroxyproline residues (21~. The two
peptides are active in the alkalinization assay with tobacco
suspension cultures at nM concentrations, and both cause a rapid
activation of a 48-kDa MAPK, similar to the 48-kDa MAPK
activated by tomato systemin in tomato cells (8~. These peptides,
supplied to young excised tobacco plants through their cut stems
at nM concentrations, induce the synthesis of TTI in leaves.
Because of their similarities to tomato systemin in signaling
properties, the two peptides were called tobacco systemin I and
II (21~. However, because of their hydroxyproline (O) contents,
they are now named tobacco hydroxyproline-rich systemin
(TobHypSys) I and II to identify them as members of a func-
tionally related systemin family (22~. Their amino acid sequences
are shown in Fig. 2. Neither peptide exhibits homology with
tomato systemin, but -OOS- motifs found in the tobacco peptides
are posttranslational modifications of the primary translation
motif -PPS- that is found in tomato systemin (14. The two new
peptides are rich in P/O residues, and in S and T residues as well.
These three amino acids make up 50~o of each peptide and are
likely involved in their recognition as defense signals.
Mass spectroscopy of the two peptides revealed that the
attached carbohydrate moieties consist of pentose residues; nine
in TobHypSys I and six in TobHypSys II. The structural prop-
erties of TobHypSys I and II (leader sequence, hydroxylation of
-P- residues, and carbohydrate decorations) indicate that they
are synthesized through the secretory system, unlike tomato
systemin, which is not glycosylated, whose prolines are not
hydroxylated, and whose precursor has no signal sequence (1, 2).
Both TobHypSys peptides originate from a single 165-aa-long
preproprotein, including a signal sequence, with the TobHypSys
I sequence near the N terminus and the TobHypSys II sequence
near the C terminus (21~. The presence of multiple signaling
peptides contained in a single preproprecursor is a characteristic
of many animal peptide hormones, but the two tobacco syste-
mins provide the first example in plants of a peptide hormone
precursor harboring multiple peptide signals.
Although tobacco does not use a tomato systemin homolog
for systemic wound signaling, TobHypSys I and II appear to
serve roles in defense signaling. Because proTobHypSys is
hydroxylated and glycosylated, like well characterized hy-
droxyproline-rich glycoproteins (23), it may be associated with
cell walls, and may be processed from the precursor at wound
sites to provide signals to amplifiy the synthesis of oxylipins
Ryan and Pearce
OCR for page 67
Peptides
Amino Acid Sequences Pentose Units
TomBypSys I RTOYXTOOOOTSSSOTHQ 8-17
1 20
TomBypSys II GRED1rvAsooooRpQDEQRQ 12-16
~ 15
TomEypSys III GREDSVLPOOSOICTD 10
Fig. 3. The amino acid sequences of tomato hydroxyproline-rich systemins,
TomHypSys 1, praline (P), TomHypSys 11, and TomHypSys 111, are shown. Hy-
droxyproline (O), threonine (T), and serine (S) residues are colored as in Fig. 2.
The number of pentose units associated to each peptide are shown in the right
column.
during long distance wound signaling. Zhang and Baldwin (24)
have elegantly shown that wounding of tobacco causes the
synthesis of jasmonic acid that acts as a systemic signal from
leaves to roots. It may be that the TobHypSys peptides help
generate jasmonic acid that is targeted to the roots of the plant
in response to wounds.
Tomato Leaf Hydroxyproline-Containing
Systemin Peptides
The isolation of 18-aa, glycosylated, hydroxyproline-containing
tobacco systemins led to an investigation of the possibility that
tomato plants may also have peptide defense signals similar to
the tobacco systemins. The alkalinization assay used to identify
and isolate the two tobacco systemins was used to analyze tomato
leaf extracts for peptide signals in addition to systemin. The assay
identified several components from tomato leaf extracts that
caused an alkalinization response. Purification and character-
ization of these components confirmed that one peptide was
tomato systemin and identified three new peptides (22) The
novel peptides exhibit several properties similar to TobHypSys I
and II, being hydroxyproline-rich glycopeptides, and ranging in
size from 15 to 20 ea. Each of the peptides contains an internal
continuous sequence of from 5 to 11 aa variously composed of
O. P. S. or T residues, and all are flanked by various charged
residues (Fig. 3~. The peptides are decorated with variable
numbers of pentose residues, but their identities and locations on
the peptides have not been determined. The amino acid se-
quences of tomato hydroxyp ro line - rich syste min (TomHyp Sys )
II and III indicated that they shared limited amino acid sequence
homology and were likely products of gene duplication-
elongation events. I~he three tomato peptides exhibit similar
biological activities as tomato systemin, indicating that they are
defense signals (22~. They all exhibit similar specific activities in
the alkalinization assays, and all are effective inducers of pro-
teinase inhibitors I and II synthesis when supplied to young
tomato plants. The tomato peptides were therefore included in
the functionally defined systemin family and named tomato
hydroxyproline-rich systemins, i.e., TomHypSys I (18 amino acid
residues), TomHypSys II (20 amino acid residues), and Tom-
HypSys III (15 amino acid residues). Although the three Tom-
HypSys peptides are powerful inducers of defense genes when
supplied to excised tomato plants, they do not serve as primary
systemic signals, because tomato plants transformed with an
antisense prosystemin gene were incapable of systemic signaling
in response to wounding (4~.
Isolation and characterization of cDNAs coding for the
tomato peptides revealed that all three were derived from the
same 146-aa preproprotein precursor that includes a sianal
sequence. This precursor, along with the precursor of the
TobHypSys peptides, provides the only examples in plants of
polyprotein hormone precursors. Box diagrams of the three
precursor proteins that harbor the six members of the systemin
Ryan and Pearce
Tom proSys (200 aa)
Tob proHypSys (165 aa)
Tom proHypSys
SYS
1 111 11
(146 aa) ~_
Fig. 4. Box diagrams of the precursors of tobacco and tomato systemin
peptides. The open boxes represent the leader sequences of the newly trans-
lated proteins. The HypSys peptides are shown as hatched boxes, with their
numeral identities above each peptide. The systemin peptide is shown as Sys.
The length of each preproprecursor is shown in parentheses.
family are compared in Fig. 4. A comparison of the amino acid
sequences of the TomHypSys precursor with the TobHypSys pre-
cursor revealed a 10-aa sequence at their N termini that were
identical at eight residues. The nucleotide sequence identity of this
sequence was 90%. The significance of this identity is not clear, but
does suggest that the two precursor genes may have a common
ancient precursor, and that this sequence may have an important
function that has been conserved. No homology was evident
between propystemin and either of the two preproprecursor pro-
teins. However, it is of interest that the sequence of tomato systemin
contains 7 of 18 residues that are P, S, and T (1~. Because
prosystemin has been found only in species of the Solaneae subtnbe
of the Solanaceae family, we speculate that prosystemin may have
been a member of the TomHypSys family and that some mutational
event may have caused the loss of the leader sequence that resulted
in the synthesis of the nascent precursor peptide to shift from a
secretory pathway ongin to a cytoplasmic ongin, providing a
powerful systemic defense si~al (systeniin) that was retained in the
evolving species of the Solaneae subtribe.
Perspectives
The multiple P, O, S, and T residues in all six members of the
systemin family in tomato and tobacco plants suggest that these
residues have important structural roles for interacting with
receptors. The P residues confer polyproline II structures (PP II)
that have distinct kinks that may be the key to receptor recog-
nition (25, 26~. PP II structures are commonly found in peptide
ligands of animals, where they appear to be important for
recognition by receptors (27~. In all five HypSys peptides, the
central P and O residues are flanked by basic or acidic amino
acids, either internally or near both the N and C termini.
The discovery of the HypSys defense signals in tomato and
tobacco raise many questions about wound signaling in these
and other plant species. The relatiorlship of the systemins to
local and systemic signaling and whether the HypSys peptides
interact with homologs of the systemin receptor or have
entirely different receptors for each peptide remain to be
determined. Also of interest is whether the different peptides
in tomato plants can activate the same complement of defense
genes as systemin in response to wounding. The presence of a
family of functionally similar HypSys defense signaling pep-
tides in tomato and tobacco that are derived from parologous
precursors introduces the possibility that, in other plant fam-
ilies, related defense signaling peptides may be present that
share a common ancestral ongin. A search for such signals is
now possible by using the same strategies that led to the
discovery of the defense signaling peptides and their genes in
tomato and tobacco that are described herein.
We thank S. Vog~man for plant growth and maintenance of plants, G.
Munske for amino acid sequence analyses, and W. Seims for matrix-
assisted laser desorption ionization mass spectrometric analyses. This
research was supported by Washington State University College of
Agriculture and Home Economics Project 1791 and by National
Science Foundation Grant IBN 0090766.
PNAS I November25, 2003 | vol. loO | suppl. 2 | ,4s79
OCR for page 68
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Ryan and Pearce
Representative terms from entire chapter:
tomato systemin
