Hormones are in the air.
Hormones are in the air
Plant Molecular and Cellular Biology Program, University of Florida, 1301 Fifield Hall, Gainesville, FL 32611
biochemical studies have identified many
of the factors that mediate hormonal
control of cell division, expansion, dif-
ferentiation, and, ultimately, death.
However, despite major advances in our
understanding of how individual hor-
mones act, we know relatively little
about how hormonal signals are inte-
grated into overall responses. For exam-
ple, the hormones indole-3-acetic acid
(IAA), gibberellin (GA), brassinolide,
and, in some circumstances, ethylene all
promote stem elongation. But what is
the role of each hormone, and how do
they interact to mediate growth? An
abundance of hormone-related mutants,
particularly in Arabidopsis, has provided
some insights into the mechanisms of
cooperative hormone action. Note that
the term ‘‘cross-talk’’ is frequently used
in this context. However, classically, this
term defines phenomena in which there
is an unwanted transfer of signals from
one circuit, channel, etc., to another
(Oxford English Dictionary). These in-
teractions are most certainly not ‘‘un-
wanted.’’ In a narrower context, cross-
talk is generally limited to common
elements within a signal transduction
scheme. Such a definition clearly ignores
a major source of phytohormone inter-
actions: effects of one input signal on
accumulation of other input signals. For
example, genetic analyses have defined a
complex relationship among sugars, ab-
scisic acid (ABA), and ethylene (1, 2)
that controls aspects of growth and de-
velopment. This interaction is regulated
at the level of both hormone synthesis
and signal transduction. However, the
molecular details of this and other inter-
actions are far from understood.
Some of the best characterized exam-
ples of the complex interactions between
phytohormones involve responses to bi-
otic stress. For example, wound re-
sponses, such as those induced by chew-
ing insects, integrate jasmonic acid (JA),
ethylene, and systemin signaling path-
ways in a manner yet to be fully estab-
lished (3, 4). Similarly, responses to
pathogenic microbes involve integration
of JA, ethylene, and salicylic acid (SA)
signaling pathways (5). Plants clearly use
a limited number of hormonal signals in
a combinatorial manner to achieve dis-
tinct outcomes. Epistatic relationships
between various mutants have defined a
provide a simple and rapid procedure to
hytohormones act to control
many aspects of plant growth,
development, and responses to
the environment. Genetic and
useful framework for testing molecular
mechanisms integrating the various hor-
mone pathways. However, genetic ap-
proaches can go only so far, and epista-
sis can be misleading. For hormones,
purely genetic approaches do not ad-
dress a major source of interactions;
there is abundant evidence that pertur-
bation of one hormone pathway can
have profound effects on synthesis and
accumulation of other hormones. Thus,
a mutation in one pathway can lead to
major alterations in other hormones. Of
course, the obvious answer to this fun-
damental problem is to measure the lev-
els of all hormones in a mutant. So why
has chemical analysis of hormones
lagged far behind genetic characteriza-
tion? Very simply, accurate measure-
ment of hormone levels in small
amounts of tissues has historically been
beyond the skill base of most laborato-
ries. Few laboratories have had the ca-
pacity to measure multiple phytohor-
mones. There is no doubt that molecular
biology and genetics have, in the last 15
years, revolutionized the field of plant
hormone biology, but we now find our-
selves in the all too familiar position of
being limited by biochemistry.
It is in this context that the article by
Schmelz et al. (6) in this issue of PNAS
should be viewed. It is not by choice
that most molecular geneticists have
largely ignored a major mechanism
whereby hormones interact; it has sim-
ply not been technically feasible for the
average laboratory to tackle the issue.
Rather, a relative handful of laborato-
ries have used technically demanding
protocols for measuring one or a few
hormones. What has been lacking is a
robust, simple technique to measure
multiple hormones in small amounts of
tissue. Although significant progress has
been made recently (7), the work of
Schmelz et al. (6) should be viewed as a
major technical advance. The authors
simultaneously quantify multiple hor-
mones. They have measured SA, JA,
ABA, IAA, the phytotoxin coronatine,
and a set of volatile organic compounds
that function as ecological signals. The
technique works with tissues from multi-
ple plant species subjected to several
biotic and abiotic stresses. It employs
readily available chemicals and stan-
dards and relies on instrumentation
available on most university campuses,
chemical ionization gas chromatography
mass spectrometry. The method is ele-
gant in its simplicity. Target compounds
are converted to their methyl esters and
measured as volatiles. The technique is,
in theory, applicable to a wide range of
primary and secondary metabolites, in-
cluding other hormones, the only limita-
tion being availability of appropriate
What does the availability of this
technique mean to the plant biologist?
It should be possible to assess the ef-
fects of perturbing one hormone signal-
ing system on a broad spectrum of other
hormones. For many of the available
mutants, this has never been done.
However, there is ample evidence that
alterations in one pathway have pro-
found consequences on other signaling
systems. An excellent example of hor-
mone alterations initiating a chain reac-
tion is found in deepwater rice (8).
Flooding of the plant causes a 50-fold
increase in internal ethylene, leading to
rapid stem elongation, so ethylene
causes stem elongation. But this is only
a small part of the story. In fact, there is
an increase in GA sensitivity mediated
by reduction of ABA levels. Thus, stem
elongation is actually the result of an
interaction among three hormones. Only
by measuring the effects of ethylene on
other hormones in the target tissue does
the complete story become evident.
Another illustration of the importance
of hormone synthesis on signal integra-
tion is the example of the ein2 mutant.
This mutant was originally isolated in
screens for ethylene insensitivity and has
been placed squarely within the ethylene
signaling pathway (9). However, ein2 has
been independently isolated in screens
for cytokinin and ABA insensitivity as
well as in screens for insensitivity to
auxin transport inhibitors (10–13). Why
does ein2 consistently show up in
See companion article on page 10552.
A robust, simple
technique to measure
multiple hormones in
small amounts of tissue
has been lacking.
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no. 18 www.pnas.org?cgi?doi?10.1073?pnas.1934350100
screens for so many different hormones?
The answer lies in the fact that ethylene
plays a critical role in mediating re-
sponses to many environmental stimuli.
Its synthesis is highly regulated. The lim-
iting step in synthesis is ACC synthase,
an enzyme encoded by a gene family of
at least 10 members in most plants (14).
Different family members are induced
by multiple factors, including cytokinin
and auxin (15, 16). Thus, high levels of
IAA or cytokinin induce ethylene syn-
thesis. Many of the phenotypic effects
associated with high IAA are actually
ethylene effects (17). Alterations in mul-
tiple hormones are manifested as sec-
ondary effects associated with ethylene
perturbation. Only because ethylene is a
very simple hormone to assay do we
know about these interactions.
The technique described by Schmelz
et al. (6) is ideally suited for examina-
tion of plant–pathogen interactions. Ge-
netic studies in Arabidopsis have defined
parallel JA?ethylene and SA pathways
mediating separate but overlapping de-
fense responses (5). We know that these
three hormones are important because
mutants in their signaling pathways pos-
sess altered pathogen responses. How-
ever, few studies have actually examined
the hormones directly. We have used
the technique of Schmelz et al. (6) to
examine the fluctuations in hormone
levels in several wild-type and mutant
lines after infection with a bacterial
pathogen (18). Results indicate that the
JA?ethylene and SA pathways are not
entirely independent of one another.
SA-deficient plants produced far less
ethylene than wild-type plants after in-
fection. An effect on ethylene synthesis
was not predicted to be a consequence
of SA deficiency. Without direct deter-
mination of hormone levels, it would be
impossible to determine whether a phe-
notype was caused by loss of SA per se.
A more complete hormone analysis also
revealed a significant increase in IAA
accumulation after infection. Although
the role of this pathogenesis-related
IAA has yet to be determined, its syn-
thesis had not been previously reported,
most likely because of the difficulties
associated with IAA quantitation. The
critical point in these studies is that si-
multaneous quantification of multiple
biologically active substances should
provide significant new insights into
mechanisms of hormone interactions. It
must be noted that the technology goes
beyond plant hormones. For example,
the authors have also measured accumu-
lation of coronatine during infection
with Pseudomonas syringae pv. tomato
(Fig. 1), This compound is an important
virulence factor that is believed to
function as a mimic of JA or one of its
So what are the implications of the
availability of a facile technique for
quantitation of several hormones in a
single assay? Hopefully, we are in a po-
sition to more fully elucidate mecha-
nisms associated with cooperative hor-
mone action. It has always been curious
that a limited number of hormones can
interact in different ways to mediate
distinct environmental responses, e.g.,
pathogen and wound responses. Both
timing and amplitude of hormone syn-
thesis must surely be important contrib-
utors to the signal output. Availability of
this technology will also justifiably raise
the bar for publication. Just as microar-
rays have raised the barrier for gene
expression studies, hormone analyses
should also be an integral part of the
characterization of a hormone-related
mutant. Why look at one hormone when
we can look at them all? Dare we call it
‘‘hormoneomics’’? We have come full
circle. Just as molecular biology reinvig-
orated hormone biology in the last de-
cade, biochemistry must now reinvigo-
rate molecular biology. We have the
genes. Now what do they do? Chemical
analysis has been a major barrier to hor-
mone biologists. This is no longer the
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P. syringae pv. tomato. Leaf tissue was extracted and methylated, and volatile analytes were collected.
Isobutane chemical ionization gas chromatography mass spectrometry results in predominantly [M?H]?
m?z ions useful in profiling the methyl esters of SA (153), JA (225), IAA (190), ABA (261), 12-oxo-
phytodienoic acid (307), and coronatine (334). A much larger number of related analytes are also
accessible by using this basic approach.
Selected ion chromatogram of Arabidopsis thaliana cv. Columbia 2 days after inoculation with
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