FLORAL ANTI-SENESCENCE FOR THE ETHYLENE RECEPTOR Progress Reports - June 2000
PROPOSAL - JUNE, 2000
FLORAL ANTI-SENESCENCE FOR THE ETHYLENE
RECEPTOR
MICHAEL PIRRUNG
DUKE UNIVERSITY
Executive Summary
This research is aimed at the development of novel floral
anti-senescence agents that halt the action of ethylene. This project involves
teams at Duke University, which will design and prepare novel compounds and
study their physiology, and the University of Wisconsin, which will study their
action against the ethylene receptor in vitro and in vivo. This
project builds upon earlier research from these groups in the structural
characterization of the receptor and its mode of action. Molecular modeling of
possible antagonists, chemical synthesis, binding assays, functional assays in
seedlings, combinatorial chemistry, and physiological studies in cut roses are
the methods that will be used. The results of this research should practically
benefit the floral industry by providing novel materials for controlling natural
and transport-induced senescence in cut flowers. These materials should
dramatically reduce economic losses due to spoilage and facilitate novel
web-based marketing strategies with “direct-from-grower” delivery.
Introduction and Literature Review
Many areas of the economy have been affected by the explosive
growth of the web, leading to national branding in many businesses that were
previously only local. National branding has long existed in cut flowers through
services like FTD, which has always relied on traditional methods of delivery
through local florists. Flower marketing has also been affected by the web, with
increased on-line ordering of flowers that are express shipped directly from the
grower or wholesaler to the customer. Some direct-shipping c-commerce sites are
listed below, along with some of their mottoes:
-
www.greatflowers.com “Flowers direct from the grower
at great prices!” -
1800flowers.com
www.proflowers.com “Guaranteed fresh for
seven days!”-
www.freshflowersource.com
-
www.flowersdirect.com “Direct from the farm
bouquet” -
www. flowerfarm.com
-
www.usatoday.pcflowers.com “We guarantee that all
our flowers will last at least 7 days or your money back.”
Express delivery is essential for these websites to compete
with local marketing, but one can readily see the difficulty with transport
time, not to mention shipping stress, reducing the quality of a very perishable
commodity. This issue is really no different from those flower marketers have
long dealt with, inventory shrinkage and reduced flower quality. A general
approach to delay wilting would permit lower-cost shipping to be used, or to
increase quality and customer satisfaction with delivered products.
Ethlylene is known to significantly limit product life in the
cut flower market, which is dominated by carnations and roses. A number of
treatments have been investigated over the years for this purpose, including the
toxic salt silver thiosuiphate (STS). STS is used by carnation wholesalers in
Central America, but the expense of silver requires re-use of STS solutions,
leading to difficulties with bacterial contamination. Silver transported
vascularly to flowers can also become photoreactive, leading to unacceptable
gray discoloration. STS is thus a quite non-optimum approach to inhibition of
ethylene action. The ability of STS to inhibit the action of ethylene has
recently been given a molecular basis by Bleecker, who showed that the ethylene
receptor uses a copper ion for ethylene binding, and that silver competes with
copper.
The shortcomings of STS have created a need for novel
approaches to control flower physiology that is mediated via ethylene. The most
recent commercial product has been Florish. an ethylene biosynthesis inhibitor.
The P1 was a consultant to Abbott during its research on Florish, which has not
lived up to early expectations because it seems much less effective on roses
than carnations, suggesting that ethylene biosynthesis may not be a universal
target. Novel ethylene antagonists (norbornadiene (NBD)) have also been
reported, most of which are pungent gases that yielded interesting science but
little in practical advances. Given agents without these drawbacks, however,
inhibiting ethylene action should be a much more general approach to inhibiting
floral senescence than inhibiting ethylene biosynthesis. This project therefore
seeks to discover small organic molecules that act as ethylene antagonists.
Ideally, they could be used in water, for vascular transport or as a spray.
Sisler has recently reported promising research in inhibiting
ethylene action using a long-known compound, 1-methylcvclopropene (1-MCP), as a
highly potent, irreversibly binding, competitive antagonist at the ethylene
receptor.’ Working with several other groups, he has shown its value in
flowers. 1-MCP inhibited senescence of carnation flowers, and abscission of
florets from penstemon flowers.2 Fumigation (6 h, 20 ppb) with 1-MCP
increased the display life of Campanula flowers from 3.3 to 9 days, and
inhibited the effects of exogenous ethylene, such as bud and flower drop, leaf
abscission, and accelerated flower senescence, in Rosa hybrida “Victory
Parade” plants. 1-MCP retarded bud and leaf abscission in
miniature roses, but had no effect on leaf yellowing. It inhibited
the normal wilting response of cut carnations exposed to 0.4 ppm ethylene, and
the vase life of 1-MCP-treated flowers exposed to 1 ppm ethylene was up to 4x
that of controls. Other groups have shown the value of 1-MCP in
inhibiting senescence in cymbidium orchids. However, a serious
limitation to 1-MCP ever moving from the research laboratory into the greenhouse
is the fact that it is a fairly unstable gaseous molecule. It is not practically
storable, so it must be prepared in a laboratory immediately before use
and, as a gas, it is difficult to handle. Thus, there is no practical way to
place 1-MCP into the hands of the flower practitioner. Floralife is currently
marketing a product called EthylBloc that is claimed to be a 1-MCP precursor,
but there is nothing in their formulation that could possibly play this role
under ambient conditions. The groups collaborating on this research program have
contributed significantly to the current state of knowledge of ethylene action
and its limitation to limit senescence. Each provides a paragraph following.
Bleecker - An understanding of the molecular mechanisms
involved in the perception and transduction of the ethylene signal by plants
provides a model for the role of developmental signals in the control of plant
life history. The genetic analysis of ethylene signal transduction has been an
ongoing interest of mine since the original characterization of the ethylene
insensitive etri mutant. The ETR1 gene was cloned and shown to code
for a protein with sequence homology to the so-called two-component regulators
of bacteria. Research in my laboratory is focused on a detailed biochemical
characterization of the ethylene receptors from Arabidopsis. Analysis of the
protein from Arabidopsis and as expressed as a recombinant protein in yeast
indicate that the protein is associated with membranes as a covalentlv-linked
homodimer. The discovery that the ETR1 protein expressed in yeast is capable of
directly binding ethylene indicated that the ETR1 protein is the bona fide
receptor for ethylene. This work was the subject of a paper in Science. More
recently we have shown that a copper cofactor mediates binding of ethylene to
the receptor, resulting in an additional paper in Science. Using the yeast
expression system as a model we are investigating the mechanism of copper
loading into the receptor. We are also studying the kinetic properties of
ethylene interaction with the ETR1 binding site using the yeast expressed
protein.
Pirrung - Our laboratory has conducted research on ethylene
biosynthesis since the early 1980s. Its focus was chemical mechanism, but we
concurrently recognized that there were significant opportunities for ethylene
biosynthesis inhibitors in applied plant physiology. We included physiological
studies as an adjunct to our basic science investigations and developed some
modest anti-senescence agents. However, the focus of our work, ethylene-forming
enzyme, did not prove to be a valuable target for senescence inhibition. Our
attention was drawn to the ethylene receptor by Bleecker’s advances in the
characterization of ETRI. The happenstance that ETR1 is related to the bacterial
two-component regulators (histidine kinases that are active only in a dimeric
form) added to our interest, as they were already the focus of a pharmaceutical
research program ongoing in my laboratory. In reviewing the literature of
two-component regulators, we proposed a mechanism for ethylene action that
involves two-point binding of two histidine kinase molecules to the essential
copper ion through N and S atoms.
Objectives and Anticipated Benefits
The first set of objectives in this program is to understand
the basic science underlying the binding of ligands to the ethylene receptor and
the molecular factors determining antagonists and agonists, which will enable
the second set of objectives, the design, preparation, and testing of novel
ethylene antagonists, leading to the third set of objectives, identifying those
antagonists with anti-senescence activity in cut flowers. We envision that the
first year of the grant will focus on basic science questions, the second year
on preparation of large numbers of compounds for testing for antagonist
properties. and the third year on testing of the most promising leads in
carnations and roses. The novel compounds we study will be designed/chosen to be
inexpensive and convenient to apply at the grower, wholesale, retail, or
consumer level. The benefit to the floral industry sought in this proposal is
increasing the vase life, transportabilitv, and quality/performance of cut
flowers, and will result primarily from the third years research results All
sectors of the industry will benefit from reduced perishability of their
products, but particularly, the opportunities for direct marketing of floral
products through electronic commerce will be enhanced by increased
transportability at lower cost and with higher quality
Materials and Methods
We are using molecular modeling, which allows us to predict
the compounds most likely to act as antagonists at the ethylene receptor’s
copper site, to develop new floral anti-senescence agents. Our proposal for the
structure and function of the receptor requires an explanation of how ethylene
triggers responses. Copper can bind up to four ligands, so if the metal binding
site is composed from the dimeric receptor, copper will be coordinately
saturated and unable to bind ethylene. This is the form of the receptor that
signals. If the receptor dirner were to “breathe” into an open form
where one monomer is non-metalated and the other metalated (perhaps with the
addition of a weak ligand such as water, to maintain 3-coordinate copper),
ethylene binding could occur, preventing “closing” of the dimer.
Ethylene would thereby stop signaling. The genetic evidence (a dominant
mutation) indicates that ETR1 actively signals in the unbound state (keeps
response pathways off) and that ethylene binding in the wild-type turns
signaling off. The dominant mutations do not bind ethylene (or even copper) but
apparently continue to signal an “off” state to the response pathways.
While this proposal does not explain the agonist/antagonist properties of
different compounds the molecular modeling we have performed does. We have
applied powerful computational chemistry software (the density functional theory
(DFT) method) to simple models of the copper site in the receptor. An ethylene
complex of copper with N and S ligands from one histidine kinase subunit is
strongly resistant to association with other ligands, as would be provided from
a second such subunit. Ethylene thereby keeps the dimeric teceptor in the
“open form, preventing signaling. On the other hand, a similar copper
complex of norbornadiene is able to asso associate with other ligands. It could
therefore bind to the receptor in the “closed” form and compete with
the binding of ethylene. We are continuing to investigate the molecular basis of
this effect, but its consequences are clear: molecules that can prevent the
association of a fourth ligand to the monomeric copper receptor site
(”open”) should be agonists; molecules that bind to the
“closed” form of the receptor and permit four ligands on copper should
be antagonists.
Modeling allows us to identify and focus on the synthesis of
compounds most likely to give the desired activity To test our model, we will
choose molecules that should be able to bind to the copper in a way similar to
the second protein molecule and thereby keep the receptor in the
“open” state. For example, molecules with two-point binding units
commonly studied in inorganic (1 corrdination chemistry would be good candidates
for agonists. Alternatively, molecules with the ability to both strongly bind
copper and hold the protein subunits together (closed”) should be
antagonists. These molecules will be tested computationallv to ensure that their
add is high and they can be accommodated within the coordination geometry of the
metal The computational evaluation is sufficiently rapid that it is impossible
in this space to enumerate all the compounds that could be “tested¬í in
silico, or novel concepts for design of antagonists that could be conceived.
The next step will use our organic chemistry expertise to
prepare designed molecules. The Duke laboratory has extensive experience with
the synthesis of novel biologically active agents and has had a program in the
preparation of anti-ethylene receptor agents since 1991. We have generated a
number of compounds for testing, including derivatives of known ethylene
antagonists. We are fully capable of preparing any molecules emerging from the
computational design process. The design will emphasize easily handled, water
soluble targets and relatively simple chemistry H can be accomplished in a step
or two from known compounds. This will ensure that any of the molecules we
discover arc readily available in quantity (unlike 1-MC, by contrast).
The next stage of the program will be testing by radioligand
binding assays of the interaction of synthetic compounds with the ETR1 binding
site using the yeast-expressed protein. The good binders identified in this
screen can be either agonists or antagonists. In order to separate these two
groups, they will be tested in an Arabidopsis seedling growth assay
earlier developed in the Bleecker laboratory. An antagonist should block the
ethylene triple response, while an agonist should induce the response.
Examination of the effects of known ethylene antagonists both in Arabidopsis seedling
growth and floral senescence can establish the value of this assay as a
surrogate endpoint for floral senescence. If there is inadequate parallel
between the activities, alternative assays can be developed using carnation
seedlings.
If these assays are as successful as we hope, we can optimize
the activity of our “hits” before moving into development.
Combinatorial chemistry, the technique of systematically preparing for testing
many variants of a lead structure using highly efficient (in some cases
automated) methods, will be used. Large families of analogues of the compounds
that emerge from the foregoing experiments can be rapidly using combinatorial
chemistry, which will aid the development of structure/activity relationships (SARs)
and optimize the biological activity. The Pirrung laboratory is one of the few
academic research groups actively practicing combinatorial chemistry, mostly for
pharmacological targets. Application to a plant growth regulator would be truly
novel.
The final and most important stage will involve physiological
studies of the compounds. We will study senescence and abscission in roses
because they are more available locally and because ethylene biosynthesis
inhibitors are ineffective in roses. Carnation senescence assays have been
previously performed in the Pirrung laboratory with anti-ethylene biosynthesis
agents. Flowers donated by local growers (Witherspoon Rose Culture, Durham, NC;
Bridges Roses, Lawndale, NC) or obtained through wholesalers (Cleveland Plant
& Flower Raleigh, NC) will be held chilled until placed under experimental
conditions (RT). The concentration and amount of ethylene antagonist will be
systematically varied with six replicates per condition in a randomized block
design. To maximize convenience in eventual application, only a single
application of antagonist will be made. The flower quality and petal count will
be monitored daily, and the overall plant life will be compared to controls
(both Floralife and no treatment). A significant part of our effort will focus
on simulated transport conditions (darkness, ethylene). Control group flowers
will be kept in darkness (1-3 d), exposed to the ethylene precursor etephon as a
pretreatment, or continuously exposed to etephon. The experimental group will be
subjected to the same conditions, but will include pre-treatments with the
ethylene antagonist under the conditions identified in the foregoing
experiments. The flowers will be scored until petal abscission is complete.
These experiments will also use six replicates per condition in a randomized
block design.
