Regulation of Gene Expression in Senscing Flowers Final Report
REGULATION OF GENE EXPRESSION
IN SENESCING FLOWERS
Final Project Report To The American Floral Endowment
Submitted by:
Randy Woodson
1140 AGAD Building Purdue University
West Lafayette, IN 47907-1140
Executive Summary
The postproduction life of many flowers is limited by premature petal
senescence. Following harvest or pollination, flowers of a number of species
exhibit a significant increase in the production of the plant hormone ethylene,
which in turn leads to an induction of the biochemical processes of senescence
(cell death). My laboratory has focused on understanding the regulation of
ethylene biosynthesis and senescence at the cellular and molecular level, with
the goal of developing genetic engineering approaches to improving the
postharvest longevity of flowers such as carnation.
In this project, we identified and characterized genes that encode the enzyme
ACC synthase from both petunia and carnation. This enzyme is central to the
regulation of ethylene biosynthesis, resulting in the production of ACC, which
is the immediate precursor to ethylene. These studies revealed that ACC synthase
is encoded by multiple genes and subject to complex patterns of regulation
within the flowers of both carnation and petunia. Inhibition of ACC synthase
with chemicals such as AVG or AOA blocks ethylene production and slows
senescence. With this in mind, we set out to genetically engineer flowers to
inhibit the expression of ACC synthase, thus blocking ethylene production
without chemicals. We also used another approach to blocking ethylene, which was
to reduce the level of ACC in flowers through the expression of a gene for an
enzyme that converts ACC to a product other than ethylene. Both approaches
appear to have promise for the industry.
This project has been successful in many ways. The data generated has been
communicated in a number of peer reviewed publications. Also, two PhD students
were educated through this project (Michelle Jones and Sven Verlinden), both of
which now enjoy independent careers in floriculture at CSU and the University of
West Virginia. Also, a postdoctoral associate (Dr. Jon Lindstron) worked on this
project and is now an Assistant Professor of Horticulture at the University of
Arkansas.
Experimental Results
Previously, we had reported on the cloning of ACC synthase and ACC
oxidase from carnation (Park et al. 1992; Wang and Woodson 1991). ACC synthase
converts s-adenosyl methionine to ACC, and ACC oxidase converts ACC to ethylene.
As part of this project, we reported the cloning of two additional ACC synthase
genes, ACS2 and ACS3 (Jones and Woodson, 1997; 1999). In addition, we identified
a novel ACC synthase gene in petunia, expressed only in the pollen (Lindstrom et
al. 1999). These steps were critical to the ultimate success of this project, as
the regulation of ACC production is quite complex and under the control of
multiple genes. Genetic engineering flowers to inhibit ethylene requires a good
understanding of this regulation.
Following up on previous research from my laboratory (Larsen et al. 1995), we
showed that pollination induced ethylene production in the flowers through
increases in the expression of both ACC synthase and ACC oxidase (Jones and
Woodson 1997). Using gene-specific probes, we were able to show that ACS3 was
responsible for early ethylene production in pollinated styles and its
expression was not inhibited by norbornadiene or MCP (Jones and Woodson 1999).
The differential regulation of members of the carnation ACC synthase gene family
following pollination and during senescence was useful in determining the role
of ACC and ethylene in interorgan communication within the flower. Our results
clearly point to the pistil playing an important role in producing ethylene that
ultimately travels to, or signals the petals to produce ethylene and senesce.
The story of pollination-induced senescence in carnation is quite
complicated. It is clear that following a compatible pollination, a signal that
coordinates ethylene production is translocated from the style to the ovary and
petals. In another study, we investigated the roles of ethylene and its director
precursor, ACC, in this signaling (Jones and Woodson 1999b). Here we showed that
ethylene and ACC increased sequentially in the styles, ovaries and petals
following pollination. Ethylene and ACC were highest initially in the stigmatic
region of the style but by 24 hours after pollination were highest in the base.
Activity of ACC synthase was correlated with ethylene production in styles and
petals, but interestingly activity was not detected in the ovary, which
accumulated significant levels of ACC. Lack of ACC synthase activity in
pollinated ovearies, coupled with high ACC content, suggested that ACC was
translocated within the gynoecium to the petals, where it was converted to
ethylene. Experiments that removed styles and petals at various times after
pollination indicated that there was a transmissible pollination signal in
carnations that reached the ovary by 12 hours and the petals by 14 to 16 hours
after pollination. This publication received the Alex Laurie Award as the best
paper published in floriculture in 1999.
Pollination of petunia flowers results in a rapid burst of ethylene from the
stigma, which leads to senescence of the petals. Petunia is an excellent model
for studying the regulation of ethylene and petal senescence as it is easy to
genetically engineer. In petunia, pollen contains considerable ACC, which has
been suggested to play a critical role in inducing ethylene. We investigated the
synthesis of ACC in petunia pollen (Lindstrom et al. 1999). We reported that a
specific ACC synthase gene was expressed in pollen, which accounted for the
accumulation of ACC. This gene was characterized and shown to have a
pollen-specific promoter that directed the expression of a reporter gene in
transgenic plants. This was the first report of ACC synthase expression in
pollen, and the first report of an ACC synthase gene promoter.
In another set of experiments, we sought to determine the role for this
pollen-borne ACC in pollination-induced ethylene. We constructed a gene
consisting of the coding region from a bacterial ACC deaminase gene and a
pollen-specific promoter from tobacco (LAT52). First, we showed that the LAT52
promoter activated the expression of a reporter gene (GUS) in transgenic
petunias. We went on to show that petunias expressing the ACC deaminase gene had
one hundred-fold less ACC in pollen. ACC deaminase converts ACC to µ -ketobutyrate, thus diverting it away from ethylene production. This large
reduction in ACC did not affect pollination-induced ethylene production from the
stigma. In addition, fertility as determined by seed set and inheritance of the
transgene were unaffected by this reduction in pollen ACC content. Our results
indicate that pollen-borne ACC does not play a role in pollination-induced
ethylene.
Future Research
This work has pointed the way for a number of approaches to genetic
engineering of flowers for postharvest longevity. First, we have identified
critical steps in the biosynthesis of ethylene in both carnation and petunia.
Also, we have shown that this regulation is complex and a single silver bullet
is not likely to control the production of ethylene. We were successful in
creating transgenic plants with the ACC deaminase gene, which converts ACC to a
-ketobutyrate, which is not used in ethylene production, thus limiting the
amount of ethylene produced. We were the first to report on a pollen-specific
ACC synthase, showing how this is regulated in petunias at the genetic level.
Given the number of genes involved in ethylene biosynthesis, future experiments
should focus on using gene-specific approaches to knock out ethylene production
in flowers. Also, it is clear that inhibiting ethylene action will be a good
strategy to prolonging the life of cut flowers.
References
Larsen PB, Ashworth EN, Jones ML and WR Woodson. 1995. Pollination-induced
ethylene in carnation: role of pollen tube growth and sexual compatibility.
Plant Physiology 108:1405-1412.
Park KY, A Dory and WR Woodson. 1992. Molecular cloning of an ACC synthase
gene from senescing carnation flower petals. Plant Molecular Biology 18:377-386.
Wang H and WR Woodson. 1991. A flower senescence-related mRNA from carnation
shares sequence similarity with fruit ripening-related mRNAs involved in
ethylene biosynthesis. Plant Physiology. 96:1000-1001.
Publications Resulting from Project
Jones, ML and WR Woodson. 1997. Pollination-induced ethylene in carnation:
role of stylar ethylene in corolla senescence. Plant Physiology 115:205-212.
Verlinden S and WR Woodson. 1998. The physiological and molecular responses
of carnation flowers to high temperatures. Postharvest Biology and Technology
14:185-192.
Linstrom JT, CH Lei, ML Jones and WR Woodson. 1999. Accumulation of ACC in
petunia pollen is associated with expression of a pollen-specific ACC synthase
late in development. Journal of the American Society for Horticultural Science
124:145-151.
Jones ML and WR Woodson. 1999. Interorgan signaling following pollination in
carnations. Journal of the American Society for Horticultural Science
124:598-604. (Alex Laurie Award)
Jones ML and WR Woodson. 1999. Differential expression of three members of
the ACC synthase gene family in carnation. Plant Physiology 119:755-764.
