Regulation of Ethylene Production During Postproduction Handling of Flower Crops 1994 proposal
Dr. William Woodson
1994
EXECUTIVE SUMMARY
The postproduction quality of many flowers is limited by the increased
synthesis and action of the plant hormone ethylene. Increased production
of ethylene plays a role in the senescence or death of flower petals, abscission
of plant parts including floral structures, and discoloration of harvested
foliage. A major improvement in the postproduction care of flower crops
was introduced from Holland in the late 70’s with the use of silver thiosulfate
to prevent ethylene responses. Because of concerns over the introduction
of increasing amounts of silver into the atmosphere the use of silver thiosulfate
has recently been banned in much of Europe including the flower auctions
of Holland. Because of this, alternative strategies are currently needed
for the regulation of ethylene responses in flower crops both in the US
and Europe. An alternative approach to inhibiting ethylene responses would
be to prevent the harvested plants from synthesizing the hormone. Recently,
we have learned a great deal about the biochemistry of ethylene biosynthesis
and have identified the genes that encode the enzymes of this pathway.
This information has been used to generate plants that fail to produce
ethylene and in many cases these plants exhibit improved postproduction
quality. We still know very little about why plant tissues suddenly exhibit
increased ethylene production at specific stages of development and during
postproduction handling. The focus of our work will be to study the regulation
of ethylene biosynthesis during postproduction handling of flower crops
in an attempt to identify critical processes involved in inducing the synthesis
of ethylene. The rationale and practical significance of this work is that
if such critical processes can be understood, then perhaps they can be
chemically or genetically modified in such a way as to prevent the increase
in ethylene that leads to postproduction loss of plant quality.
INTRODUCTION AND LITERATURE REVIEW
The biosynthesis of the phytohormone ethylene is under strict regulation
in plant tissues [15]. An increase in ethylene production is associated
with several stages of development including flower senescence and abscission
[3], and in response to both biotic [2] and abiotic stresses [4]. The ethylene
biosynthetic pathway was elucidated by Adams and Yang [1] and is:
Met —–> SAM —–> ACC ——> Ethylene
In most tissues ethylene production is low and the rate limiting step
in this pathway is the conversion of SAM to ACC by ACC synthase [5]. In
presenescent carnation flower petals, ethylene production is limited by
low activities of both ACC synthase and ACC oxidase, which converts ACC
to ethylene [14]. These enzymes increase in a coordinate fashion concomitant
with the rise in ethylene production associated with petal senescence.
Flowers and fruits that exhibit this increase in ethylene during postharvest
handling are classified as being climacteric. In climacteric flowers such
as carnation, the basal level of ethylene production which precedes induction
of senescence is referred to as system I ethylene, whereas the increased
ethylene produced during senescence is system II ethylene [3]. System II
ethylene is autocatalytic in nature, for example ethylene stimulates its
own synthesis. The induction of system II ethylene appears to be under
the control of developmental signals. The transition to system II is characterized
by an increase in the abundance of mRNAs encoding ACC synthase and ACC
oxidase [14]. Interestingly, the production of system II ethylene is completely
dependent on the continued perception of ethylene. Treatment of flowers
with 2,5-norbornadiene, a competitive inhibitor of ethylene action, results
in a rapid inhibition of ethylene production [11]. In addition, inhibition
of ethylene action with 2,5-norbornadiene inhibits the activities of both
ACC synthase and ACC oxidase [11], and the expression of their corresponding
mRNAs is prevented [14]. Clearly, the expression of system II ethylene
synthesis is under the regulation of ethylene.
The question remains as to the developmental signals responsible for
the transition from system I to system II ethylene production during flower
development In many flowers the developmental switch from system I to system
II ethylene is hastened by pollination [7]. Pollination has been found
to result premature petal senescence, changes in petal pigmentation and
abscission of floral structures [3,10]. The increase in ethylene production
resulting from pollination has been implicated in the regulation of all
of these postpollination events. Pollination has been found to result in
increases in ethylene production by styles, ovary, receptacles and petals
[7,14]. The increase in ethylene production by the various organs is sequential
in nature with the styles producing ethylene most rapidly (within 30 minutes
of pollination). The interaction of the reproductive structures and the
petals, along with the sequential nature of pollination- induced ethylene
production, suggests that a transmissible factor is involved in regulating
petal ethylene production and the postproduction losses associated with
this increase in ethylene. In the work proposed here we will address the
nature of the local and systemic pollination signals that lead to increased
ethylene production throughout the carnation flower.
OBJECTIVES AND ANTICIPATED BENEFITS
The overall objective of my research at Purdue is to understand the
basic mechanism by which flower crops regulate the production of ethylene
during postproduction handling. The basic question addressed by my research
is why do flower petals and other floral organs suddenly switch to a situation
of high ethylene production at certain developmental stages, following
pollination or in response to postproduction handling practices. Should
this research successfully identify the developmental signals and cellular
factors that lead to increased ethylene production it is anticipated that
chemical or genetic control measures will lead to improved postproduction
quality of our high value flower crops. Within the team framework of AFE,
it is anticipated that this work will complement the efforts of Dr. Nell
and his colleagues. Our research will provide a basic understanding of
the regulation of ethylene synthesis. This information will be made available
to others in the postproduction study team in an effort to assist in the
development of novel techniques for improving postproduction handling of
flower crops. Also I would encourage AFE to consider making a small amount
of funds available to PI’s for yearly meetings at various institutions
to discuss research. The will increase the effectiveness of the team approach.
The specific objectives of this work are as follows: (1) To identify the
endogenous cellular factors and exogenous environmental signals that lead
to increased expression of the genes encoding ACC synthase and ACC oxidase.
(2) To transform carnations with antisense ACC synthase and ACC oxidase
genes in an attempt to inhibit ethylene production. (3) To identify genes
involved in the developmental switch from system I to system II ethylene.
MATERIALS AND METHODS
Objective 1 The availability of cDNAs representing ethylene biosynthetic
pathway transcripts win allow us to address the endogenous cellular factors
and exogenous environmental signals that lead to increased expression of
ACC synthase and ACC oxidase genes. My lab was one of the first to isolate
these genes from any plant and to date we have cloned ACC synthase from
carnation [14] and ACC oxidase from carnation [12] and petunia [13]. An
analysis of the expression patterns for the carnation ACC synthase cDNA
clone indicates this represents a transcript that increases during petal
senescence [14] and does not detect ACC synthase mRNA in wounded tissue,
auxin-treated tissue or styles shortly after pollination. In order to understand
the factors that regulate ethylene biosynthesis it will be necessary to
isolate other ACC synthase genes and study there expression. We will take
several approaches in an attempt to isolate and clone other ACC synthase
mRNAs from carnation. We will use RNA-based PCR in an attempt to amplify
a cDNA for ACC synthase from pollinated styles and ovaries. Oligonucleotides
will be synthesized based on regions of conservation between ACC syntheses.
These sense and antisense oligos will be employed as PCR primers using
cDNA synthesized from RNAs a DNA template. The PCR derived cDNA clones
will likely be less than 500 bp in length, therefore, it will be necessary
to isolate full length clones for the purpose of complete sequence comparison
and identification of regions of sequence divergence for making gene specific
probes. These gene specific probes will in turn be used to study the regulation
of different ACC synthase genes in carnation flowers during postproduction
handling and following pollination.
Objective 2 The ability to create null mutations of the cloned ethylene
biosynthetic pathway genes could prove useful in improving the postproduction
quality of flower crops by limiting their capacity to produce ethylene
genetically. This has the advantage over chemical control measures in that
it is permanent and requires no treatment by growers, shippers or wholesalers.
This technology is based on genetically engineering plants to express a
complementary, or antisense (backward) strand of a given mRNA that in turn
anneals to the endogenous transcript preventing translation into a functional
protein. This approach has proven to be useful in controlling ethylene
production in tomato fruits where the expression of an antisense ACC synthase
completely blocked fruit ripening [9]. These fruits were also much less
responsive to exogenous ethylene since they were unable to induce their
own ethylene synthesis autocatalytically. We will conduct experiments to
prevent or reduce expression of ACC synthase in transgenic carnation by
expressing their antisense RNAs. It is anticipated that research outlined
in objective 1 will identify other candidate ACC syntheses for these experiments.
Depending on the divergence between ACC synthase sequences it should
be possible to generate gene specific antisense constructs that target
a specific ACC synthase for inhibition. For these experiments a portion
of the cDNA clone will be cloned into the binary vector pKYLX71. The resulting
plasmid will be mobilized into Agrobacterium tumefaciens, which in turn
will be used to transform carnation as previously described [8]. The effects
of expression of specific antisense ACC syndiases, on ethylene biosynthesis
and postproduction quality will be assessed.
Objective 3 As previously mentioned, the nature of the developmental
signals that lead to the induction of system II ethylene, and in turn to
petal senescence, are unknown. Several groups have taken a molecular genetic
approach to elucidate the regulation of ethylene biosynthesis, and action
using Arabidopsis thaliana. Recently, Kieber et al. [6] isolated a recessive
Arabidopsis mutant, ctr1, that constitutively exhibits an ethylene response
phenotype. The mutated gene was subsequently cloned and found to encode
a member of the Raf family of protein kinases and is thought to be involved
in the signal transduction pathway leading to ethylene responses. The conclusions
drawn from these experiments is that normally ethylene responses are inhibited
by the CTR1 gene and the mutation prevented this. The transition to system
II ethylene is an autocatalytic response to ethylene. Therefore a possible
explanation for the transition from the low basal rates of ethylene production
associated with system I to the increased production of system II could
be a down-regulation of the CTR1 gene. To test this we will clone the CTR1
homolog from carnation and determine if its expression pattern is indicative
of being down-regulated during the transition to system II ethylene. If
this is indeed the case, then a possible control measure would be to over-express
this gene in the petals of transgenic: plants and thereby prevent the increase
in ethylene production. We have recently received a cDNA clone for CTR1
from Joe Ecker at the University of Penn. This clone will be used to screen
our cDNA library in an attempt to isolate its homolog from carnation.
LITERATURE CITED
1. Adams DO, Yang SF: Ethylene biosynthesis: identification of 1-aminocyclopropane-1-
carboxylic acid as an inter-mediate in the conversion of methionine to
ethylene. Proc Natl Acad Sci USA 76:170-174 (1979).
2. Boller T: Ethylene in pathogenesis and disease resistance. In: Mattoo
AK, Suttle JC (eds) The Plant Hormone Ethylene, pp. 293-314. CRC Press,
Boca Raton, FL (1991).
3. Borochov A, Woodson WR: Physiology and biochemistry of flower petal
senescence. Hortic Rev 11:15-43 (1989).
4. Hyodo H: Stress/wound ethylene. In: Mattoo AK, Suttle JC (eds) The
Plant Hormone Ethylene, pp. 43-63. CRC Press, Boca Raton, FL (1991).
5. Kende H: Ethylene biosynthesis. Ann Rev Plant Physiol Plant Mol
Biol 44:283-307 (1993).
6. Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR: CTR1, a
negative regulator or the ethylene response pathway in Arabidopsis, encodes
a member of the raf family of protein kinases. Cell 72:427-441 (1993).
7. Larsen PB, Woltering EJ, Woodson WR: Ethylene and interorgan signalling
in flowers following pollination. In: Schultz J, Raskin E (eds) Plant Signals
in Interactions with Others, American Society of Plant Physiologists, Rockville,
MD (in press).
8. Lu C-Y, Nugent G, Wardley-Richardson T, Chandler SF, Young R, Daffing
MJ: Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus
L.). Bio/Technology 9:864-868 (1991).
9. Oeller PW, Lu M-W, Taylor LP, Pike DA, Theologis A: Reversible inhibition
of tomato fruit senescence by antisense RNA. Science 254:437-439 (1991).
10. Stead AD: Pollination-induced flower senescence: a review. Plant
Growth Regul 11:13-20 (1992).
11. Wang H, Woodson WR: Reversible inhibition of ethylene action and
interruption of petal senescence in carnation flowers by norbornadiene.
Plant Physiol. 89:434-438 (1989).
12. Wang H, Woodson WR: A flower senescence-related mRNA from carnation
shares sequence similarity with fruit ripening-related mRNAs involved in
ethylene biosynthesis. Plant Physiol 96:1000-1001 (1991).
13. Wang H, Woodson WR: Nucleotide sequence of a cDNA encoding the
ethylene- forming enzyme from petunia corollas. Plant Physiol 100:535-536
(1992).
14. Woodson WR, Park KY, Drory A, Larsen PB, Wang H: Expression of
ethylene biosynthetic pathway transcripts in senescing carnation flowers.
Plant Physiol 99:526- 532 (1992).
15. Yang SF, Hoffman NE: Ethylene biosynthesis and its regulation in
higher plants. Annu Rev Plant Physiol 35:155-189 (1984)
BUDGET
Year 1 Year 2 Year 3 Total
Graduate Assistant 12,000 12,500 13,000 37,500 G
rad Fee Remission 2,004 2,124 2,124 6,252
Fringe Benefits; 216 225 234 675
Supplies and Expenses 6,000 6,000 6,000 18,000
Total 20,220 20,849 21,358 62,427
LEADER QUALIFICATIONS
(William R. Woodson)
A. Education
B.S. Horticulture University of Arkansas 1979
M. S. Horticulture Cornell University 1981
Ph.D. Plant Physiology Cornell University 1983
B. Academic Positions
Professor Department of Horticulture 1993-present Purdue University
Associate Professor Department of Horticulture 1989-1993 Purdue University
Assistant Professor Department of Horticulture 1985-1989 Purdue University
Assistant Professor Department of Horticulture 1983-1985 Louisiana
State University
C. Awards and Honors
1994 Agriculture Research Award, Purdue University, School of Agriculture
Pi Alpha Xi, National Floriculture Honor Society
Sigma Xi, The Scientific Research Society
C. Editorial Position
Associate Editor (Postharvest Biology of Flowers) Journal of the American
Society for Horticultural Science
Associate Editor Postharvest Biology and Technology
Associate Editor (Horticulture Education) Journal of Natural Resources
and Life Sciences Education
