Improving Floral Scent Production in Flowers
PROPOSAL - JUNE 1, 2000
IMPROVING FLORAL SCENT PRODUCTION IN FLOWERS
NATALIA DUDAREVA
PURDUE UNIVERSITY
EXECUTIVE SUMMARY
A Iarge number of cut flowers have lost their scent during the selection and
breeding processes due to, on the one hand, a focus on maximizing post-harvest
shelf-life, shipping characteristics, and visual esthetic values (i.e., color,
shape), and on the other hand, to the lack of selection for the scent. trait.
This loss of scent has long been recognized as a major problem in the
floriculture industry. Engineering transgenic plants with improved scent quality
would ameliorate this problem. The goal of the proposed research is to create
transgenic plants with modified scent quality and to transfer this technology to
the floriculture industry. Crops with improved scent quality and newly
introduced aromas offer possibilities to expand markets and increase grower’s
income.
Bioengineering of floral scent requires an understanding of the molecular and
biochchemical basis of floral scent production, along with the availability of
cloned genes encoding enzymes involved in the biosynthesis of floral volatiles
and promoters directing the expression of these genes to the proper tissue
(petals and at the proper time (stage of development Until ntil recently. the
majority of floral scent research has been concentrated mainly on the chemical
composition of floral fragrance or emitted volatiles but has tailed to identify
specific enzymes or genes involved in the biosynthesis of these components. We
have recently isolated a cDNA encoding S-adenosyl-L-methionine: benzoic acid
carboxyl methyltransferase (BAMT), the final enzyme in the biosynthesis of the
volatile ester methylbenzoate, a major component of snapdragon floral scent. The
availability of a cloned biosynthetic gene places us in a unique position to
make a detailed study of the regulation of floral scent production and is the
first step towards engineering transgenic plants with modified volatile
composition. This project will employ molecular, genetic. and biochemical
approaches to understand how production of volatiles could he manipulated in
plants using an important floricultural plant Antirrhinum majus (snapdragon),
as a model system.
INTRODUCTION and LITERATURE REVIEW
Plants did not naturally evolved to produce their scent for the benefit of
humans; nevertheless, it is clear that humans find an esthetic value in certain
types of floral scents, and the presence of floral scent may have contributed in
some cases for the decision by humans to cultivate and propagate specific plant
species. While there is certainly a wide variation in human taste, most people
prefer the scents of bee-pollinated, and especially moth-pollinated, flowers,
which they often describe as “sweet-smelling” (Knudsen and Tollsten,
1993).
Scent emitted by flowers is typically a complex mixture of small
(approximately 100-150D) volatile compounds, which gives the flower its unique,
characteristic fragrance. Floral fragrances are dominated by mono- and
sesquitepenoid, phenylpropanoid, and benzenoid compounds and vary widely among
species in terms of the number, type and relative amounts of constituent
volatiles (Crotcau and Karp, 1991: Knudsen et al., 1993; Knudsen and Tollsten,
1993). The chemical composition of floral scents has been extensively
investigated for hundreds of years because of the commercial value of floral
volatiles in perfumery. Several thousand compounds have been identified from
various floral scents and the chemical structures of most are known today
(Knudsen et al., 1993), however until recently there have been few studies
concerning the biochemical synthesis of floral scent compounds. and of the
enzymes and genes that control these processes. In fact, recent investigations
into the biogenesis of floral scent production in Clarkia, and our even
more recent work on the scent of snapdragon (Antirrhinwn majus represent
the only examples to date in which isolation of enzymes and genes responsible
for the formation of scent volatiles in the flower have been accomplished (Dudareva
et al., l999; Dudareva and Pichersky, 2000). The enzymes LIS linalool synthase),
IEMT (SAM:(iso) eugenol 0-methyltransferase), BEAT (acetvl CoA: benzylalcohol
acetyl transferase). SAMT (S-adenosyl-L-methionine: salicylic acid carboxyl
methyltransferase), and BAMT (S-adenosyl-L-methionine: benzoic acid carboxyl
methyltransferase) catalyzing the formation of linalool, methyl(iso)eugenol,
benzylacetate, methylsalicylate, and methylbenzoate, respectively - and their
corresponding genes have been isolated and characterized (Dudareva et al., 1996;
1998a, b; 2000; Pichersky et al., 1994; 1995: Wang et al., 1997; Wang and
Pichersky, 1998; Ross et al., 1999). It has been shown that Clarkia and
snapdragon flowers synthesize their scent compounds de novo in the
tissues from which they are emitted, and that their emission levels,
corresponding enzyme activities, and mRNA levels are all spatially and
temporally correlated. Expression of these genes is relatively uniform, being
highest in petals, and restricted to surfaces of floral tissues (epidermal
cells), from which volatile compounds can easily escape into the atmosphere
after being svnthesized (Dudareva et al., 1996; 1998a,b: Pichersky et a!., 1994:
1995: Ross ct al.. 1999: Wang et al.. 1997; Wang and Pichersky, 1998).
The many decades of classical breeding of ornamentals have seen a reduction
in the scent of cut flowers due to a focus on maximizing post-harvest
shelf-life, shipping characteristics. and visual esthetic values (i.e., color,
shape) of flowers (Zuker et al., 1998; Dudareva et al., 1999). Engineering
transgenic plants with improved scent quality would overcome this genetic
regression. Success in creation of scented flowers from non-scented ones depends
in each case on many different variables and is therefore difficult to predict.
A major consideration is the availability of substrates in the same cell, and in
the same compartment within the cell, where the enzymes themselves are
localized. Indeed, our recent investigations of floral scent emission in
snapdragon clearly show that amount of substrate in the cell is involved in the
regulation of biosynthesis and emission of flower volatiles (Dudareva et al.,
2000). However, for the majority of substrates req required for the synthesis of
scent compounds, little information is at present available about their own
synthesis, cellular, and subcellular localization. In the future, in order to
achieve the production of noticeable amounts of new floral scent compounds, it
could be necessary to increase the cellular concentration of substrates required
for their production. Results from this research will clarify this major
concern.
OBJECTIVES and ANTICIPATED BENEFITS
The focus of this project is on developing detailed studies on the regulation
of floral scent production for engineering transgenic plants with modified
volatile composition. To avoid undesirable effects of the introduced genes, it
is necessary to have promoters that target expression of these introduced genes
to specific cell and tissue types in the flower during plant growth and
development. This research will specifically address the questions of how plants
regulate the production of volatile esters, what is missing in non-scented
plants and how the quality of floral scent can be improved. The specific
objectives of this proposal are:
1 . Determine what is missing in Antirrhinum varieties that do not
emit methylbenzoate.These experiments will provide information about the molecular changes that
affect the level of scent emission in plants and will be completed during
first year.2. Characterize temporal and spatial expression of BAMT promoter using a
reporter gene. The objective is to establish the target of expression of the
introduced gene under the control of BAMT promoter during flower development.
This promoter can be potentially used to produce transgenic cut flowers with
novel scents. This part will be completed during second year.3. Genetically engineer floral scent production in non-scented plants via
gene transfer The generation of transgenic snapdragon plants requires from
10-12 months. Transgenic plants will be generated using BAMT promoter obtained
in objective 2 during second year and analyzed during the final third year of
the project.
Results from this research will provide the knowledge base for manipulation
of the output of volatile compounds by recombinant DNA technologies. By
transforming originally scentless species with scent genes, we can possibly
create plants more attractive to the consumers. These plants will be a novelty,
a combination of color, shape and scent not seen before. Clearly, new crops with
modified composition of volatiles and newly introduced aroma would benefit US
agriculture by increasing crop productivity and the value of ornamentals.
MATERIALS and METHODS (Project description)
Objective 1.
Determine what is missing in Antirrhinum varieties that
do not emit methylbenzoate. Headspace analysis in combination with gas
chromatography and mass spectrometry of volatiles emitted from flowers of 37
different terent Antirrhinum majus cultivars revealed that the volatile
ester methylbenioate is the most abundant scent compound detected in the
majority of snapdragon varieties. Out of 37 cultivars analyted. only three -
Sonnet White, Potomac White and Potomac Pink - do not emit methylbenzoate or
emit it at very low levels. The availability of snapdragon varieties that differ
in the emission of methylbenzoate gives us a unique opportunity to determine the
molecular changes that affect the level of emission of this volatile. We
examined the BAMT gene expression in these methvlbenzoate nonemitting varieties.
Weak or no detectable hybridization signals were detected in Potomac White and
Sonnet White petals. respectively. indicating that changes have occurred at the
level of transcription of BAMT gene however, we found accumulation of BAMT
transcripts in Potomac Pink petals. These results taken together suggest that
different terent regulatory mechanisms are involved in the regulation of’
methylhenzoate production in different snapdragon varieties. To determine why
Potomac Pink flowers do not emit methylbenzoate although they contain and
express BAMT gene RT-PCR approach will be used to isolate the BAMT eDNA clone.
eDNA will be sequenced to determine possible changes in the coding region of the
gene and activity of this protein will be analyzed after overexpressing it in
the expression vector pET-I la. The level of BAMT protein in Potomac Pink
flowers will be determined by Western blot analysis to find if BAMT protein is
still being made (active or inactive, if some mutations had occurred in the
gene). A discrepancy between protein and enzyme activity levels could suggest
post-translational regulation as well as protein stability differences or some
form of enzymatic regulation.
Since we found that in Sonnet White and Potomac White the changes had
occurred at the mRNA level we will further examine the regulation of expression
of the BAMT gene in these varieties. The BAMT genes will be isolated and
characterized from both varieties and also from scented Maryland True Pink. The
promoter regions of active and inactive genes will be compared and used in in
viva assays (described below) to detect the presence of regulatory elements
that determine its mode of expression. 2-kb promoter region from scented
snapdragon has been already isolated and sequence is now in progress.
Objective 2. Characterize temporal and spatial expression of the
BAMT promoter. To date, most investigators have used the 35S promoter from a
plant virus to regulate the expression of the gene inserted into the plant. This
promoter gives rise to gene expression in most tissues at all stages of
development, therefore it lacks targeted regulation. Since HAMT gene is only
expressed in flower petals and its expression is developmentally regulated (Dudarcva
et al., 2000), its promoter is of potential interest to regulate the expression
of the introduced scent genes. A fragment of at least 1.5 kb containing the
promoter region of the BAMT gene (obtained in objective 1) will be fused in
frame with the GUS reporter gene in the binary vector pBI101 This plasmid will
be introduced into snapdragon and tobacco plants by Agrobacteriurn
tumefaciens-mediated transformation (Firoozabady and Kuehnle, 1995; Heidmann
et al., 1998). In each case, minimum 20 transgenic plants will be generated and
analyzed for GUS expression using standard fluorometric techniques (Jefferson et
al., 1987). These experiments will reveal if the BAMT promoter fragment retains
its expected tissue- and development-specific regulation patterns in homologous
and heterologous expression systems and can be used in future experiments for
specific and programmed expression of other genes in flower tissues.
Objective 3. Genetically engineer floral scent production in miami
scented plants via gene transfer. The BAMT gene from snapdragon will he the
first choice in trying to achieve a new scent in a plant. The BAMT gene
will be introduced into snapdragon varieties that do not emit methvlhenzoate and
to tobacco plants using the binary system of the pBI vectors and the Ti plasmid
of Agrobacterium (Firoozabady and Kuehnle, 1995: Heidmann et a!.. 1998).
We have recently shown that amount of benzoic acid, substrate for methylbenzoate,
in the cell is involved in the regulation of biosynthesis and emission of
mnethylhenzoate (Dudareva et al, 2000). Therefore, we will first test for the
presence of benzoic acid in snapdragon varieties that do not emit
rncthylhcnzoatc amid tobacco plants. Our preliminary results show that Potomac
Pink flowers that do not emit methylhenzoate contain substantial amount of
benzoic acid in petal tissue. Several promoters including BAMT promoter,
obtained in objective 2, and 35S promoter of CaMV will be used in these
experiments. Since the 35S promoter is transcribed relatively uniformly
throughout the plant, this construct will help us assess the availability of
substrates throughout the plant, as well as the toxicity effects of the
expression of the introduced gene. Transgenic plants will be confirmed by
Southern blot anal anaylysis The level of protein expression will be determined
by Western blotting using antibodies against BAMT and! by measuring BAMT enzyme
activity in crude extracts prepared from transgenic tissues. The final
evaluation will be the characterization of volatiles emitted by transgenic
plants by hcadspace analysis and GC-MS.
