Improving Floral Scent Production in Flowers Progress Report- June 2002
ANNUAL PROGRESS REPORT
JUNE 2002
Project Title:
Improving Floral Scent Production In
Flowers
Researcher/Institution Information:
Natalia Dudareva, Purdue University, Department of Horticulture
and Landscape Architecture, West Lafayette, IN 47907-1165 Phone: 765-494-1325
Fax: 765-494-0391
E-mail: dudareva@hort.purdue.edu
Industry Needs And Project Objectives:
Ornamental crops represent a highly important economic
commodity with a world market value of over $30 billion. Unfortunately, many
modern floricultural varieties, including cut flowers, foliage and potted
plants, have lost their scent during selection and breeding processes.
Traditional breeding of ornamental crops has resulted in the production of
cultivars with improved vase life, shipping characteristics, and visual esthetic
values (i.e., color, shape), however it has sacrificed an other important
commercial trait, floral scent. Thus, the manipulation of flower scent would
obviously have a great economic impact by increasing crop productivity and the
value of ornamentals. Crops with improved scent quality and newly introduced
aromas offer possibilities to expand markets and increase grower’s income. The
objective of this three-year research project is to improve scent quality of cut
flowers by manipulation of the output of volatile compounds using recombinant
DNA technologies. To achieve this objective, we are investigating the molecular
changes that affect the level of scent emission in plants and isolating the BAMT
promoter, which can potentially be used to produce transgenic cut flowers with
novel scents.
Summary Of Work Completed:
Objective 1
. Determine what is missing in Antirrhinum
varieties with low to moderate emission of methylbenzoate.
We have previously shown that methylbenzoate is produced in
upper and lower petal lobes of snapdragon flowers by the action of the
biosynthetic enzyme S-adenosyl-L-methionine: benzoic acid carboxyl
methyltransferase (BAMT) (Dudareva et al., 2000). During the first year of the
project we found that Potomac Pink cultivar emits methylbenzoate at a low level,
although it contains a high level of BAMT transcripts. We used RT-PCR and found
that Potomac Pink contains two forms of the BAMT gene, one is an active form,
which is capable of producing methylbenzoate, and the other is an inactive form
in which10 changes in the amino acid sequence led to the elimination of
enzymatic activity. To study the genetic control of methylbenzoate emission in a
variety with low methylbenzoate emission, a Potomac Pink plant, which is an F1
hybrid, was self-pollinated, and seeds were grown to generate an F2 population.
We now have 14 individual plants from the F2 population. Emission of
methylbenzoate was analyzed in all individual plants and is shown on Figure 1.
BAMT activity in petal lobes of flowers from individual plants of the F2
population was determined. It was very low when compared with Maryland True
Pink, the cultivar with the highest emission of methylbenzoate, and also did not
show any correlation with amount of emitted ester. To check whether BAMT
expression co-segregates with methylbenzoate emission, the BAMT mRNA expression
was analyzed in individual plants. Since the BAMT probe used in RNA blot
analysis recognized both types of BAMT (altered- inactive or normal-active),
quantitative RT-PCR with type -specific primers was used to determine the
contribution of each type of BAMT mRNA in total BAMT expression in the F2
population. We found that both types of the BAMT gene are expressed in petal
tissue at approximately equal levels. We have also analyzed the amount of BAMT
protein in individual plants by Western blot analysis using rabbit polyclonal
anti-BAMT antibodies. The amount of the BAMT protein was undetectable by Western
blot analysis, indicating that the formed protein is 
probably
quickly degraded and that the amount of normal active BAMT, which could be
responsible for low activity, is too low for detection.
Figure 1.
Methylbenzoate emission of Potomac Pink F2
generation. Potomac Pink F1 and MTP are included for comparisons. Headspace
analysis of emitted volatiles was done on the fifth day after anthesis in
controlled growth camber conditions.
Another possibility is that the low amount of emitted
methylbenzoate in Potomac Pink plants could be made by another gene which is,
for a example, S-adenosyl-L-methionine:salicylic acid carboxyl
methytransferase (SAMT). It has been previously shown that Clarkia breweri
SAMT, although it is highly specific for salicylic acid, does methylate benzoic
acid with relatively high efficiency (Ross et al., 1999). To find whether the
SAMT gene is involved in methylbenzoate production in Potomac Pink flowers, we
have isolated the SAMT gene from snapdragon using a genomic approach. The
function of the SAMT gene was biochemically determined using an Escherichia
coli expression system. Kinetic parameters of SAMT for salicylic acid,
benzoic acid and methyl donor SAM were determined using substrate interaction
and saturation kinetics. We found that the apparent Km values for
benzoic acid for snapdragon SAMT and BAMT are very close, indicating that SAMT
can potentially participate in methylbenzoate biosynthesis in snapdragon
flowers.
Objective 2.
Characterize temporal and spatial
expression of BAMT promoter using a reporter gene.
The focus of this part of the proposal was to determine
whether the BAMT promoter is capable of driving transgene expression in a
“scent specific” manner (in proper tissue and at the proper stage of
development) in other plant species such as petunia and tobacco. Using Agrobacterium
tumefaciens we have successfully transformed leaf disc explants with a
construct containing the putative full-length BAMT promoter region (2kb)
translationally fused to the GUS-NOS reporter gene. After shoot and root
induction on kanamycin-containing media, plants were transferred to the soil and
kept in a greenhouse. For unknown reasons petunia plants died after transferring
them to soil. We generated 15 transgenic tobacco plants, but used 10 plants for
further analysis.
For functional mapping of cis-acting motifs
responsible for the temporal and spatial expression of BAMT gene we generated a
series of three 5’deletions derived from the full-length BAMT promoter, 1.5,
1.0, and 0.6 kb in size, and translationally fused them to the GUS-NOS reporter
gene. These chimeric genes were transformed into Petunia Mitchell and Nicotiana
tabacum. Similar to the construct containing a full-length promoter region,
plants were transferred to soil after shoot and root induction on kanamycin-containing
media and kept in a greenhouse. We generated 10 tobacco plants per construct
with the exception of the 0.6-kb plants, where only three plants survived the
transfer to soilless media. The presence of the promoter-GUS transgene in
individual transformed plants was confirmed by PCR using primers designed to
recognize the kanamycin resistant gene NPTII that was introduced along with the
BAMT:GUS gene. DNA was isolated from each primary transgenic plant and a PCR
reaction was performed on each sample. Ten independent transgenic tobacco plants
were identified for each of the 2, 1.5, and 1kb BAMT:GUS promoter plants, and
all three of the 0.6-kb that survived were transgenic.
Tobacco plants were grown under normal greenhouse conditions
until flowering, and flowers were then collected for histochemical analysis of
GUS activity. GUS activity was found at a low level in the tobacco corollas and
only in the plants containing the full-length (2.0 kb) promoter region. Although
GUS activity in the corollas was limited to the outer area and no activity was
detected in the tube, which is similar to BAMT expression in snapdragon, these
results show that the 2-kb promoter region does not contain sufficient
regulatory sequences, which are necessary to reach endogenous levels of BAMT
expression. The low level of GUS activity suggests that an enhancer element is
missing in the 2-kb BAMT-GUS construct. Additionally, in contrast to BAMT
expression in snapdragon, GUS activity was identified in the sepals, ovaries,
and receptacles of the 2.0-kb tobacco plants indicating that a suppressor of
spatial promoter activity is also missing in the full-length (2.0 kb) promoter
region. Transgenic tobacco plants containing the 1.5-kb and the 1.0-kb promoter
region of BAMT both showed GUS activity in the ovaries and receptacles whereas
plant with the 0.6-kb construct resulted in no GUS activity. Taken together,
obtained results show that a 2-kb BAMT promoter region is necessary to direct
the transcription of transgene in petal tissue. Additionally, more than one cis-element
seems to be responsible for the transcriptional regulation of BAMT.
Objectives For The Coming Year:
Recent isolation and characterization of the first several
genes encoding scent biosynthetic enzymes opens the door for metabolic
manipulation of floral scent to improve its quality. In the last two years
isolated scent genes have already been used for the metabolic engineering of
plant volatile composition (Lewinsohn et al., 2001; Lucker et al., 2001),
however no significant increase in the amount of volatile compounds has been
reported (Vainstein et al., 2001). These results clearly show that in addition
to the availability of cloned genes encoding enzymes involved in the
biosynthesis of floral volatiles, an understanding of the biochemical, molecular
and other events controlling the
production and emission of volatiles from
plant tissues is absolutely required for the bioengineering of floral scent.
These results also show the importance of the proposed research. Our objectives
for the coming year are to:
1. Complete the investigation of the molecular changes
that have occurred in Antirrhinum varieties with low to moderate emission
of methylbenzoate.
Previously we have shown that BAMT protein is enzymatically
active as a homodimer (Murfitt et al., 2000). Since Western blot analysis did
not detect the BAMT protein in crude extracts from 14 Potomac Pink individual
plants from the F2 population despite the high level of BAMT mRNA expression, it
is possible that altered-inactive BAMT interacts with normal- active BAMT
forming an inactive protein, which is probably degraded quickly. To check this
hypothesis, we will coexpress normal and altered BAMT proteins in E. coli
and check BAMT activity of the resulting recombinant protein. Co-transformation
with two plasmids (normal BAMT in pET 11a and mutant BAMT in pET 28a) will be
performed in a single transformation event. Positive transformants will be
screened for multiple resistances with kanamycin (pET 28a) and ampicillin (pET
11a). Crude extracts of sonicated transformed cells after IPTG induction will be
tested for BAMT activity and E. coli cells transformed with normal BAMT
in pET 11a alone will be used as the control.
Since we have proven that SAMT can potentially participate in
methylbenzoate emission in snapdragon flowers, we will analyze expression of the
SAMT gene in upper and lower petal lobes of 14 individual plants from the F2
population by RNA gel blot analysis and its possible co-segregation with
methylbenzoate emission. Total RNA will be isolated from 4-day-old flowers, one
day before the maximum of methylbenzoate emission. If SAMT transcripts are
undetectable in floral tissues by RNA-blot hybridization, we will use RT-PCR in
the presence of SAMT-specific primers to detect the low levels of SAMT gene
expression. Co-segregation of SAMT expression with emission of methylbenzoate
will indicate that this gene is responsible for variations in methylbenzoate
emission in Potomac Pink plants.
Complete characterization of BAMT promoter in transgenic tobacco plants using a
reporter gene.
Each primary transformant will be self-pollinated to generate
a T1 population to determine the inheritance of the transgene and also for
further investigation of the BAMT promoter. This part of work is now in
progress. To examine at which stage of flower development the appearance of BAMT
expression is initiated and reaches the maximum, GUS activity will be measured
in extracts from petals at different stages of flower development.
The discrepancy between the BAMT-driven GUS expression in
tobacco transformants and BAMT expression in snapdragon could be due to the
absence of specific regulatory proteins required for the expression of genes
involved in scent production because Nicotiana tobacum used for
transformation does not emit methylbenzoate. To check this hypothesis transgenic
plants containing the full-length BAMT promoter region –GUS construct from T1
population will be crossed with Nicotiana svuaveolens, which has been
shown to emit methylbenzoate (Loughrin et al., 1991; Kolosova et al., 2001), and
GUS expression will be analyzed in the progeny.
We will also check whether expression of the BAMT-GUS
construct could be induced by benzoic acid. In these experiments cut flowers
will be placed in solution containing different concentrations of benzoic acid
in addition to sucrose and GUS activity will be analyzed in floral tissue. These
results could be very important in the cut flower industry for increasing scent
production by the induction of scent genes.
Literature Cited:
Dudareva N, Murfitt LM, Mann CJ, Gorenstein N, Kolosova N,
Kish CM, Bonham C, Wood K (2000) Developmental regulation of methyl benzoate
biosynthesis and emission in snapdragon flowers. Plant Cell 12: 949-961.
Kolosova N, Gorenstein N, Kish CM, Dudareva N (2001)
Regulation of circadian methyl benzoate emission in diurnally and nocturnally
emitting plants. The Plant Cell 13: 2333-2347.
Lewinsohn E, Schalechet F, Wilkinson J, Matsui K, Tadmor Y,
Nam KH, Amar O, Lastochkin E, Larkov O, Ravid U, Hiatt W, Gepstein S, Pichersky
E (2001) Enhanced levels of the aroma and flavor compound S-linalool by
metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol
127: 1256-1265.
Loughrin JH, Hamilton-Kemp TR, Anderson RA, Hildebrand DF
(1991) Circadian rhythm of volatile emission from flowers of Nicotiana
sylvestris and N. suaveolens. Physiol Plant 83: 492-496.
Lucker J, Bouwmeester HJ, Schwab W, Blaas J, Van der Plas LHW,
Verhoeven HA (2001) Expression of Clarkia S-linalool synthase in
transgenic petunia plants results in the accumulation of S-linalyl-beta-D-glucopyranosid.
Plant J 27: 315-324.
Murfitt LM, Kolosova N, Mann CJ, Dudareva N (2000)
Purification and characterization of S-adenosyl-L-methionine:benzoic acid
carboxyl methyltransferase, the enzyme responsible for biosynthesis of the
volatile ester methyl benzoate in flowers of Antirrhinum majus. Arch
Biochem Biophys 382: 145-151.
Ross JR, Nam KH, D’Auria JC, Pichersky E (1999) S-adenosyl-L-methionine:
salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent
production and plant defense, represents a new class of plant methyltransferases.
Arch Biochem Bioiphys 367: 9-16.
Vainstein A, Lewinsohn E, Pichersky E, Weiss D (2001) Floral
fragrance. New inroads into an old commodity. Plant Physiol 127: 1383-1389.
Professional/Published Information:
- Negre F, Kolosova N, Knoll J, Kish CM, Dudareva N. (2002) Novel S-adenosyl-L-methionine:salicylic
acid carboxyl methyltransferase, an enzyme responsible for bios6ynthesis of
methylsalicylate and methylbenzoate, is not involved in floral scent
production in snapdragon flowers. Arch Biochem Biophys, in press. - Dudareva N, Faldt J, Kolosova N, Kish CM, Gorenstein N, Bohlmann J.
Myrcene synthase from snapdragon, a new gene involved in floral scent
biosynthesis. Submission will be to Plant J by 11/1/2002. - Goodwin SM, Kolosova N, Kish CM, Wood KV, Dudareva N, Jenks MA.
(2002)Cuticle involvement in floral emission of methylbenzoate by Antirrhinum
majus L. Physiologia Plantarum, in press. - Dudareva N, Kolosova N, Kish CM, Gorenstein N. (2002) Floral scent –from
compounds to genes. Plant, Animal and Microbe Genomes X Conference, 12-16 of
January 2002, San Diego, CA (Oral Presentation). - Knoll J, Kish CM, Gorenstein N, Boatright J, Dudareva N. (2002) Snapdragon
cultivars with low to moderate production and emission of methylbenzoate:
Regulatory aspects. Second Gordon Research Conference on the Biology,
chemistry and evolution of floral scent, March 3-8, 2002, Ventura, CA(poster) - Boatright J, Peel G, Rhodes D, Kish CM, Gang D, Dudareva N. (2002)
Biosynthesis of benzoic acid in petunia and snapdragon flowers. XII
International Antirrhinum Meeting, 17-21 April 2002, Porticcio – Corsica,
France, (Oral Presentation). - Dudareva, N, Kolosova, N, Kish, CM, Boatright, JL, Peel, G, Rhodes, D.
(2002) Floral scent – from compounds to metabolic pathways and their
regulation. Plant Biology Canada 2002, Annual Meeting of the Canadian
Society of Plant Physiologits, June 8-12, 2002, Calgary, Alberta (Plenary
Lecture). - Boatright J, Peel G, Rhodes D, Kish CM, Gang D, Dudareva N. (2002)
Biosynthesis of benzoic acid in petunia and snapdragon flowers. Plant
Biology 2002, August 3-7, 2002 Denver, Colorado (Oral Presentation). - Dudareva N (2001) “Floral scent ¬ñfrom compounds to metabolic
pathways and their regulation”, Department of Biology, Michigan State
University, East Lansing, MI, October 31, 2001(Invited Seminar). - Dudareva N (2202) Floral scent –from compounds to metabolic pathways and
their regulation”, Department of Biological Sciences, Lehman College,
City University of New York, Bronx NY, April1, 2002 (Invited Seminar).
