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Chelsea Young
Special Project Lab Report
Introduction:
Largely conserved throughout the plant kingdom, flavonoids serve multiple functions in
most plant families, including protection from mutagenic UV rays, pollinator-attraction, feeding
deterrance and microbe defense (Winkell-Shirley 2001). The flavonoid biosynthetic pathway
begins with chalcone-isomierized condensation of one molecule of 4-coumaroyl-CoA and 3
molecules of 3 Malonyl-CoA to yield chalcone (Figure 1). Chalcone is subsequently isomerized
to naringenin by chalcone isomerase, at which point it can either be maintained as such or
shuttled into several different pathways to give rise to six different classes of flavonoids: the
chalcones, flavones, flavonols, flavandiols, anthocyanins, and condensed tannins (WinkelShirley 2001).
Figure 1: The flavonoid pathway (Winkell-Shirley 2001)
In some cultivars of dahlia, transposon insertions have rendered certain critical genes in
the flavonoid pathway nonfunctional. In this case, the compound preceding the earliest disrupted
step will build up in the flower, affecting pigment color. Such gene disruption often leads to a
variegated pigment pattern. This variegation pattern depends on whether the transposon
maintains its location within the gene or excises and, if it excises, at what developmental stage.
A transposon that excises early enough in plant development could give rise to a flower
in which the fully functional pigment pathway is restored. A flower resulting from this
phenomenon is commonly referred to as a revertant. This paper seeks to analyze the
biochemistry and genetics responsible for one such revertant in the popular cultivar Dixie
Winedot.
Results:
Two Dixie Winedot flowers, one wildtype (variegated) and one revertant, were picked
from the greenhouse. The wildtype displayed a pale yellow background with purple spots (Figure
2). The revertant displayed entirely purple pigmentation, suggesting that an excision event very
early in development or at the prezygotic stages had undergone an transposon excision event,
thereby restoring the flavonoid pathway in the entire plant.
Figure 2: A traditional Dixie Winedot flower. The revertant is entirely purple.
I. Biochemical Analysis
Using a mortar and pestle, extracts of petal tissue were prepared using 1% acidic
methanol as a solvent. Spots of pigment extract were dotted onto nitrocellulose paper. A TLC
was run using a solution of acetic acid: hydrochloric acid: water at 8:2:3. Separation was
achieved after 41 minutes (Figure 3).
Figure 3: The results of the TLC separation.
Band 2
Band 1
Bands 1 and 2 were subsequently scraped off of the TLC plate and resuspended in acidic
methanol. The tubes were centrifuged and the solution was decanted to remove any
nitrocellulose particles. These pigments were analyazed in a spectrophotomer, reading every four
nanometers from 250 to 550 nanometers to obtain an absorbancy spectrum (Figure 3).
Figure 3: The absorpancy spectrum of the wildtype and revertant pigments contained in Band 1
and Band 2.
Wild Type and Revertant Pigments
3
Absorbance
2.5
WT band1(100%)
2
REV band1(100%)
1.5
WT band2(100%)
1
REV band2(100%)
0.5
0
250
350
450
Wavelength (nm)
550
WT Band 1 and Revertant Band 1, which appear yellow on the TLC plate, gave a reading
suggestive of chalcone. WT Band 2 and Revertant Band 2, purple on the TLC plate, gave a
reading characteristic of a flavonol (Figure 4).
Figure 4: Various flavonoid absorbancy spectrums with which the unknowns were compared.
Wild Type and Revertant Pigments
3
2.5
2
1.5
1
0.5
0
250
300
350
400
450
500
550
600
The pigment solution extracted from WT Band 1, suspected to be chalcone, was
compared to those of the available standards: kaempferol, kaempferride and resokaempferol to
compare the spectrums (Figure 4).
Figure 4: WT Band 1 compared to known standards Kaempferol, Kaempferide and
Resokaempferol.
Comparison of Unknown with Known Flavonols
3
Absorbance
2.5
Kaempferol
2
Kaempferide
1.5
Resokaempferol
1
WT band1(100%)
0.5
0
250
350
450
550
Wavelength
These results suggest that in wildtype flowers, there is a buildup of chalcone, whereas in
revertant flowers, the primary pigment is a type of flavonol.
II. Genetic Analysis
Based on these biochemical analyses, it was suspected that in the wildtype Dixie
Winedot flower, a step in the pathway leading to the synthesis of flavonol was disrupted,
resulting in the buildup of a yellow pigment suspected to be chalcone. PCR was deemed to be an
effective way to test this hypothesis.
cDNA sequences of chalcone isomerase in four members of the Asteraceae family were
obtained through the NCBI database. The sequences were aligned using the program Prifi,
available online. Because genomic DNA sequences of chalcone isomerase were not available for
any members of the Asteraceae family, the placement and length of introns were estimated based
on the CDS of petunia (Figure 5). Introns were inserted into the degenerate sequence in order to
approximate overall gene length (around 700 bases).
To test the validity of using petunia to estimate intron placement, the amino acid
sequence of chalcone isomerase from petunia and chrysanthemum were compared. The two were
shown to be largely conserved (Figure 6). Based on the sequence alignment for the four
sequences from Asteraceae, two setes of degenerate primers were prepared and ordered from
Invitrogen (Figure 5).
Figure 5: A schematic depicting nucleotide cDNA comparison with proposed introns inserted.
Areas of homology (black) and non-matching areas (white) are depicted. The overlapping primer
pairs are also show below.
Intron 1
~107bp
Intron 2
~100bp
Intron 3
~167bp
Primer Pair 1
Primer Pair 2
Figure 7: Conserved (black) and non-conserved (white) amino acids in chalcone isomerase
amino acid sequence from petunia and chrysanthemum.
Total genomic DNA was extracted using a standard CTAB extraction protocol. The
resulting genomic DNA was run on a 1.2% Invitrogen E-Gel and found to be sufficiently good
quality to perform PCR.
For the PCR reaction, because degenerate primers were being used, the same reactions
were run at twelve different temperatures in a gradient from 50 to 60 degrees Celsius. After the
reaction was completed, the PCR products were run out on a gel (Figure 7). Binding was seen at
all temperatures. The sequence amplified by Primer Pair 1 was a smear, so a second gel was run
to distinguish the Primer Pair 1 reaction products (Figure 8).
Figure 7: A gel showing the PCR reaction products of WT Primer Pair 1, WT Primer Pair 2,
Revertant Primer Pair 1 and Revertant Primer Pair 1.
WT Primer Pair 1
WT Primer Pair 2
Revertant Primer Pair 1
Revertant Primer Pair 2
50C
60C
50C
60C
Figure 8: In a second gel, PCR products from the reaction involving Primer Pair 1 at 58 and 52
degrees Celsius for WT and Revertant and the reaction involging Primer Pair 2 at 58 degrees for
WT and revertant were run on a gel.
WT
Rev
WTRev
58C 52C 58C 52C 58C
There are approximately tend bands present from both the WT and the revertant PCR
reactions for Primer Pair 2, suggesting non-specific pairing.
For Primer Pair 2, two identical bands are present in both the wildtype and the revertant.
Discussion:
For the biochemical analysis, based on the combined spectrophotometric analyses, it
seems likely that Band 1 in the wildtype plant contained a number of compounds, one of which
is chalcone. In the absence of chalcone isomerase, the chalcone can be acted on by other
enzymes, including CGT and AUC. Thus, the imperfect alignment of the WT band 1 may result
from the presence of compounds beside chalcone.
To get a better idea of which compounds are present, HPLC should be performed. This
would give a clear idea of exactly which flavonoids are present in each specimen.
Figure 10: The enzymes that can act on chalcone.
As for the PCR reaction, the ten bands present for the PCR reaction for Primer Pair 1
strongly suggest non-specific pairing. This could be a side effect of the special precautions taken
to counteract the failure to bind that is common with degenerate primers. Specifically, the
primers were designed to be longer than usual (around 30 nucletotides) and the recommended
temperature was higher than usual (70 degrees Celsius). To encourage annealing, the PCR
reactions were run at a lower temperature than was called for, which likely contributed to
nonspecific binding. Additionally, the first estimated intron in the chalcone isomerase sequence
was shown to be located in the middle of Primer Pair 1, which makes it seem likely that Primer
Pair 1 was not able to amplify the chalcone isomerase gene at all, but instead annealed to other
similar sequences.
For the two bands present in the wildtype and revertant reactions, there are three possible
hypotheses. First, it’s possible that one of the bands results from nonspecific pairing. Second, it’s
possible that dahlia contains more than one chalcone isomerase gene. This is not unlikely
because maize has been shown to have three copies of the chalcone isomerase gene. Third, it’s
possible that dahlia, being octaploid, contains multiple alleles of the chalcone isomerase gene.
Assuming that a transposon were present, one would have expected to see the following
gel pattern from the PCR reaction of the wildtype flower: (1) a higher molecular weight, darker
band representing the allele disrupted by the transposon, and (2) a lower molecular weight,
lighter band representing the relatively small percentage of alleles from which the transposon
excised during development (generating purple spots). Judging from lane 10 of Figure 7, this
appears to be the case; however, comparing fluorescence intensity after PCR reactions is not an
accurate means by which to compare the prevalence of the gene in the total DNA extraction.
Therefore, no definite conclusions can be drawn based on this preliminary data.
To definitely ascertain whether or not a transposon inserted into the chalcone isomerase
gene is responsible for wildtype function, two experiments may prove informative. First,
sequencing the two bands produced from the PCR reaction of the wildtype and revertant flower
would be the most accurate means to judge the presence or absence of a transposon in the allele.
Second, comparing these PCR reaction products to those obtained from other nonvariegated dahlia varieties could prove informative. In the non-variegated cultivars, transposons
are probably not present. Therefore, if these varieties display only one band, whereas Dixie
Winedot continues to display two bands, it would seem likely that the higher-molecular weight
amplified sequence does indeed contain a transposon.
The authors hope that some motivated students from next year’s Biology 129B class will
adopt this topic and continue the investigation. The Dahlia field is truly one of the greatly
unknown areas of knowledge in the world today.