Lab 4 Meta-Report
Assessing the Effects of Metals on Dahlia Pigmentation
Introduction
Petal coloration is important in attraction of pollinators to flowers. The diversity
of flower color has been intensively studied and utilized in genetic research since Mendel.
Anthocyanin pigments are of central importance in flower coloration in many
angiosperms, including dahlias and other Composites, although pigment molecules alone
are often insufficient to produce the colors seen in many flowering plants. Within a cell
anthocyanin is synthesized in the cytoplasm, but only after transfer to the vacuole does
the pigment attain a bright color. Among the accessory factors that determine coloration
is the pH of vacuoles of floral cells, anthocyanin assembly with flavonols and flavones
and other organic molecules (collectively called co-pigments) (Reuveni et al. 2001, Mol
1998), and anthocyanin assembly into complexes coordinating a metal ion such as
aluminum and copper (Sarma et al. 1997) or magnesium and manganese (ShakedSachray et al. 2002). Some bright blue flowers coordinate a chromate ion with 4
anthocyanin molecules. Depending on the protonation state, anthocyanins will absorb
different wavelengths of light; in acidic conditions, anthocyanins typically appear red
while in basic conditions they are usually green or blue. For instance, in petunia, low
vacuolar pH correlates with red coloration, whereas high pH correlates with purple
(Reuveni et al. 2001). However, the availability of metals ions generated by pumps that
selectively transport specific ions into the vacuole can also have an effect on flower
pigment. Hydrangeas flowers are blue when grown in acidic soil and red when grown in
alkaline conditions, which is contrary to what would be expected based on pH affecting
anthocyanins. The mechanism which causes this color change was found to be pH
affecting the solubility of aluminum ions; those aluminum ions in turn were responsible
for floral pigmentation (Yoshida et al. 2008, Takeda et al. 1990, Takeda et al. 1985). To
assess whether environmental pH or metal ion conditions might modulate flower color in
dahlias, metal or pH treatments were applied to several Dahlia cultivars and the effects on
flowers examined. Metals selected were:
 nickel (as NiCl; Tso et al. 1972)
 tin (as StCl2 and StSO4; Cohen 1940)
 copper (as CuCl2; Brenchley 1938)
 cobalt (as CoCl2; Brenchley 1938)
 zinc (as Zn2+; Sagasser et al. 2002)
 magnesium (as MgSO4; Mazza and Miniati 1993, Takeda and Hayashi
1977, Hayashi and Takeda 1970)
 manganese (as MnSO4; Asrar et al. 2005, Mazza and Miniati 1993,
Takeda and Hayashi 1977)
 iron (as FeSO4)
 molybdenum (as H2MoO4)
This report synthesizes the analysis of the effects of metals ions and pH in different
Dahlia cultivars conducted by students in the Spring 2008 quarter of Biological Sciences
137; Dahlias in Plant Genetics: E. Abrash, A. Christian, R. Kamber, J. McCallen, A.
Schultink, W. White, and C. Young.
Objectives
The objectives of this study followed the outline of metals used above; analysis of
which metals or pH affected dahlia floral pigments was the primary objective of this
experiment, and results will be defined based on the individual researcher and the metals
or pH values used.
Materials and Methods
In this investigation, groups of plants were dosed with the various metal
treatments in differing concentrations. Doses were applied twice during a week to plants
in single watering trays (for each treatment) such that subsequent watering would
promote uptake of additional metal or hydrogen ions. In each case, about 125 mL of total
solution was given to each tray over seven days. Alternately, for plants in individual pots,
metal solutions were applied on the first day and the fourth day of the experiment.
Applications of acids were typically in the pH range of 4-5, through addition of citric acid.
Application of bases were in the 8-9 pH range through addition of sodium hydroxide.
Copper, iron, magnesium, manganese, molybdenum and cobalt were given using 10 μM
solutions. Zinc was dosed using 1 μM and 5 μM solutions as separate treatments. Tin
and nickel were given using 50 μM solutions.
Photographs of flowers were taken at the time of initial treatment. One week later,
plants were re-examined for changes in flower color and compared to individuals of the
same cultivar that had not received the treatments, and compared to ADS color chips.
Summary Table of Results
Name
Dixie's Winedot
Pinot Noir
Dixie's Winedot
Pinot Noir
Dixie's Winedot
Pinot Noir
Teesbrooke Red
Eye
Dixie's Winedot
Clear Choice
Lakeview Premier
Clear Choice
Lakeview Premier
Clear Choice
Classic Poeme
Smooth Operator
Fire Mountain
Moray Susan
Moray Susan
Treatment
1 uM zinc
1 uM zinc
5 uM zinc
5 uM zinc
pH 4 citric acid
pH 4 citric acid
CG Elegance
Lakeview Premier
10 uM cobalt
10 uM iron
Lakeview Premier
10 uM manganese
10 uM copper (as CuSO4)
10 uM copper (as CuSO4)
pH 4 citric acid
pH 4 citric acid
pH 8 sodium hydroxide
pH 8 sodium hydroxide
50 uM nickel
50 uM nickel
50 uM tin
50 uM tin
10 uM copper (as CuCl2)
10 uM cobalt
Effect on Color
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
unknown
increase in saturation of magenta,
followed by increase in yellowing
centrally
unknown
none
increase in saturation of pink, pk-19 to
pk-22
Other Effects
premature senescence
premature senescence
premature senescence
premature senescence
premature senescence
premature senescence
plant death
plant death
Lakeview Premier
Lakeview Premier
10 uM magnesium
10 uM molybdenum
increase in saturation of pink, pk-19 to
pk-23
none
Results and Discussion
Plants in most treatment groups displayed very little pigmentation differences
compared to control plants. Instead, flowers on most treated cultivars appeared to senesce
prematurely, displaying a dry and crinkled appearance despite apparently adequate health
of the vegetative tissues. Certain treatments exacerbated this effect: copper in particular
as well as zinc and basic solutions seemed to cause premature senescence. The observed
senescence phenotypes might reflect a specific effect of the treatments on floral
progression; alternatively, floral senescence might be symptomatic of an overall
reduction in plant health, with flowers being sensitive “indicator” organs that display
phenotypes before leaves and other tissues. On the other hand, better control of flower
age might help to demonstrate the effects of metals on senescence, as some of the control
flowers aged significantly during the duration of the experiment. Additionally, natural
variation in senescence phenotypes, which would be especially prevalent in the vastly
differentiated floral forms of dahlias, has previously been observed in Arabidopsis
thaliana (Levy et al. 2005).The experimental observations suggest that at the
concentrations used, only magnesium, manganese, and to a lesser extent cobalt affect
flower color.
Cobalt treatment followed three phases of pigmentation. The flowers of the
cultivar Moray Susan before treatment were pink with yellow coloring in the interior.
About a week into treatment the flowers minimized their yellow color but the pink
became more red, resembling a magenta pigment. After extensive cobalt treatment, the
flowers regained their pink color, but only in the outer ray florets. The inner ray florets
did not show any pink but were completely yellow.
Lakeview Premier plants receiving magnesium and manganese displayed
significant differences in ray floret coloration. Both magnesium and manganese have
been shown in the past to complex with anthocyanin pigments (Takeda and Hayashi 1977,
Mazza and Miniati 1993). Magnesium has furthermore been shown in a recent study to
stabilize anthocyanin pigments in flower petals, not by increasing anthocyanin synthesis,
but most likely by forming stable anthocyanin/magnesium complexes (Shaked-Sachray et
al. 2002). If manganese and magnesium from the watering solution are translocated to
developing flowers, it seems plausible that anthocyanin synthesis and/or accumulation
would be upregulated in the presence of Mg2+ or Mn2+. Magnesium has shown to be a
component of the blue pigment commelinin from cornflowers (Hayashi and Takeda
1970) and manganese toxicity has been correlated with increased anthocyanin
accumulation in spearmint (Asrar et al. 2005). Definitive determination of the presence of
manganese and magnesium in darkened flowers should be established. If present, the
nature of the metal-anthocyanin interaction(s) could also be more fully investigated. Of
particular interest would be identification of the anthocyanin pigments present in
Lakeview Premier through UV-visible spectrophotometry.
Certainly the results shown here represent only an initial step in investigating
confounding effects of anthocyanin pigmentation. Further research might help determine
exactly which anthocyanins are present, their degree of complexation to metal ions, and
the effects of ranges of metal ion concentrations and pH levels. Could we treat at a lower
dose of metal for a longer period of development and see more changes in flower color
and avoid the senescence response?
One experiment performed in the lab was to extract anthocyanin in acidic
methanol (1 mL HCl in 100 mL in methanol) from several pink and purple cultivars. The
extracts were bright pink or purple. When extracts were mixed with Dowex chelating
resin, anthocyanins were converted to a pale yellow-pink color. Dowex can extract
divalent metal ions (depending on the affinity of the pigment for the metal). Adding back
metals could restore some color to the anthocyanin in the pink-purple range; adding
ferrous iron caused the anthocyanin to be reddish brown. In the future, it would be
interesting to explore this further by identifying which ions are bound to Dowex from the
extract. Do all dahlias complex the same metal ions? Another interesting question would
be could we treat at a lower dose of metal for a longer period of development and see
more changes in flower color?
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