Use the Case Study file attached below, and answer the red tabs questions 1-18 from the yellow boxes that appear of pages 1-7
Title
Authors and author
information
Abstract:
A summary
written by the
authors
Citation for this paper
Introduction: Not all
journals mark it with
a subheading
Footnotes, including
contact information for
corresponding author
and funding sources
The first page of a typical article from Plant Physiology.
(See text for more information about each section)
In-text citation:
Full citation: is
found at the end of
the article
Indicates
footnotes
Indicates
footnotes
Case study: Reading a Primary Research Article
from Plant Physiology
This case study examines a recent article published in the journal
Plant Physiology. The full article is appended to this PDF. Because
of space constraints, only the major points from the paper are
covered in the case study, and the biochemical pathway is
presented in simplified form.
Copyright (2013) American Society of Plant Biologists. www.aspb.org
It’s like learning to ride a bike
It takes some practice to learn to read a
scientific paper, but with a little effort you
should be able to navigate your way around
with confidence. This is a guided walk through
a recently published article to get you started.
The Title
The title of the paper needs to be factual,
informative, and concise. Most journals have a
strict character limit; Plant Physiology’s limit is
150 characters. You could think of the title as
the abstract in a tweet.
Q1. What is the title of this article?
The Authors
Single-author papers are rare, especially in
biology. Most papers report the efforts of a
team and so have two or more authors. Each
author must have made a significant
contribution to the research and writing of the
paper (for guidelines about authorship, see
www.plantphysiol.org/site/misc/ifora.xhtml#Aut
horship). Minor contributors can be recognized
in the Acknowledgments section at the end of
the paper. The order in which the authors are
listed is important. Typically, the first person
listed (the “first author”) conducted much of the
research and gets the most credit. Sometimes
two or more people are given co–first
authorship, which is usually indicated in a
footnote of the paper. First authors are typically
graduate students or postdoctoral researchers
carrying out their research in the lab of a more
senior scientist, who is typically listed in the last
position. If the research involved a
collaboration among more than one lab group,
the senior authors are typically all listed at the
end. In between the first-author position and
the senior-author position, others who made
significant contributions to the paper are listed.
Some papers list the contributions of each
author at the end of the paper. The
corresponding author is indicated in the
footnotes and is the contact person for the
paper; usually the first or last author is the
corresponding author. The institutional
affiliations of each author are listed in this
section.
Q2. How many authors does this paper
have? How many institutions and
departments are represented? Who is
the corresponding author?
Q3. Which organization provided the
funds used to carry out the research,
and which author was awarded the
funds?
The Abstract
The abstract is a summary of the entire study.
The authors highlight the question that they
addressed, the methods they used, the
hypotheses they tested, and the results of their
experiments, and explain what their results
mean, all in one paragraph (for Plant
Physiology, a paragraph of no more than 250
words).
Q4. After reading the abstract, what do
you understand about this paper?
What is the experimental organism
being studied? In your own words,
what question is being addressed in
this paper?
The Introduction
In some journals, including Plant Physiology,
the heading “Introduction” is not used, but the
introductory information is always the first part
of the article after the abstract. The introduction
provides the background and justification for
the experiment. The introduction describes why
the study was carried out and the question
being investigated or hypotheses being tested.
Statements of facts should be supported with a
citation to another published article.
Moreau et al.
(2012) introduces the
pigment molecules found in flowers called
anthocyanins, which are types of a category of
chemicals called flavonoids. The article states
that the biochemistry and genetics of
anthocyanin production have been studied in
peas and other plants. The first paper cited in
the Introduction is a review article by
Grotewold, published in 2006, called “The
Genetics and Biochemistry of Floral Pigments,”
published in the Annual Review of Plant
Biology. This review article gathers together a
lot of information from many articles. It
describes the biochemical pathway for
anthocyanin synthesis and the reactions that
are catalyzed by the enzymes discussed in
Moreau et al. (2012) and so provides important
background information. A simplified version of
the anthocyanin biosynthetic pathway is shown
below.
As shown in the figure above, a colorless
precursor, Naringenin, can be hydroxylated at
the 3′ position by the enzyme F3′H, or at the 3′
and 5′ positions by the enzyme F3′5′H. These
compounds, as well as the unmodified
precursor, are subsequently converted into
pigments. The presence or absence of the
hydroxyl groups affects the color of the
pigments. Delphinidin (and related compound
petunidin, not shown), are purple and are
hydroxylated at both the 3′ and 5′ positions.
Moreau et al. (2012) states that the b mutant of
pea, which has pink flowers rather than purple
flowers, resembles some purple-turned-pink
mutants in other plants (Petunia and Gentiana)
that resulted from mutations in the F3’5’H gene.
However, in Glycine max (soybean) plants
mutated in the F3’5’H gene, the flowers are
white, not pink like the flowers of the
corresponding mutants of Petunia and
Gentiana.
Moreau et al. (2012) wonder if the b mutant of
pea, which has pink flowers, might arise from a
loss-of-function mutation in the F3’5’H gene.
Another possibility is that pea, which is a
legume related to soybean, might produce
white flowers when the F3’5’H gene is mutated.
To help clear up this apparent discrepancy, the
authors decided to investigate the b mutant in
pea. They set out to determine which gene is
mutated in the b mutant and to address the role
of the mutated gene on flower pigment
synthesis.
Q5. How many references are cited in
the Introduction? How many of them
would you want to look up to fully
understand the study?
Q6. Do the authors clearly state what
questions they addressed in this
study?
The Results
The results section includes a description of
the research conducted and the results
obtained. Results can be presented as tables,
large datasets, and figures, which can include
graphs, videos, diagrams, and photographs.
In the article by Moreau et al. (2012), the
results are presented in four figures.
Figure 1 shows photographs of the
experimental and control organisms to
demonstrate the phenotypic effect of the b
mutation. Figure 1A shows the pigmentation
pattern of a wild-type pea. Figure 1B shows a
flower from a b mutant of pea that is less
pigmented, and Figure 1C shows an unstable b
mutant in which the gene is active in some
parts of the petal (the darker parts) and inactive
in others (the lighter parts).
Figure 2 shows a chromatographic separation
of pigments extracted from wild-type (A) and b
mutant (B) petals. This instrument detects
chemicals based on their size and chemical
properties. The peaks labeled 611 and 635
represent delphinidin and petunidin, which are
present in the wild-type sample and absent in
Glycine
(soybean)
the b mutant sample. (Panels C and D, and
supplemental Figure 1, show additional assays
that identify the pigment profiles of wild-type
and mutant petals).
Using a gene cloning method, the authors
isolated the F3’5’H gene from wild-type peas
based on homology to the gene from related
plants. The sequence of the pea protein was
compared to those of other plants. The
sequence alignments are shown in
Supplemental Figure 2, and a phylogenic
representation is shown in Figure 3.
The pea enzyme (indicated by a red arrow) is
most closely related to the enzyme found in
another legume, Medicago truncatula,
indicated as CU651565 9.
Finally, the authors show that the b mutant they
identified and characterized has a defect in the
expression of the F3’5’H gene. Figure 4 shows
a characterization of this gene and a cDNA
copy of the mRNA transcript of the gene. The
authors were able to amplify the gene from wild
type and b mutant DNA (lanes 1 and 3). They
were able to amplify the cDNA from wild-type
plants (lane 2) but not b mutant plants (lane 4).
The lower bands show the amplification
product of a control gene (Ago gene, lanes 1
and 3) and cDNA (Ago cDNA, lanes 2 and 4).
The presence of the AGO cDNA band in lane 4
(which lacks the b shows that there is nothing
wrong with this sample; the lack of a band is
specifically because of the lack of the b cDNA,
not a problem with the cDNA in the sample in
general. Lane 5 is another control, which used
no DNA or cDNA; the smudge at the bottom of
the gel comes from residual PCR primers used
in the assay. The lane on the right shows size
standards.
Q7. How straightforward was it to
understand the data presented in the
figures?
Q8. What information do the figure
legends provide?
Q9. Where do you find information
about the experimental methods used?
Q10. What information is presented in
the Supplemental Materials? Why do
you think some information is put in
this supplementary section?
The Discussion
This section summarizes the finding of this
study and interprets how the new information
integrates with previous knowledge.
Here is the key finding of Moreau et al. (2012):
“In this paper, we have presented genetic and
biochemical evidence to show that b mutants
lack a functional F3′5′H gene that results in a
rose-pink flower color due to the presence of
cyanidin- and peonidin-based anthocyanins.”
The first part of the discussion analyzes the
type of mutations the authors identified in the b
mutants, including the nature of the unstable b
mutant shown in Figure 1C.
The next part compares the F3′5′H genes in
legumes. An interesting observation the
authors make is that although the pea gene is
most closely related to the gene from Medicago
truncatula, this plant makes yellow flowers!
This observation points to the complexity of the
biochemical pathway of anthocyanins, as well
as the possibility that a single amino acid
mutation in the Medicago gene might make the
enzyme it encodes non-functional (something
interesting to follow up on!).
The last part of the discussion talks about
flower color in soybean, starting with a
discussion of the F3H gene and mutations of it.
The third-to-last paragraph of the discussion
observes, “However, it is not clear why a w1
encoding a defective F3′5′H gene would
condition white flower color in soybean, when
the pea b mutant and other F3′5′H mutants
derived from purple-flowered wild-type plants
have pink flowers.” The authors point out that
although the soybean study showed that the
w1 mutation lies very close to the F3′5′H gene,
its identity has not been proven to be the
F3′5′H, leaving open the possibility that the w1
mutation is in a different gene. If w1 is not a
mutation of the F3′5′H gene, then we no longer
have the puzzling issue of different phenotypes
for F3′5′H mutations in different species
(something interesting to follow up on!). Thus,
by characterizing the F3′5′H gene in pea, these
authors have contributed some clarity to a set
of puzzling observations and provided a new
hypothesis to follow up on.
Q11. In your own words, how does the
discussion section differ from the
results section?
Q12. Find the places in the discussion
that specifically refer to the results and
the data presented in the supplemental
materials. Is each result discussed
similarly, or are different types of data
discussed differently?
Q13. What information is conveyed by
the final paragraph of the Discussion?
The Materials and Methods
In this section, the authors describe the
sources of the biological materials they used
and the conditions of their growth and the
experimental procedures that they followed.
Additional information about their methods,
including the sequence of the DNA primers
they used for sequencing, can be found in the
Supplemental Materials section of the paper.
Q14. In what way is the font of this
section different from the remainder of
the paper? Why do you think this
information is presented differently?
The Acknowledgments
People who helped out with photography and
plant care and technical assistance are given
thanks here.
Q15. Where do you find guidelines that
specify what kinds of contributions are
necessary for authorship versus those
that are recognized in this section?
The References
This section lists articles that were cited in the
text. Some journals list them in alphabetic
order, and others list them in the order in which
they appear in the text.
Q16. How many references are listed?
How many include one or more of the
authors who contributed to this paper?
Q17. What kinds of articles are listed in
the references section, and what kinds
of sources are not included?
Q18. How would you go about finding
these references? How does reading
the HTML online version of the article
facilitate accessing references?
For more information
For more information about the format and
style of a scientific paper, you might find the
“Instructions for Authors” guidelines interesting.
Every journal provides specific guidelines
about the content and format of the papers it
will publish, and reading the instructions for
authors will help to familiarize you with this
format. The complete Instructions for Authors
for Plant Physiology is found at
http://www.plantphysiol.org/site/misc/ifora.xhtml
.
See also:
American Society of Plant Biologists. (2013). How
to read a scientific paper
http://journalaccess.aspb.org/ReadaSciPaper.
Carpi, A., Egger, A.E., and Kuldell, N.H. (2008).
Scientific communication: Understanding scientific
journals and articles,” Visionlearning Vol. POS‐1
(9).http://www.visionlearning.com/
library/module_viewer.php?mid=158
Pechenik, J. (2013). A Short Guide to Writing about
Biology. Prentice Hall, New Jersey.
Written by Mary E. Williams (2013) for the
American Society of Plant Biologists.
www.aspb.org
The b Gene of Pea Encodes a Defective Flavonoid
39,59-Hydroxylase, and Confers Pink Flower Color1[W][OA]
Carol Moreau, Mike J. Ambrose, Lynda Turner, Lionel Hill, T.H. Noel Ellis, and Julie M.I. Hofer*
Department of Metabolic Biology (C.M., L.H.) and Department of Crop Genetics (M.J.A., L.T.), John Innes
Centre, Norwich NR4 7UH, United Kingdom; and Institute of Biological, Environmental, and Rural Sciences,
Aberystwyth University, Gogerddan Campus, Aberystwyth, Ceredigion SY23 3EB, United Kingdom (T.H.N.E.,
J.M.I.H.)
The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes
corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are
generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher
plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 59 position of the B ring of
flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 39,59-hydroxylase (F3959H), the
enzyme that hydroxylates the 59 position of the B ring, was a good candidate for B. In order to test this hypothesis, we
examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a
variety of lesions in an F3959H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and
petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the
finding that the F3959H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the
B gene of pea corresponds to a F3959H gene. The molecular characterization of genes involved in pigmentation in pea provides
valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that
lead to variation in pigmentation among legume species.
Flavonoids are a large class of polyphenolic second-
ary metabolites that are involved in pigmentation, de-
fense, fertility, and signaling in plants (Grotewold,
2006). Their basic skeleton consists of two six-carbon
aromatic rings, A and B, connected by ring C, a three-
carbon oxygenated heterocycle. Flavonoids are divided
into different subclasses according to the oxidation state
of the C ring, and compounds within each subclass are
characterized by modifications such as hydroxylation,
methylation, glycosylation, and acylation. Anthocya-
nins, for example, the major water-soluble pigments in
flowers, have a fully unsaturated C ring and are usually
glycosylated at position 3. Two important determinants
of flower color are the cytochrome P450 enzymes
flavonoid 39-hydroxylase (F39H; EC 1.14.13.21) and fla-
vonoid 39,59-hydroxylase (F3959H; EC 1.14.13.88). These
hydroxylate the B ring of the anthocyanin precursor
molecules naringenin and dihydrokaempferol, generat-
ing substrates for the production of cyanidin-3-glucoside
and delphinidin-3-glucoside, which can be seen in a
variety of pigmented flowers (Grotewold, 2006).
The study of genetic loci regulating floral pigmen-
tation has a long history, beginning with crosses made
between white- and purple-flowered varieties of gar-
den pea (Pisum sativum; Knight, 1799; Mendel, 1866).
Later crosses made between white-flowered P. sativum
and rose-pink-flowered Pisum arvense defined two
factors conferring flower color as A and B, respectively
(Tschermak, 1911). The white flowers of pea anthocyanin-
inhibition (a) mutants lack anthocyanins and flavones
(Statham et al., 1972), in accordance with the role of A
as a fundamental factor for pigmentation (Tschermak,
1911; De Haan, 1930). Another locus in pea, a2, similarly
confers a white-flowered phenotype lacking anthocya-
nins and other flavonoid compounds (Marx et al., 1989).
It was shown that A and A2 regulate the expression of
genes encoding flavonoid biosynthetic enzymes (Harker
et al., 1990; Uimari and Strommer, 1998), and recently
they were identified as a basic helix-loop-helix (bHLH)
transcription factor and a WD40 repeat protein, respec-
tively (Hellens et al., 2010). They are likely to be com-
ponents of the Myb-bHLH-WD40 transcription factor
complex that regulates flavonoid biosynthesis in all plant
species studied so far (Koes et al., 2005; Ramsay and
1 This work was supported by the European Union FP6 Grain Le-
gumes Integrated Project (grant no. FOOD–CT–2004–506223 to J.M.I.H.)
and by the Department for Environment, Food, and Rural Affairs Pulse
Crop Genetic Improvement Network (grant no. AR0711 to C.M., L.T.,
T.H.N.E., and M.J.A.).
* Corresponding author; e-mail jmh18@aber.ac.uk.
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-
scribed in the Instructions for Authors (www.plantphysiol.org) is:
Julie M. I. Hofer (jmh18@aber.ac.uk) and Mike J. Ambrose (mike.
ambrose@jic.ac.uk).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscrip-
tion.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.197517
Plant Physiology�, June 2012, Vol. 159, pp. 759–768, www.plantphysiol.org � 2012 American Society of Plant Biologists. All Rights Reserved. 759
mailto:jmh18@aber.ac.uk
http://www.plantphysiol.org
mailto:jmh18@aber.ac.uk
mailto:mike.ambrose@jic.ac.uk
mailto:mike.ambrose@jic.ac.uk
http://www.plantphysiol.org/cgi/doi/10.1104/pp.112.197517
Glover, 2005). The gene encoding the Myb component
of this complex in pea, as well as genes at other loci in-
volved in pigment production, such as Clariroseus (B),
Roseus (Ce), and Fuscopurpureus (Cr; Statham et al., 1972),
remain to be identified.
The major anthocyanins found in wild-type pea
lines that contribute to their purple flower color are
delphinidin-, petunidin-, and malvidin-3-rhamnoside-
5-glucosides (Statham et al., 1972). Rose-pink b mutants
(Blixt, 1972) produce a different range of anthocyanins
(pelargonidin-, cyanidin-, and peonidin-3-rhamnoside-
5-glucosides), suggesting that the B gene controls hy-
droxylation of the anthocyanin B ring (Statham et al.,
1972) and encodes a hydroxylase. Pink-flowered mutants
identified in species that are typically purple flowered,
such as Petunia 3 hybrida (Snowden and Napoli, 1998;
Matsubara et al., 2005) and Gentiana scabra (Nakatsuka
et al., 2006), were found to have resulted from the in-
sertion of transposable elements into the gene encod-
ing F3959H. If anthocyanin biosynthesis in pea were to
conform to the enzymatic steps elucidated in other
plant species (Grotewold, 2006), then the activity
missing in b mutants would be predicted to corre-
spond to that of a F3959H.
In soybean (Glycine max), however, the wp locus,
which conditions a change in flower color from purple
to pink (Stephens and Nickell, 1992), was reported to
encode a flavanone 3-hydroxylase (F3H; EC 1.14.11.9;
Zabala and Vodkin, 2005). Furthermore, an insertion/
deletion mutation in a gene encoding a F3959H was
associated with the white-flowered phenotype of the
soybean w1 mutant (Zabala and Vodkin, 2007). These
results suggested that anthocyanin biosynthesis in le-
gumes, or at least in soybean, may differ from that in
other plant species studied, where F3959H mutations
result in pink flowers (Snowden and Napoli, 1998;
Matsubara et al., 2005; Nakatsuka et al., 2006) and F3H
mutations result in white flowers (Martin et al., 1991;
Britsch et al., 1992). More recently, a Glycine soja ac-
cession carrying a w1-lp allele was described as having
pale pink banner petals and a flower color designated
as light purple (Takahashi et al., 2010). Our analysis
here of the b mutant of pea, which is also a legume,
addresses the complexity of these findings in soybean.
Transposon-tagged mutations have facilitated the
isolation of genes involved in anthocyanin biosynthesis
in numerous plant species, and transposon tagging is a
useful technology for gene identification that remains
particularly relevant for species without sequenced ge-
nomes, such as pea. Endogenous retrotransposons and
DNA transposons have been identified in pea, but the
transposition rate of those studied to date has been too
low to be exploited for gene tagging (Shirsat, 1988;
Vershinin et al., 2003; Macas et al., 2007). The identifi-
cation of active DNA transposons usually occurs when
sectors are found on pigmented flowers or seeds. Be-
cause most cultivated pea crop varieties have white
flowers, any chance identification of sectored flowers
in the field is extremely limited. A secondary purpose of
this study was to carry out a screen for sectors on
purple-flowered peas with the aim of identifying an
active transposon.
We generated pink-flowered fast neutron (FN) de-
letion mutants and used these to identify the gene
corresponding to B. Among the pigmentation mutants
we obtained were several new b alleles, including
pink-sectored mutants, which we characterized fur-
ther. Stable pink b mutants were shown to carry a
variety of lesions in an F3959H gene, including com-
plete gene deletions. Analysis of one of these deletion
lines showed that it lacked delphinidin and petunidin,
the major anthocyanins of the progenitor wild-type
pea variety. These results, combined with the finding
that the F3959H gene cosegregates with b in a genetic
mapping population, strongly support our hypothesis
that the pea gene b corresponds to a F3959H.
RESULTS
Generation of New b Mutant Alleles
We used FN mutagenesis to generate pigmentation
mutants in line JI 2822, which is wild type at the flower
color loci A, A2, Albicans (Am), B, Ce, and Cr. The fully
open petals of JI 2822 flowers are nonuniformly pig-
mented (Fig. 1A); the adaxial standard petal is pale
purple, the two wing petals are dark purple, and the
two fused abaxial keel petals are very lightly pig-
mented. The standard and wing petals fade to a blue
purple. The JI 2822 flower is described here as purple
to conform with previous naming conventions (De
Haan, 1930).
M2 and M3 progeny from the mutagenized popu-
lation were screened for flower color variants that
differed from the wild type. Six FN lines were iden-
tified with pale pink standards, rose-pink wing
petals, and lightly pigmented keel petals (Fig. 1B).
Backcrosses to JI 2822 showed that four of these lines,
FN 1076/6, FN 2160/1, FN 2255/1, and FN 2438/2,
carried stable recessive mutations that determined the
pink flower trait. These lines yielded rose-pink F1
progeny when crossed to the b mutant type line, JI
118, confirming that they carried allelic mutations.
Two further lines, FN 2271/3/pink and FN 3398/
2164, were stable rose-pink and allelic to b; however,
sibling individuals carried flowers with pink sectors
on a purple background (Fig. 1C), suggesting they
were unstable at the b locus.
The b mutation is also known to confer paler stem
axil pigmentation than the wild type and paler pod
color in genotypes carrying the purple-podded Pur
allele (De Haan, 1930; Statham et al., 1972). All six FN b
alleles likewise differed from JI 2822 in having paler
axillary rings. No effect on pod color was observed in
the FN alleles, because JI 2822 is a green-podded
genotype (pur). The FN b mutants are described here as
rose pink to incorporate previous conventions
(Tschermak, 1911; De Haan, 1930) yet distinguish them
from cerise-pink ce and crimson-pink cr mutants.
760 Plant Physiol. Vol. 159, 2012
Moreau et al.
The b Mutant Lacks Delphinidin and Petunidin
Methanol-HCl extracts of anthocyanins from the wing
petals of line JI 2822 and a stable pink M3 plant, FN
2271/3/pink, were analyzed using liquid chromatogra-
phy (LC) coupled with mass spectroscopy (MS). Chro-
matograms with two major peaks showed that JI 2822
contained two major anthocyanins (Fig. 2A; 611 and 625
atomic mass units [amu]). MS data averaged across the
peaks indicated that these were anthocyanins isomeric to
delphinidin and petunidin glycosylated with deoxyhex-
ose and hexose sugars (Supplemental Fig. S1). Frag-
mentation of the sugar moieties as mass losses of 146 and
162 amu were consistent with Rha and Glc, respectively.
Fragmentation consistent with the loss of both mono-
saccharide moieties individually was observed, which
suggested that the anthocyanidins delphinidin (303 amu)
and petunidin (317 amu) were monoglycosylated at two
different positions (Supplemental Fig. S1). These results
agree with earlier studies that identified delphinidin-3-
rhamnoside-5-glucoside and petunidin-3-rhamnoside-5-
glucoside among the anthocyanins present in wild-type
pea (Statham et al., 1972).
The peaks indicating glycosylated delphinidin and
petunidin were absent from FN 2271/3/pink samples
(Fig. 2B). A range of ions consistent with glycosylated
cyanidin and peonidin were present in FN 2271/3/pink
and absent from JI 2822 (Fig. 2, C and D). These were
isomeric to cyanidin glycosylated with deoxyhexose and
hexose sugars (595 amu), peonidin glycosylated with
deoxyhexose and hexose sugars (609 amu), and cyani-
din glycosylated with a pentose and two hexose sugars
(743 amu; Fig. 2C). Fragmentation of the sugars attached
to cyanidin (287 amu) as mass losses of 162 , 294, and
456 amu was consistent with a pentose moiety buried
beneath a Glc moiety (Supplemental Fig. S1). No single
loss of 132 amu, expected of an exposed pentose, was
observed. These results confirmed earlier studies that
identified cyanidin-3-sambubioside-5-glucoside among
the anthocyanins present in b mutants (Statham et al.,
1972). Fragmentation of the sugars attached to cyanidin
and peonidin (301 amu) as mass losses of 146 and 162
amu was consistent with cyanidin-3-rhamnoside-5-
glucoside and peonidin-3-rhamnoside-5-glucoside, also
previously identified in b mutants (Statham et al., 1972).
The conversion of cyanidin and peonidin to del-
phinidin and petunidin requires hydroxylation at the
59 position of the B ring of the precursor flavonoids.
Because the products of this conversion were not ob-
served in b mutants, it was presumed that the B gene
controls the hydroxylation of the anthocyanin B ring
(Statham et al., 1972). Our studies confirmed this
conclusion and suggested to us that the gene encoding
F3959H was a good candidate for B.
Isolation of a Pea F3959H Gene from a Purple-Flowered
Wild-Type Plant
We performed PCR on cDNA derived from JI 2822
wing petals using primers based on aligned Medicago
truncatula and soybean F3959H sequences. This yielded
a product encoding a partial open reading frame (ORF)
with extensive sequence similarity to F3959H. We used
primers based on this new pea sequence together with
primers based on the Medicago sequence for adaptor-
ligation PCR (Spertini et al., 1999), which enabled us to
isolate genomic DNA sequences and a larger cDNA
product including a TAG stop codon. Amplification
and sequencing of a single PCR product, using primers
at the 59 and 39 ends of the surmised contig, confirmed
that a 1,548-bp cDNA encoded a cytochrome P450
monooxygenase 515 amino acids long.
A BLASTP search of Medicago genome pseudomo-
lecules (version 3.5) using the chromosome visualiza-
tion tool CViT (http://www.medicagohapmap.org)
identified CU651565_9 on bacterial artificial chromo-
some (BAC) CU651565, a F3959H 515 amino acids in
length, as the most similar sequence, with 89% iden-
tity. The predicted pea protein sequence is 79%, 78%,
and 75% identical to predicted full-length F3959H se-
quences from lotus (Lotus japonicus; LjT34E09.40),
soybean (AAM51564, ABQ96218, and BAJ14024), and
butterfly pea (Clitoria ternatea; BAF49293), respectively.
The soybean sequences are classified as CYP75A17
cytochrome P450s (Nelson, 2009). The Arabidopsis
(Arabidopsis thaliana) sequence most closely related to
the pea F3959H (48% identity) is the cytochrome P450
monooxygenase CYP75B1, encoded by TRANSPAR-
ENT TESTA7 (At5g07990; GenBank accession no.
NP196416). This 513-amino acid protein has been
demonstrated to have F39H activity (Schoenbohm
et al., 2000), and it lies within a separate clade when
compared with other plant F3959H sequences (Fig. 3).
Figure 1. Pea b mutant phenotypes. A,
Purple-flowered wild-type line JI 2822.
B, Rose-pink-flowered b mutant line
FN 2271/3/pink. C, Unstable b mutant
line FN 2271/3/flecked with rose-pink
sectors on a purple background.
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A 3,231-bp genomic DNA sequence was obtained
from PCR products amplified from JI 2822 DNA using
primers spanning the cDNA sequence and adaptor-
ligation PCR products corresponding to the promoter
and 39 untranslated region (GenBank accession no.
GU596479). The position of a single 530-bp intron, 915
bp downstream of the ATG start codon, was deter-
mined by alignment of the genomic DNA and cDNA
sequences. A single intron is predicted in Medicago
CU651565_9 at the same position, but in other legumes,
such as soybean (Zabala and Vodkin, 2007) and lotus
(LjT34E09.40), two introns are reported or annotated. In
both these species, the position of the predicted second
intron is coincident with the position of the pea intron.
The first introns are predicted in different positions, 331
and 348 bp downstream of their ATG, for lotus and
soybean, respectively.
Genetic Mapping of F3959H Reveals Cosegregation with b
A cleaved-amplified polymorphic sequence (CAPS)
marker for F3959H that distinguished the JI 15 and JI
73 alleles was generated by TaqI cleavage of the PCR
products amplified from genomic DNA. Cosegregation
of the CAPS marker with b was tested directly in a JI 15 3
JI 73 recombinant inbred population of 169 individuals,
because JI 73 carries the recessive b allele. JI 73 also carries
k, the homeotic conversion of wing petals to keel petals,
and d, the absence of pigmentation in foliage axils,
whereas JI 15 carries ce, an independent crimson-pink
flower trait. The b, ce double mutant is almost white,
so single and double mutants can be distinguished easily,
except in a k mutant background, where only the pale
standard petal gives a clue to flower color. The genotypes
Figure 2. LC-MS analysis of anthocyanins present in the wild type and
b mutant lines. A, Extracted ion chromatograms showing the summed
intensities of ions with masses corresponding to delphinidin and
petunidin, each glycosylated with Rha and Glc, present in JI 2822.
These masses are m/z = 611 (delphinin) and m/z = 625 (petunin). B,
Masses corresponding to delphinin and petunin absent from FN 2271/
3/pink. A and B are plotted to the same scale. C, Extracted ion chro-
matograms showing the summed intensities of three alternative an-
thocyanin ions, with masses based on glycosylated cyanidin (m/z =
743, m/z = 595) and peonidin (m/z = 609), present in line FN 2271/3/
pink. D, Masses corresponding to cyanin and peonin absent from JI
2822. C and D are plotted to the same scale. Chromatographic peaks
are annotated with m/z of the mass responsible for the peak.
Figure 3. Phylogenetic analysis of cytochrome P450 sequences. The
optimal neighbor-joining tree derived from the multiple sequence
alignment in Supplemental Figure S2 is drawn to scale, with the sum of
branch lengths = 4.7. The Jones-Taylor-Thornton amino acid substitution
model was used in phylogeny construction, and the scale bar indicates
the number of amino acid substitutions per site. Percentage support
for 1,000 bootstrap replicates is shown at the branch points. Labeled
lines show GenBank accession numbers as follows: LjT34E09_40,
L. japonicus; BAJ14024, soybean; BAF49293, C. ternatea; ADW66160,
P. sativum; CU651565_9, M. truncatula; ABH06585, Vitis vinifera;
BAE86871, G. scabra; P48418, Petunia 3 hybrida; CU651565_21, M.
truncatula; NP_001064333, Oryza sativa; NP196416, Arabidopsis;
ABH06586, V. vinifera; BAB83261, soybean; NP182079, Arabidopsis;
NP775426, Rattus norvegicus.
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at b and ce are particularly difficult to distinguish in a k, d
background, where axillary pigmentation is also absent.
For these reasons, the cosegregation analysis was re-
stricted to a subset of 160 of the 169 recombinant inbred
lines. The b phenotype cosegregated exactly with the JI 73
F3959H CAPS marker lacking a TaqI restriction enzyme
site (b:B = 71:89; x2 = 2.0, not significant), consistent with
our hypothesis that this F3959H identifies a single gene
that corresponds to B.
Identification of Lesions in F3959H Alleles from
Pink-Flowered b Mutants
In order to provide further evidence of a corre-
spondence between the pea gene encoding F3959H and
B, we sequenced alleles from known mutants. The b
mutant type line, JI 118, carries a single nucleotide
polymorphism 332 bp downstream of the ATG. This
G/A transition would result in a single amino acid
change, G111E (Supplemental Figs. S2 and S3). Line JI
73, the b mapping parent used above, carries a 23-bp
deletion in the ORF, 291 bp from the ATG start. This
deletion would introduce a change in the reading
frame at position 98, resulting in the inclusion of 29
residues unrelated to the wild type followed by a
premature stop codon (Supplemental Fig. S3). PCR
analysis using primers that spanned the F3959H gene
showed that lines FN 2160/1, FN 2255/1, and FN
2438/2 as well as the stable pink line FN 2271/3/pink
all carry complete gene deletions (Supplemental Fig.
S4). FN 1076/6 contains a genomic rearrangement that
is consistent with a reciprocal break and join between
the F3959H gene and a predicted Ogre retroelement
(Neumann et al., 2003). The 59 segment of the Ogre el-
ement lies 1,330 bp downstream of the F3959H start
codon, whereas the 39 segment lies upstream of position
1,330 at the 39 end of the F3959H gene (Supplemental
Fig. S4).
Characterization of an Unstable Pink-Sectored b Mutant
Unstable b mutants occurred in the M3 families FN
2271/3/flecked (Fig. 1C) and FN 3398/2164. It was
found that sectored pink M3 siblings gave rise to sec-
tored or stable pink M4 progeny, whereas stable pink
M3 plants gave rise to stable pink M4 progeny only.
Wild-type purple M3 siblings gave rise to either stable
wild type, or a mix of stable wild type and stable pink,
or a mix of stable wild type, stable pink, and sectored
pink M4 progeny. Sectored pink M4 progeny gave rise
to sectored or stable pink M5 plants in the following
generation. In order to study this instability further,
PCR analysis was carried out on individual flowers
and progeny plants of line FN 2271/3/flecked/8.
Primers 39pinkS1 and 39pinkS2comp amplified 693
bp of genomic DNA and reported on exon 1 and the
intron of the F3959H gene. Primers 39pinkS2 and 39extR
amplified 683 bp of genomic DNA or cDNA and
reported on exon 2. Both pairs of primers were used in
conjunction with control primers designed to a pea
Argonaute gene, which verified that PCR amplification
had occurred, even in the absence of a F3959H PCR
product. Genomic DNA and cDNA were prepared
from the purple petals of a JI 2822 wild-type flower
and from the petals of an entirely pink flower on a FN
2271/3/flecked/8 plant that carried purple/pink-
sectored flowers at other nodes. PCR using primers
39pinkS2 and 39extR showed the presence of the
F3959H gene in JI 2822 and pink flower FN 2271/3/
flecked/8 genomic DNA samples; however, cDNA
amplification occurred in line JI 2822 only, suggesting
that the F3959H gene was present but not expressed in
the entirely pink FN 2271/3/flecked/8 flower (Fig. 4).
Stable pink-flowered M4 progeny were grown from
seed set on that entirely pink FN 2271/3/flecked/8
flower. When these were analyzed by PCR, exon
1 and exon 2 of F3959H failed to amplify from genomic
DNA, suggesting that the gene was deleted in these
progeny, as was observed previously in the stable
pink-flowered line FN 2271/3/pink.
DISCUSSION
The early part of anthocyanin biosynthesis from
chalcone to anthocyanidin is well conserved in higher
plants and has been studied in detail (Grotewold,
2006). One of the key enzymes responsible for blue-
purple coloration in flower petals is F3959H, which
catalyzes hydroxylation at the 39 and 59 positions of
the B ring of naringenin and dihydrokaempferol,
yielding flavanone and dihydroflavonol precursors
of the chromophore delphinidin (Grotewold, 2006;
Yoshida et al., 2009). Flowers that lack this enzyme,
Figure 4. F3959H gene expression in unstable b mutant line FN 2271/
3/flecked. PCR amplification of F3959H and Argonaute (Ago) genes
from JI 2822 genomic DNA (lane 1), JI 2822 cDNA (lane 2), FN 2271/
3/flecked/8 genomic DNA (lane 3), FN 2271/3/flecked/8 cDNA (lane
4), and no-DNA control (lane 5) is shown. Lane 6 shows 100-bp
markers. The top Ago band represents PCR amplification products
spanning two introns from genomic DNA, and the bottom Ago band
represents PCR amplification products (without introns) from cDNA.
The F3959H primers do not flank an intron; therefore, F3959H PCR
products from genomic DNA and cDNA are the same size.
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such as rose (Rosa hybrid) and carnation (Dianthus
caryophyllus), contain only cyanidin and/or pelargo-
nidin chromophores, so their natural coloration is re-
stricted to yellow, pink, and red but not purple or blue.
Flower color also can be affected by pH, the presence
of copigments, and whether the anthocyanidin chro-
mophores are polyacetylated or held in metal com-
plexes (Yoshida et al., 2009). For example, hydrangea
(Hydrangea macrophylla) sepals can be red, mauve,
purple, violet, or blue, yet only one anthocyanin, del-
phinidin 3-glucoside, is present. It has been proposed
that the anthocyanin and copigments in hydrangea
sepals are held in a metal complex and that color de-
pends on the concentrations of these components and
the pH conditions (Kondo et al., 2005). In wild-type
pea, the F3959H gene is intact and F3959H activity
produces delphinidin-based anthocyanidins, which
confer a purple flower color. In this paper, we have
presented genetic and biochemical evidence to show
that b mutants lack a functional F3959H gene that re-
sults in a rose-pink flower color due to the presence
of cyanidin- and peonidin-based anthocyanins. The
presence of these latter 39-hydroxylated compounds in
b mutants suggests that a F39H exists in pea, contrary
to previous conclusions (Statham et al., 1972).
Lesions Present in F3959H Alleles
Plant P450 monooxygenases have not been charac-
terized structurally because they are extremely insol-
uble when purified; however, membrane-associated
mammalian P450s have been studied by homology to
the crystal structure of a soluble bacterial P450 (Ferrer
et al., 2008). P450s have only three absolutely con-
served residues: a Cys that serves as a ligand to the
heme iron, and an EXXR motif that is thought to sta-
bilize the core around the heme (Werck-Reichhart and
Feyereisen, 2000). The Cys lies within the P450 con-
sensus sequence FXXGXRXCXG in the heme-binding
loop, corresponding to FGAGRRICAG in the pea
F3959H (Supplemental Fig. S2). Another consensus se-
quence, A/GGXD/ETT/S, corresponds to a proton-
transfer groove, and this corresponds to AGTDTS in
the pea F3959H (Supplemental Fig. S2). The G111E
mutation in the b type line, JI 118, does not occur in
these conserved motifs, but the change in size and
charge at this residue presumably affects protein
function. Alignment of the pea F3959H sequence with
homologous plant proteins (National Center for Bio-
technology Information BLASTP) shows that substi-
tutions occur at the G111 residue; however, none of the
substitutes are charged residues, supporting our pro-
posal that G111E is a detrimental change.
Line JI 73 carries a b allele with a spontaneous 26-bp
deletion that is predicted to encode a truncated version
of the F3959H protein. At the 39 end of the 26-bp de-
leted sequence, there is a 10-bp motif, ATTTCTCAAA,
that is repeated at the 59 end of the deletion break
point (Supplemental Fig. S3). This repeat pattern sug-
gests that this stable b allele may have arisen from a
spontaneous deletion event as a result of recombina-
tion and unequal crossing over. The same 26-bp dele-
tion was found in lines JI 17, JI 132, and JI 2160 in the
John Innes Pisum germplasm collection.
A genomic rearrangement consistent with a trans-
location event involving a retroelement was evident in
line FN 1076/6. Here, sequencing showed that a break
occurred in the F3959H gene, between nucleotides 1,329
and 1,330 downstream of the ATG, but we do not know
whether the two fragmented portions of the F3959H gene
remain on the same chromosome (Supplemental Fig. S4).
The 59 end of the genic disjunction was 95% identical
to nucleotides 77,728 to 78,111 of a Ty3-gypsy Ogre-
like retroelement (Neumann et al., 2003) identified in
pea BAC clone JICPSV-297I9, whereas the sequence at
the 39 end of the disjunction was 95% identical to nu-
cleotides 77,213 to 77,726 of the same retroelement.
This indicates that a break occurred in the Ogre ele-
ment between nucleotides 77,726 and 77,728 and that
nucleotide 77,727 was missing from this copy of Ogre
or was lost during the rearrangement. The presence
of this retroelement does not necessarily implicate it in
the mechanism of translocation but more likely reflects
the abundance of the Ogre retroelement family. Data
from 454 sequencing of cv Carerra estimated that
copies of Ogre represent up to 33% of the pea genome
(Macas and Neumann, 2007).
We gathered evidence of independent, recurring,
spontaneous deletion events derived from unstable b
alleles carried by lines FN 2271/3/flecked and FN
3398/2164. These sectored flowers carried an F3959H
gene, presumably in nonepidermal tissue where it is
not expressed, but repeatedly gave rise to stable pink
deletion alleles in their progeny (Fig. 4). One possible
explanation of these unstable b alleles is that FN 2271
M1 seed carried both a deletion of the b gene and a
rearrangement of the chromosome carrying the wild-
type B allele. This rearranged chromosome would be
prone to the generation of acentric fragments that
would fail to segregate properly at mitosis, generating
sectors with a haploinsufficiency for many loci, in-
cluding b. Individuals with the unstable phenotype
would give rise to pink homozygous deletion progeny
(with a wild-type karyotype). They would also gen-
erate progeny that are homozygous or heterozygous
for the unstable chromosome, but the transmission of
this unstable chromosome may be inefficient, or those
that are transmitted efficiently may be selected for
stability. In this scheme, the pink-flowered FN 2271
mutants derive from a simple deletion segregating in
the population and the instability is not specifically
associated with the b locus.
Alternatively, the unstable alleles at the b locus in the
FN 2271 lineage may be prone to deletion, perhaps be-
cause of the action of a nearby transposon activated in
the FN mutagenesis. Deletion of the b gene at one allele
would be masked by the presence of the other, wild-type
B allele, but the presence of such a deletion would reveal
subsequent deletions of the B allele, which would be
seen as pink sectors. In this scheme, deletion of b is not
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generated directly by mutagenesis and the instability is
associated specifically with the b gene. Pink flowers of
this type could be indicators of a captured insertion el-
ement, but in no case did we find a stable pink mutant
with the F3959H gene detectably present, even when
these derived from seed set from an entirely pink flower
on an unstable plant where the gene, but not the tran-
script, had been detected by PCR.
F3959H Homologs in Legumes
Cytochrome P450s are one of the largest enzyme
families in plants. A search of annotated Medicago
pseudomolecules (http://www.medicagohapmap.org)
reveals 142 F3959H homologs (BLASTP, P . 1e-40), with
approximately one-third of these located on chro-
mosome 5. Gene clusters are found in many other
organisms, and in Medicago, BACs containing five or
more homologous ORFs occurred on chromosomes 2
(AC130800), 3 (AC145061), 5 (FP102223 and AC137079),
and 6 (AC157489), although some of these may be
pseudogenes. The soybean genome contains 712 cyto-
chrome P450s, of which 380 are denoted pseudogenes
(Nelson, 2009). Medicago BAC CU651565 carrying
CU651565_9, the most similar intact ORF to pea F3959H,
is unanchored in version 3.5 of the Medicago genome
pseudomolecules; therefore, we were unable to gain
any further evidence of orthology by analyzing collin-
earity with b gene-flanking markers. In the previous
version of annotated Medicago pseudomolecules (ver-
sion 3.0), BAC CU651565 was located on chromosome
3, which is syntenic with pea linkage group III, where b
maps.
Another predicted Medicago F3959H gene, CU651565_21
(Fig. 3), lies only 52 kb from CU651565_9. The coding
sequence of CU651565_21 corresponds to a protein
522 amino acids in length, which is anomalous com-
pared with the lengths of related F3959H sequences
(Supplemental Fig. S2). Multiple sequence alignment
(Supplemental Fig. S2) suggests that CU651565_21
may in fact correspond to a 506-amino acid protein
that would be 63% identical to CU651565_9 and 62%
identical to the pea F3959H. An alternative intron-
splicing model derived from ORFs annotated in
Medicago pseudomolecule version 3.0 is presented
(Supplemental Fig. S5).
It is not clear whether the closest related lotus and
soybean sequences are orthologous to the pea F3959H,
because they have two introns; therefore, they are
structurally dissimilar to the pea and Medicago genes.
The Petunia 3 hybrida F3959H also has two introns,
whereas the G. scabra F3959H has one, indicating that
intron number is a variable feature of these genes.
Diversity of exon-intron structure has been noted
among genes encoding P450 enzymes, with multiple
gains and losses in their evolutionary history (Werck-
Reichhart and Feyereisen, 2000).
The amino acid sequence of CU651565_9, 89% iden-
tical to pea F3959H, is the closest match; however, the
yellow (rather than purple/blue) pigmented flowers of
M. truncatula suggest that there are differences in an-
thocyanin biosynthesis between these two species. All
of the conserved P450 motifs are intact in CU651565_9,
but a comparison with homologous sequences from
other plant species shows differences that may be sig-
nificant. For example, residue Phe-350, which is Leu or
Val in aligned homologs (Supplemental Fig. S2), may
disrupt F3959H function in M. truncatula. In support of
this possibility, overexpression of the Myb transcription
factor LAP1 in M. truncatula induced anthocyanin pig-
ments, which were identified as glycosylated cyanidins
and pelargonidins but not delphinidins (Peel et al.,
2009). The absence of glycosylated delphinidins in these
transgenic plants suggests a defect in F3959H activity,
especially because glycosylated delphinidins were ob-
served in white clover (Trifolium repens) overexpressing
LAP1 (Peel et al., 2009).
Three soybean sequences (AAM51564, ABQ96218,
and BAJ14024) are all 78% identical to pea F3959H;
however, they are themselves nonidentical. ABQ96218
(Zabala and Vodkin, 2007) and AAM51564 (from cv
Chin-Ren-Woo-Dou) are 99% identical and 509 and
508 amino acids long, respectively. They encode a
CYP2 subfamily cytochrome P450, also classified as
a CYP75A17 cytochrome P450 (Nelson, 2009), at lo-
cus Glyma13g04210 on linkage group F of soybean
(http://soybase.org). ABQ96218, originating from cv
Lee 68 and cloned from the Williams isoline L79-908,
carries a G305D amino acid substitution (Zabala and
Vodkin, 2007) in the conserved P450 proton-transfer
groove motif that would likely render this allele
nonfunctional (Supplemental Fig. S2). BAJ14024
(Takahashi et al., 2010) is a predicted F3959H from
soybean cv Clark, 509 amino acids long, with in-
variant conserved motifs and 99% identical to both
ABQ96218 and AAM51564.
Flower Pigmentation in Pea and Soybean
Soybean is believed to have been domesticated from
purple-flowered G. soja (Takahashi et al., 2010). Studies
of the standard (banner) petals of purple-flowered
soybean cultivars show that these have a different
sugar moiety at the 3 position of the C ring of their
anthocyanidins compared with pea: the primary an-
thocyanins detected in soybean cv Clark (W1W1 w3w3
W4W4 WmWm TT TdTd) and cv Harosoy (W1W1
w3w3 W4W4 WmWm tt TdTd) were malvidin, delphi-
nidin, and petunidin 3,5-di-O-glucoside and delphini-
din 3-O-glucoside (Iwashina et al., 2008), whereas
delphinidin and petunidin-3-rhamnoside-5-glucoside
were the major anthocyanins found in the wing petals
of pea line JI 2822 in this study, consistent with pre-
vious studies on line L 60 of pea (Statham et al., 1972).
As the intensity of coloration in pea petals indicates
(Fig. 1), the concentration of total anthocyanins in
standard petals is less than in wing petals of pea at all
stages of flower development (Statham and Crowden,
1974), whereas soybean flowers often have wing petals
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that are less intensely pigmented than their standard
petals.
The Wp gene of soybean lies on linkage group D1b,
corresponding to chromosome 2 (http://soybase.org).
The wp allele is reported to contain a 5,722-bp CACTA
transposable element in intron 2 of a F3H gene, F3H1,
with down-regulated expression (Zabala and Vodkin,
2005). A null mutation would result in a lack of the
substrates dihydromyricetin, dihydrokaempferol, and
dihydroquercetin required for conversion into antho-
cyanins (Grotewold, 2006; Iwashina et al., 2008);
therefore, a null mutant would be expected to have
white flowers and, indeed, white-flowered mutants
have been observed in other plant species (Martin
et al., 1991; Britsch et al., 1992). Analysis of a wp ge-
notype obtained by back-crossing to soybean cv Loda
showed that the wp line had a low flavonoid content:
9% of the total flavonol glycosides, no detectable
kaempferol 3-O-glucoside, and 28% of dihydro-
flavonols compared with cv Clark (Iwashina et al.,
2008). The presence of dihydroflavonols indicates that
F3H activity occurs in the wp mutant, suggesting that it
is not a null allele. Alternatively, if the CACTA ele-
ment insertion does render F3H1 null, a second F3H
gene, F3H2, may be functional (Zabala and Vodkin,
2005).
Although the presence of anthocyanins in the wp
mutant can be explained by the considerations above,
the pale pink coloration (instead of pale purple) re-
mains unexplained. Many factors such as copigments
and vacuolar pH could influence soybean flower color,
but the presence of an additional defective pigmenta-
tion gene, such as the ABQ96218 allele of F3959H, for
example, would also cause pink flower color. A com-
parison of flower color and flavonoid content in
available Wp and wp near-isogenic lines (Iwashina
et al., 2008) and cosegregation analysis of F3H1 and wp
would help to confirm which structural genes were
defective.
The soybean w1 gene on chromosome 13 confers
white flower color; accordingly, no HPLC peaks cor-
responding to anthocyanins were observed in a Clark-
w1 near-isogenic line (L63-2373, w1w1, w3w3, W4W4,
WmWm, TT, TdTd; Iwashina et al., 2007). However, it is
not clear why a w1 encoding a defective F3959H gene
would condition white flower color in soybean, when
the pea b mutant and other F3959H mutants derived
from purple-flowered wild-type plants (Snowden and
Napoli, 1998; Matsubara et al., 2005; Nakatsuka et al.,
2006) have pink flowers. Genetic linkage analysis of an
F2 population segregating for w1 showed that 12
white-flowered individuals out of 39 F2 progeny car-
ried an F3959H allele containing a tandem repeat in-
sertion that would result in premature termination of
the protein (Zabala and Vodkin, 2007). This linkage
evidence is consistent with w1 being less than 1.1
centimorgan (Kosambi, 1944; Allard, 1956) from the
tandem repeat-containing F3959H gene but with a high
SE: the F3959H homozygotes in the purple flower class
were not shown to be W1 homozygotes by progeny
testing, and the population size is small. Thus, it is not
clear that a mutated F3959H gene conditions white
flower color in soybean.
One possibility is that w1 is a separate nonfunctional
pigmentation locus, distinct from, but tightly linked to,
the F3959H gene. This w1 locus is predicted to be
functional in a G. soja line carrying the w1-lp allele,
which has pale pink banner petals (Takahashi et al.,
2010), and nonfunctional in Clark-w1. A cross between
these two lines produced purple-flowered F2 progeny
at a frequency of 0.9% (Takahashi et al., 2010), which is
consistent with recombination between a distinct w1
gene and the F3959H gene. Soybean orthologs of genes
encoding components of the Myb-bHLH-WD40 tran-
scription factor complex that regulates anthocyanin
biosynthesis (Koes et al., 2005; Ramsay and Glover,
2005), such as a and a2 (Hellens et al., 2010), have not
yet been identified. These are good candidates for the
proposed F3959H-adjacent w1 gene.
Pigmentation loci in pea, which have been studied in
crosses for more than 100 years (Mendel, 1866;
Tschermak, 1911), represent historic anchor markers
that will aid comparative genomics between legume
species as more physical maps are generated from
sequenced genomes. Further biochemical studies,
combined with genetic and genomic analyses, will
help to elucidate the differences in anthocyanin bio-
synthesis that lead to variation in pigmentation among
legume crop species such as soybean as well as im-
portant legume forage species such as alfalfa (Medicago
sativa) and clover.
MATERIALS AND METHODS
Plant Material
The garden pea (Pisum sativum) type line for b, JI 118, also known as WBH
22 (Blixt, 1972), multiple marker line JI 73 (genotype b, also known as WBH
1238), multiple marker line JI 15 (genotype B, also known as WBH 1458), F13
recombinant inbred mapping population JI 15 3 JI 73, and all FN mutant lines
were obtained from the John Innes Pisum Germplasm collection. Plants were
grown in 16-h daylength in John Innes No. 1 compost with 30% extra grit.
DNA was prepared from leaves according to Vershinin et al. (2003), and RNA
was prepared from flowers according to Hofer et al. (2009).
Mutagenesis
A total of 1,400 seeds of line JI 2822 were subjected to 20 Gray FN irradiation
from a 252Cf source at Oak Ridge National Laboratory. Irradiated M1 plants
were self fertilized, and M2 families of up to four plants were screened for
variant flower color phenotypes. Rose-pink mutants were backcrossed to JI
2822 to generate lines FN 1076/6, FN 2160/1, FN 2255/1, FN 2438/2, FN
2271/3/pink, and FN 3398/2164. These stable pink lines segregated purple:
pink in a 3:1 ratio after backcrossing, indicating that the pigmentation muta-
tions were recessive.
LC-MS
Purple (JI 2822) and pink (FN 2271/3/pink) wing petal tissue was harvested from
10 fully open flowers, ground in liquid N2, and stored in methanol at 220°C. Sample
aliquots of 10 mL containing 300 mg of tissue in methanol and 0.1 M HCl were
analyzed by LC-MS using a Surveyor HPLC apparatus attached to a DecaXPplus
ion-trap mass spectrometer (Thermo Fisher). Anthocyanins were separated on a
766 Plant Physiol. Vol. 159, 2012
Moreau et al.
(http://soybase.org
100- 3 2-mm, 3-mm Luna C18(2) column (Phenomenex) using the following gra-
dient of methanol (solvent B) versus 2 mM trifluoroacetic acid in water (solvent A),
run at 230 mL min21 and 30°C: 0 min, 2% B; 40 min, 70% B; 41 min, 2% B; 50 min,
2% B. Anthocyanins were detected by UV A520 and by positive electrospray ioni-
zation MS. Spray chamber conditions were 50 units of sheath gas, 5 units of aux-
iliary gas, 350°C capillary temperature, and 5.2-kV spray voltage. In order to
investigate the structure of anthocyanins, data-dependent secondary fragmentation
(MS2) spectra were collected at an isolation width of mass-to-charge ratio (m/z) = 4.0
and 35% collision energy.
Isolation of Pea F3959H cDNA and Genomic DNA
Total RNA was extracted from JI 2822 wing petals using the Qiagen RNeasy
Plant Mini kit. DNA was removed from RNA samples by digestion with DNA-
free DNaseI (Ambion) in buffers according to the manufacturer’s protocol. Two
micrograms of RNA was reverse transcribed with SuperScript reverse tran-
scriptase (Invitrogen) from an oligo(T) primer in a 20-mL reaction. Amplifi-
cation of a F3959H cDNA fragment from pea was achieved using 1 mL of 1:20
diluted first-strand cDNA in 20-mL PCRs containing 0.25 mM primers
mtF35HF1 and mtF35HR2 (Supplemental Table S1) for 35 cycles with an
annealing temperature of 62°C. Products were separated by electrophoresis on
a 1% agarose gel in 13 Tris-borate/EDTA buffer. A 794-bp sequence obtained
from this fragment was used to design additional primers for the amplification
of 3,231-bp genomic DNA using successive rounds of adaptor ligation PCR
(Spertini et al., 1999). The genomic DNA sequence was used to design primers
pinkmtF1 and 39extR for the amplification of a 1,595-bp cDNA clone, minus
the ATG start codon and extending 50 bp beyond the TAG stop codon. This
was cloned into a Topo4 vector (Invitrogen).
Mutation Analysis
Genomic DNA from JI 2822 and FN mutant lines was analyzed using pairs
of primers that spanned the F3959H gene sequence in order to determine the
size of deletion alleles (Supplemental Table S1). Primers PsAGO1 and
PsAGO2, flanking introns 19, 20, and 21 of a pea Argonaute1 cDNA clone
(accession no. EF108450), were included in the reactions as internal controls.
For the analysis of unstable lines, wing petal cDNA and genomic DNA from JI
2822, plant FN 2271/3/flecked/8, and its progeny were analyzed. Touch-
down PCR was performed using 250 nM primers 39pinkS2 and 39extR, 250 mM
deoxyribonucleotide triphosphates, and 1 unit of Taq polymerase in a 10-mL
volume of PCR buffer. Primers PsAGO1 and PsAGO2 were included in the
reactions as internal controls. Components were denatured at 95°C for 180 s,
before being subjected to one cycle of 94°C for 45 s, 62°C for 45 s, and 72°C for
90 s, followed by 10 further cycles with the annealing temperature 1°C lower
at each cycle. Twenty-nine further cycles of 94°C for 45 s, 50°C for 45 s, and 72°
C for 90 s were terminated at 72°C for 300 s. Reactions were held at 10°C for
300 s prior to analysis by agarose gel electrophoresis (Supplemental Fig. S4;
Supplemental Table S1).
Genetic Mapping
A CAPS marker for F3959H was generated by TaqI cleavage of the 363- and
340-bp PCR products amplified from 100 ng of genomic DNA from parental
lines JI 15 and JI 73, respectively, using primers pinkmtF1 and psf35hF2comp.
Reactions contained 250 nM primers, 250 mM deoxyribonucleotide triphos-
phates, and 1 unit of Taq polymerase in a 20-mL volume of PCR buffer.
Components were denatured at 94°C for 120 s, cycled through 94°C for 30 s,
55°C for 60 s, and 72°C for 120 s for 35 cycles, and finally incubated at 72°C for
5 min. Cleavage products of 293 bp from line JI 15 and 340 bp from line JI 73
were separated on a 2% agarose gel. Cosegregation of b with the 340-bp
F3959H CAPS marker was tested for 160 lines out of 169 in total at the F13
generation of a recombinant inbred population derived from the cross JI 15 3
JI 73. A total of 71 lines were b/b and carried the 340-bp marker, and 89 in-
dividuals were B and carried the 293-bp marker.
Sequencing
Sequencing was performed using the BigDye Terminator version 3.1 cycle
sequencing kit (Applied Biosystems) at the John Innes Centre Genome Lab-
oratory. Genomic DNA sequence was obtained from line JI 2822 using the
primers listed in Supplemental Table S1. A 3,232-bp overlapping DNA se-
quence contig was generated using the program BioEdit (http://www.mbio.
ncsu.edu/bioedit/bioedit.html). Overlapping DNA sequence contigs from b
mutant lines JI 118, JI 73, and FN 1076/6 and cDNA sequences from lines JI
2822, JI 118, JI 73, and FN 1076/6 were obtained in the same way.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: JI 2822 F3959H cDNA se-
quence, GU596478; JI 2822 F3959H genomic DNA sequence, GU596479.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Ion fragmentation analysis of anthocyanins pres-
ent in the wild type and b mutant lines.
Supplemental Figure S2. F3959H sequence analysis.
Supplemental Figure S3. Sequence characterization of mutant b alleles.
Supplemental Figure S4. Characterization of mutant b alleles by PCR.
Supplemental Figure S5. Proposed splicing model for Medicago gene
CU651565_21.
Supplemental Table S1. Primers used for PCR and sequencing.
ACKNOWLEDGMENTS
We thank Andrew Davis for photography, Ruth Pothecary and Hilary Ford
for plant care, and a Nuffield scholarship student, Priyanka Tharian, for
technical assistance.
Received March 22, 2012; accepted April 3, 2012; published April 6, 2012.
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Moreau et al.
- CSPage1.ppt
- CSpage2_NW
- CSpage3_NW
- CSpage4_NW
- CSpage567_NW
- b gene of pea