Download Detection of a True Breeding Homeotic Gene Mutant Pps

Journal of Heredity 2007:98(4):373–377
doi:10.1093/jhered/esm037
Advance Access publication July 9, 2007
ª The American Genetic Association. 2007. All rights reserved.
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Detection of a True Breeding Homeotic
Gene Mutant Pps-1 with Partially
Petaloid Sepals in Opium Poppy (Papaver
somniferum L.) and Its Genetic Behavior
OM PRAKASH DHAWAN, MUKESH KUMAR DUBEY,
AND
SUMAN PREET SINGH KHANUJA
From the Division of Genetics and Plant Breeding, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP,
Lucknow 226015, India (Dhawan and Dubey); and the Division of Genetic Resources and Biotechnology, Central Institute of
Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India (Khanuja).
Address correspondence to O. P. Dhawan at the address above, or e-mail: [email protected].
A spontaneous true breeding homeotic gene mutant Pps-1
with distinct partial petaloid sepals was detected in the population of downy mildew (DM)–resistant elite accession I-14
during our studies for the identification of disease resistance
sources in opium poppy. The trait was found to be stable
and inherited truly in the subsequent generations. Genetic
studies were carried out through systematic reciprocal crosses
with the parental wild-type genotype I-14, and segregation pattern of phenotypic characteristics in F1 and F2 populations
clearly indicated single recessive nuclear gene control of the
mutant character. The studies have demonstrated that the mutant phenotype is due to mutations at the Pps-1 locus that possibly corresponds to B-class function (according to ABC model)
with negative control function. The mutant Pps-1 being singlewhorl homeotic mutant might greatly help in providing insight
into mechanisms of flower development in opium poppy.
The information on floral developmental genetics has exploded in recent years (Albert et al. 2005) and extensive documentation of the developmental morphology of Arabidopsis
flowers has facilitated the linkage of genes and morphology.
These studies served as the benchmark by which mutant
defects have been identified and gene functions defined (Coen
and Meyerowitz 1991; Weigel 1995; Ma and dePamphilis
2000). Parallel studies in Arabidopsis and Antirrhinum identified
several homeotic gene mutations in which dramatically altered
identity of specific floral organ types were found and in which
conversions of one organ identity to another were characterized (Ma and dePamphilis 2000; Theissen et al. 2000). Both
these model organisms have been used to study the role of
homeotic genes in regulating floral organ development. Specific groups of homeotic genes are expressed in successive
pairs of whorls. These groups of genes interact to specify floral organ identity, such that the A function alone promotes
sepal development, A and B petal development, B and C stamen development, and C carpel development (Coen and
Meyerowitz 1991; Soltis et al. 2002; Kramer and Jaramillo
2005). A recent study of the expression of floral identity genes
in poppies, buttercups, and their relatives have shed more
light on the evolutionary origin of petals (Baum and Whitlock
1999; Kramer et al. 2003).
Opium poppy (Papaver somniferum L.) is one of the oldest
medicinal plants cultivated for life-saving alkaloids such as
morphine and codeine (Weid et al. 2004). Morphine is a preferred analgesic for cancer patients and codeine is cough depressant (Kapoor 1995). Besides these 2, opium poppy
produces approximately 80 more alkaloids belonging to various tetrabenzylisoquinoline-derived classes (Weid et al.
2004). Seeds obtained from the capsules are edible and used
in confectionary (Nergiz and Otles 1994), and fatty oils in
seeds are used for culinary and pharmaceutical purposes
(Krist et al. 2005). It is an annual winter herb and the plants
are highly variable in morphological characteristics. In addition to these, many spontaneous mutants with altered floral
structures like complete conversion of petals into stamens
and stamens into petals has also been identified (Belyaeva
and Nevkrytaya 1979). All these spontaneous mutants suggest that there is a considerable mutability of genes controlling floral behavior. Papaver somniferum and several of its
relatives are currently studied in terms of developmental genetics, and have become model organisms (see Discussion).
We recovered a distinct partial petaloid sepal spontaneous
mutant in which nearly half the area of sepals on the margins
is modified into petal-like structures. The studies have demonstrated that the mutant phenotype is due to mutations at
the Pps-1 locus. Here, we report the phenotype of Pps-1 and
its genetic behavior through systematic reciprocal crosses
with the wild type indicating its probable location in B function with negative control function.
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Journal of Heredity 2007:98(4)
Figure 1. (A) Partial petaloid mutant (Pps-1) lines of opium poppy (2 rows on right) along with the parent line I-14 (1 row on the
left). (B) A row of Pps-1 mutant showing partial petaloid sepal phenotype on the flower. (C) Close-up of the single flower of the
parent I-14 (left) and Pps-1 mutant (right). (D) Another close-up of the flower of Pps-1 mutant showing the conversion of sepal
margins into petal-like structures.
Materials and Methods
The plant materials consist of a true breeding flower mutant
Pps-1 with partial petaloid sepals (Figure 1) spontaneously
originated in the downy mildew (DM)–resistant accession
I-14 of opium poppy. The mutant line Pps-1 has been inbred
for 6 selfing cycles and is completely distinct, uniform, and
stable. The parental genotype I-14 has also been inbred for 9
selfing cycles and is being used in crossing programs as the
main source of DM resistance in opium poppy.
The experiments were carried out at the research fields of
Central Institute of Medicinal and Aromatic Plants, Lucknow, India, during Rabi season (November–April). The parental accession I-14 has narrow leaves with very deep leaf
incisions and white petals. The mutant genotype Pps-1 differs
from its parent I-14 in only one character where margins of
both the sepals are modified into petal-like structures.
We crossed Pps-1 mutant line with the parental line I-14
taking it both as male and female parents during 2002–2003
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crop seasons. The F1 seeds were then sown in the field along
with their parents to collect seeds of the F2 generations and
raising reciprocal F1s simultaneously with F2 during 2003–
2004 crop season. Different generations (reciprocal F1s
and F2s) and parents (Pps-1 and I-14) were sown in the field
in randomized block design with 3 replications during 2004–
2005 crop season and observations were collected on singleplant basis. Row-to-row distance of 50 cm and plant-to-plant
distance of 10 cm was maintained. Normal agronomic practices were carried out to have good crop population. Chisquare analysis was applied to test the goodness-of-fit for frequency distributions in the F2 generations.
Results and Discussion
Homeotic mutations in flowers have been related to the deviated development of floral organs in abnormal locations.
In this study, the typical Pps-1 expression has been observed
Brief Communications
Table 1. Segregation pattern of homeotic gene mutant in different generations of the reciprocal crosses involving Pps-1 mutant and
parental accession I-14 having wild-type phenotype
Number of observed plants
Genotype/cross
Generation
Wild type
Mutant type
Segregation
ratio
v2
P
Pps-1
I-14
Pps-1 I-14
I-14 Pps-1
Pps-1 I-14
I-14 Pps-1
P1
P2
F1(P1 P2)
rF1(P2 P1)
F2(P1 P2)
rF2(P2 P1)
0
180
165
172
185
170
140
0
0
0
55
50
—
—
—
—
3:1
3:1
—
—
—
—
0.554
0.605
—
—
—
—
0.50–0.30
0.50–0.30
to be a true breeding homeotic gene mutation of opium
poppy in which sepals are partially converted into petaloid
structures on the margins. An opposite effect has been
reported from PISTILLATA floral mutant of Arabidopsis
thaliana where petals have been replaced by sepal-like organs
(Hill and Lord 1989). The mutant Pps-1 is interesting because
as a naturally occurring mutation it affects only the first whorl
of floral organs producing partially petaloid sepals. The character is stable and inherits truly as a typical recessive trait in
the subsequent generations. A true breeding inbred line
named Pps-1 with this mutant character has been stabilized
through subsequent selfing and selection for 6 generations.
In the present study, the phenotypic characteristics of the
Pps-1 in comparison to wild-type population of I-14 have
been analyzed genetically and correlated to the possible
mechanism parallel to ABC model reported in other systems.
Phenotypic Characteristics of Pps-1
Mutant
In a selfed population of the genotype I-14 of P. somniferum
while being screened for disease resistance characters, a true
breeding spontaneous flower mutant Pps-1 with partial petaloid sepals was observed. This mutant spontaneously originated in the DM-resistant accession I-14 of opium poppy
(Figure 1). It was found that Pps-1 was fully fertile and there
was no effect in other 3 floral whorls. The mutant line Pps-1
like the parental line I-14 had narrow leaves with very deep
leaf incisions. It also had white petals and with same level of
resistance to DM caused by Peronospora arborescence as the
parents I-14. Pps-1 mutant thus differed from its parental
genotype I-14 only in this understandably homeotic gene
mutation at Pps-1 locus in which nearly half the area of both
the sepals turns into petal-like structures along the margins
of sepals, whereas the central portion remains wild type
(Figure 1).
Genetic Analysis
Genetic analysis can provide the basis to understand gene
functions and regulation and has been successful in identifying many floral genes (Baum et al. 2002). With the distinct
morphotypic mutant Pps-1 differing only for the sepal devel-
opment, the genetic analysis through F1 and F2 analysis was
carried out in this study.
The phenotypic characteristics of all the F1 plants in reciprocal crosses involving Pps-1 and I-14 during both the
years (2003–2004 and 2004–2005 crop seasons) showed normal sepals indicating thereby that the typical mutant character
(Pps-1) was recessive in nature and there was no cytoplasmic
control for the mutant trait. Segregation pattern of the F2
populations of both the reciprocal crosses also gave good
fit of the monogenic Mendelian ratio (P 0.50–0.30) of normal wild type (I-14) and the mutant characters (Pps-1) indicating that mutant character is perhaps controlled by a single
recessive nuclear gene ‘‘Pps-1’’ (Table 1). Phenotypic characteristics of both the parents during both the years also
showed that the mutant character is true breeding and
may be used in breeding and molecular studies to understand
the mechanisms of homeotic gene expression in flowering
plants.
The classical ABC model of flower development put forward by Coen and Meyerowitz (1991) outlines the existence
of 3 classes of gene functions termed A, B, and C that work
through in overlapping domains to determine specific organ
identity. As sepal identity is reported to be encoded by A
function genes alone, mutants in this class exhibit complete
homeotic transformations of organ identity. The genes corresponding to these classes have been well characterized and
in Arabidopsis are represented by APETALA 1 (AP1) and
APETALA 2 (AP2) in the A class, APETALA 3 (AP3)
and PISTILLATA (PI) in B class, and AGAMOUS (AG)
in the C class (Bowman et al. 1989, 1991). As the A function
is poorly defined and seems to largely depend on the suppression of C function, studies of petal development and evolution have generally been focused on the B-group genes.
Kramer and Irish (1999) carried out a detailed study of
the ontogenetic changes in B-group gene expression in 4 species namely, icelandic poppy (Papaver nudicaule), bleeding heart
(Dicentra eximia) and buttercups (Ranunculus bulbosus and Ranunculus ficaria). The functions of AP1 gene, generally contributing to sepal development have not been found to be highly
conserved (Theissen et al. 2000). This suggests that the regulation of A function and their interaction with B and other
transfactors need to be re-evaluated through more genetic
and molecular studies involving various mutants of sepals
and petals showing homeotic transformations. This can be
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Journal of Heredity 2007:98(4)
achieved better if novel mutants differentially and morphologically become available like Pps-1.
On the other hand, such distinct mutants may be used as
potential morphological markers in breeding programs for
crop improvement. The I-14 genotype has been found previously to be highly resistant to DM disease caused by P. arborescence (Sattar et al. 1995; Dhawan et al. 1998) and has been
involved in breeding experiments for the transfer of DM resistance to develop high-yielding DM-resistant varieties of
opium poppy. Genetically, the DM resistance has not been
found to be linked to the Pps-1 mutation as all the progenies
of the parental line (I-14) and the mutant line (Pps-1) observed were invariably showing disease resistance phenotype.
However, the linkage of this marker character can be studied
with other important traits and subsequently can be used as
an effective morphological marker in the breeding programs
for crop improvement.
Mutant Pps-1 obtained and isolated to stability level is
significant not only because it represents a deviation in Awhorl about which very little is known at the moment but
also because it is not a laboratory-induced mutation as is
the case in most of the commonly studied floral mutants.
Probably this is the first report of selection of a true breeding
partial homeotic gene mutant of A-function genes of flower
development spontaneously arising in natural populations.
It has been demonstrated during earlier reports that Afunction genes (Coen and Meyerowitz 1991; Soltis et al.
2002; Buzgo et al. 2004; Kramer and Jaramillo 2005) and
all 4 SEPALLATA genes (SEP1, SEP 2, SEP3, and SEP4)
(Ditta et al. 2004; de Folter et al. 2005) are responsible for
sepal formation. However, it needs to be further ascertained
looking at the morphology and breeding behavior of Pps-1
whether only A-class genes or interference of B-class genes is
also there that are known to be involved in the development
of petals and stamens only. One of the reasonable hypotheses
might be that this phenotype is due to simple outward shift in
the B-domain boundary resulting in partial petaloid sepal mutant phenotypes.
However, the role of a specific ‘‘B’’ function in suppressing the expression of ‘‘A’’ and thus limiting it to only sepal
formation by transfactors lying in a separate domain not interacting with other part of A for petal formation should not be
ruled out. In this study, however, there is no indication for an
antagonism or a down regulation of A function by B function.
Single-whorl homeotic mutant condition is not a common
occurrence, and existence of these types of mutations suggests
that further pursuits of the Pps-1 locus in opium poppy could
thus prove very useful to other studies. Continued investigation of the partial petaloid sepal mutant (Pps-1) of opium
poppy and other similar mutants will help us to compare
and contrast the relationships between the different floral
whorls and may help to provide insight into the evolution
of the flower itself. By integrating data on gene expression
with functional studies of various plant mutants, it is expected
to get clearer picture of flowers’ development in plants.
Papaveraceae and Ranunculaceae are both members of
the order Ranunculales (Angiosperm Phylogeny Group II
2003). Ranunculales occupy an important position in angio-
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sperm phylogeny: they are the basalmost eudicots. Therefore,
P. somniferum and several of its relatives are currently studied
in terms of developmental genetics and have become model
organisms. These taxa include P. somniferum (Irish 2006),
Eschscholzia californica (California poppy, Papaveraceae;
Becker et al. 2005; Groot et al. 2005; MacLeod et al.
2006), and Aquilegia (Ranunculaceae; Kramer et al. 2003).
Funding
Junior Research Fellowship of the Council of Scientific and
Industrial Research (CSIR), Government of India, New
Delhi, to M.K.D. (2-56/2002(I)EU.II).
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Received February 7, 2006
Accepted February 27, 2007
Corresponding Editor: Prem Jauhar
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