Download Identification of the ABCE floral gene orthologs

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Authors: Yunjing Wang, Harvey E. Ballard, R. Ryan McNally, and Sarah E. Wyatt
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Affiliation: Environmental and Plant Biology Department, Ohio University
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Title: Differential expression of the ABCE floral gene orthologs in the chasmogamous and
cleistogamous flowers of Viola pubescens
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Corresponding author: Sarah E. Wyatt
Address: 315 Porter Hall
Environmental and Plant Biology Department
Ohio University
Athens, Ohio, 45701
Email:
[email protected]
Tel:
740-593-1133
Wang, Ballard, McNally and Wyatt. - 2 -
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Abstract
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Plants with the chasmogamous-cleistogamous mixed breeding system have reproductive
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advantages because the outcrossing chasmogamous flowers and the self-pollinating
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cleistogamous flowers collaborate to ensure seed output and increase genetic diversities.
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Reductions in floral organs and precocious sexual maturation characterize cleistogamous
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flowers, but the molecular determination of the differences between the flower types has
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never been investigated.
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of floral organs by regulating various downstream targets.
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orthologs in Viola pubescens Aiton were studied, in an effort to address the molecular
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mechanisms underlying the mixed breeding system. Two A class, two B class, one C
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class and one E class floral genes were identified by reverse transcriptase polymerase chain
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reaction (RT-PCR), and they were expressed in both types of flowers.
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RT-PCR showed that the ABCE floral genes were expressed at different levels in the two
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flower types.
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increased expression of the C class gene in cleistogamous flowers, may contribute to the
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developmental and morphological differences between chasmogamous and cleistogamous
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flowers in V. pubescens.
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control of the mixed breeding system and lays the foundation for future investigations into
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the regulation of the violet floral genes.
The ABCDE floral organ identity genes control the development
The ABCE floral gene
Semi-quantitative
The differential expression of the ABCE floral genes, especially the
This study provides the first step to understanding the molecular
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Keywords: ABCDE model; chasmogamous flowers; cleistogamous flowers; Violaceae;
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Viola pubescens
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Introduction
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Plants with both open chasmogamous and closed cleistogamous flowers on the same
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individual occur in at least 50 angiosperm families (Lord 1981; Culley and Klooster 2007).
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Chasmogamous flowers have a showy appearance and are prone to outcrossing, while
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cleistogamous flowers are usually reduced with some underdeveloped floral parts and
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produce seeds by self-pollination. The chasmogamous-cleistogamous mixed breeding
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system ensures seed production and represents an evolutionarily successful reproductive
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strategy (Uphof 1938; Lord 1981; Masuda et al. 2001; Lu 2002). Scientists have been
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interested in plants with the mixed breeding system for a long time, and a large body of
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morphological, ecological and population biological work has been conducted (Beattie
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1971, 1974; Mayers and Lord 1983a, 1983b; Masuda and Yahara 1992; Culley 2002).
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the molecular basis underlying the floral dimorphism remains largely unknown.
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But
As compared to chasmogamous flowers, cleistogamous flowers exhibit various degrees
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of reductions and modifications in different floral parts, and precocious sexual maturation
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is another distinct character associated with cleistogamous flowers (Uphof 1938; Lord 1981;
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Campbell et al. 1983).
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sizes in cleistogamous petals result in a reduced cleistogamous corolla (Minter and Lord
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1983b). On the other hand, pollen meiosis occurs significantly earlier in the
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cleistogamous flowers than in the chasmogamous flowers (Minter and Lord 1983a).
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For example, in Collomia grandiflora, small cell numbers and
The ABC model of flower development states that three classes (A, B, and C) of
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homeotic transcription factors are essential for the initiation of different floral organs (Coen
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and Meyerowitz 1991; Theissen 2001). Specifically, class A genes specify sepals and
Wang, Ballard, McNally and Wyatt. - 4 -
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work together with class B genes to control petal development.
Class B and C collaborate
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to give rise to stamens while carpels are determined by C class genes (Coen and
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Meyerowitz 1991; Theissen 2001). Additional research extended the ABC model to the
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ABCDE model, with D class genes specifying ovules and E class genes contributing to the
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determination of all four floral organs (Theissen and Saedler 2001; Jack 2004; Krizek and
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Fletcher 2005).
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(AP2); the B class consists of APETALA3 (AP3) and PISTILLATA (PI); AGAMOUS (AG) is
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the only known member of the C class. The SEEDSTICK, SHATTERPROOF1 and 2 are
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considered as D class genes in Arabidopsis based on their functions in ovule development
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(Pinyopich et al. 2003; Favaro et al. 2003); and SEPALLATA1, 2, 3, 4 (SEP1, SEP2, SEP3,
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SEP4) belong to the E class (Pelaz et al. 2000; Homna and Goto 2001; Ditta et al. 2004).
In Arabidopsis, the A class genes are APETALA1 (AP1) and APELATA2
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Besides their organ-identity functions, ABCDE floral genes activate different
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downstream effectors to control the elaborate morphogenesis and growth programs of
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different floral organs (Jack 2004; Krizek and Flecher 2005). Quite a few such effectors
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were identified in recent years (Sablowski and Meyerowitz 1998; Ito et al. 2004; Takeda et
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al. 2004; Lee et al. 2005; Szecsi et al. 2006). The expression of RABBIT EARS (RBE),
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which encodes a SUPERMAN-like zinc finger protein, is downstream of AP1.
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affects the organ shape on the second whorl, regardless of the organ identity (Takeda et al.
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2004). NAC-LIKE, ACTIVATED BY AP3/PI (NAP) is directly induced by the AP3/PI
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heterodimer and is functional in the transition between cell division and cell expansion in
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developing stamens and petals (Sablowski and Meyerowitz 1998).
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on microsporogenesis by activating different downstream regulators such as the
RBE
AG exerts its function
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SPOROCYTELESS (SPL) gene (Ito et al. 2004).
AP3, PI, AG and SEP are also suggested
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to be positive regulators of the CRABS CLAW gene, which is required for nectar and carpel
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development (Lee et al. 2005).
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control petal size by interfering with cell expansion.
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regulate BPEp while AG has a negative influence on its expression (Szecsi et al. 2006).
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With more and more labs identifying downstream targets of the ABCDE floral genes, the
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list will continue to be extended.
Very recently, BIGPETALp (BPEp) was discovered to
AP1, AP3/PI, and SEP positively
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Considering that chasmogamous and cleistogamous flowers have floral organs
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adopting different morphologies and growth rates and that the ABCDE floral genes are
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master regulators of floral organ development, it was hypothesized that differential
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expression of the ABCDE floral genes plays a role in the production of the two flower
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types.
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cleistogamous violet, Viola pubescens Aiton (commonly known as yellow-stemmed violet),
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to test our hypothesis.
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pubescens belongs to a genus famous for the large number of species with the
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chasmogamous-cleistogamous mixed breeding system (Lord 1981; Culley and Klooster
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2007). This species was chosen for investigation because it is easily available, produces
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an abundance of chasmogamous and cleistogamous flowers over the growing season, and is
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putatively diploid with n=6 chromosomes (Ballard 1996).
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herb, commonly found in the understory of mesic forests in eastern North America. The
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plant produces chasmogamous flowers in the early spring (mid-April to May).
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forest canopy has fully leafed out, cleistogamous flowers are produced (May to September)
The present research focused on the ABCE floral gene orthologs in a
The D class genes were not examined in this investigation.
V.
V. pubescens is a perennial
After the
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(Culley and Wolfe 2001; Culley 2002).
The ABCE floral gene orthologs of V. pubescens
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were identified by RT-PCR from the floral RNA of V. pubescens. Semi-quantitative
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RT-PCR was conducted to compare the expression of these floral genes in the two flower
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types, in an effort to understand the roles of the ABCE floral genes in the mixed breeding
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system.
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Materials and Methods
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Plant materials
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Chasmogamous and cleistogamous flowers (young buds and mature flowers) of V.
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pubescens, as well as leaves and stems, were collected from the Ridges Land Laboratory of
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Ohio University, Athens, Ohio.
Plant materials were preserved in RNAlater RNA
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Stabilizing Reagent (Qiagen, Valencia, California, USA) immediately after collection.
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Morphology
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Some RNA-later preserved flowers were spared for morphological study.
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flowers at full anthesis and cleistogamous flowers right before seed-set were dissected
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under a binocular microscope by sequentially peeling away whorls of floral organs to
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expose the inner ones.
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camera (Nikon, Japan) attached to the microscope.
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were compared by Image J software (http://rsb.info.nih.gov/ij/).
Chasmogamous
Images of each set of floral organs were captured using a digital
Sizes of flowers and floral organs
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RT-PCR and semi quantitative RT-PCR
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Total RNA was extracted from the young floral buds of both types of flowers, leaves and
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stems with the RNeasy Plant Mini Kit (Qiagen, Valencia, California, USA) and treated with
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DNase (Qiagen, Valencia, California, USA).
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RT-PCR Kit (Qiagen, Valencia, California, USA).
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were designed from the conserved regions of the known ABCE orthologs in other plant
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species obtained from GenBank.
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The RT-PCR program included a reverse transcription step at 50ºC for 30min followed by a
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step to inactivate the reverse transcriptase at 94ºC for 15min, and then 35 cycles of 30s at
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94ºC for denaturation, 1 min for annealing (the annealing temperatures vary, depending on
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the primer pairs, and can be found in Table 1.) and 1min at 72ºC for extension.
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controls without reverse transcriptase were conducted to evaluate the possibility of
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genomic DNA contamination.
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cloning vector (Invitrogen, Carlsbad, California, USA) and then sequenced on an ABI 310
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genetic analyzer.
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Tool (BLAST) provided by NCBI to confirm their identities.
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constructed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html).
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RT-PCR was conducted using the OneStep
Degenerate primers for the RT-PCRs
Sequences of the designed primers are listed in Table 1.
Negative
The RT-PCR products were cloned into the TOPO TA
The sequences were analyzed with the Basic Local Alignment Search
The phylogenetic trees were
For the semi-quantitative RT-PCR, the 26S rRNA gene of V. pubescens was used as a
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loading control.
Products of the semi-quantitative RT-PCR were analyzed on 1.5%
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agarose gels by electrophoresis. Relative abundance of each floral gene mRNA was
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normalized to that of the control gene using Image J software (http://rsb.info.nih.gov/ij/).
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Results
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Morphological differences between the two flower types
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Both types of flowers had five well-developed sepals, although the sepals of cleistogamous
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flowers were smaller (Fig. 1a, 1b). Chasmogamous flowers had five large and showy
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petals with the lowest one protruded slightly at the base into a “spur”, characteristic of
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Viola flowers (Fig. 1c).
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surrounding the pistil, and the lowest two stamens had noticeable nectar glands attached to
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them (Fig. 1d).
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rudimentary in appearance. The “spur” on the lowest petal was scarcely evident, and no
Five stamens of chasmogamous flowers formed a cone
In contrast, the petals and stamens in cleistogamous flowers were quite
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nectar glands were observed on the stamens (Fig. 1e). Some petals were reduced to
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filamentous structures, and some stamens had no pollen sacs (pictures not shown).
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Although the overall size of cleistogamous flowers was about 1/3 that of chasmogamous
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flowers (Fig. 1a, 1b), the ovary of cleistogamous flowers was only slightly smaller than
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those of chasmogamous flowers (Fig. 1f, 1g). Thus ovaries constitute a larger proportion
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of the flowers in cleistogamous flowers than in chasmogamous flowers.
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curved style of cleistogamous flowers has been reported to facilitate self-pollination
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(Beattie, 1971) (Fig. 1g).
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Identification of the ABCE floral gene orthologs
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RT-PCR products about 300-500bp in length were obtained from floral RNAs with specific
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degenerate primer pairs.
The tightly
The identities of the RT-PCR products were verified by
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sequencing and BLAST analysis. The genes generating RT-PCR products that share high
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identities (>70%) with the ABCE floral genes in other plants were considered ABCE floral
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gene orthologs in V. pubescens.
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assignment of them as ABCE floral gene orthologs.
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VAP2, VAP3, VPI, VAG and VSEP3 respectively, with V representingViola. The control
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gene, 26S rRNA in V. pubescens, was identified in a similar way and tentatively designated
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V26S. The sequences of VAP1, VAP2, VAP3, VPI, VAG, VSEP3 and V26S are available in
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Genbank under the accession numbers: EF442781, EF453370, EF453371, EF564798,
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EF453372, EU169457 and EF564797.
Phylogenetic tree analysis (Fig. 2) also supported the
They were tentatively named VAP1,
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Expression of VAP1, VAP2, VAP3, VPI, VAG and VSEP3 in V. pubescens
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The expression of VAP1, VAP2, VAP3, VAPI, VAG and VSEP3 were studied in young floral
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buds, leaves and stems of V. pubescens by semi-quantitative RT-PCR, with the 26S rRNA
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gene of V. pubescens used as a loading control (Fig. 3).
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chasmogamous and cleistogamous flowers; however, their expression levels in vegetative
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tissues (leaves and stems) were different (Fig. 3).
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barely detectable in leaves (Fig. 3). The RNA of VAP2 was observed in both leaves and
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stems, although to a lesser degree than in flowers (Fig. 3). For the B, C and E classes of
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genes, little expression was detected in leaves and stems (Fig. 3).
All genes had expression in both
VAP1 was expressed in stems but was
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Expression of VAP1, VAP2, VAP3, VPI, VAG and VSEP3 in chasmogamous and
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cleistogamous flowers
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Differential expression of the ABCE floral gene orthologs of V. pubescens were observed
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between chasmogamous and cleistogamous flowers. The A class genes VAP1 and VAP2
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had similar expression level in the two types of flowers (Fig. 3) The expressions of the B
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class genes VAP3 and VPI were more pronounced in chasmogamous than in cleistogamous
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flowers, while the C class gene VAG was expressed to a much weaker degree (about three
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times less) in chasmogamous flowers as compared to in cleistogamous flowers (Fig. 3).
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For the E class gene VSEP3, its expression level was higher in chasmogamous flowers than
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in cleistogamous flowers (Fig. 3)
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DISCUSSION
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VAP1, VAP2, VAP3, VPI, VAG and VSEP3 are ABCE floral gene orthologs in V. pubescens
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The ABCDE model of flower development, derived initially from studies of two
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eudicot model plants Arabidopsis and Antirrhinum, explained how four whorls of floral
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organs are controlled by three classes of homeotic genes (Coen and Meyerowitz, 1991;
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Theissen, 2001). The simplicity of the ABCDE model promoted the identification and
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characterization of ABCDE orthologs in many other plant species, and conserved roles of
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the ABCDE orthologs were confirmed across angiosperms and gymnosperms (Rigola et al.
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2001; Whipple et al., 2004; Zhang et al. 2004; Krizk and Fletcher, 2005).
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Two A class genes (VAP1 and VAP2), two B class genes (VAP3, VPI), one C class gene
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(VAG), and one E class gene (VSEP3) from V. pubescens, a violet species with the
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chasmogamous-cleistogamous mixed breeding system, were identified in this study.
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Sequence analysis and phylogenetic trees indicated high levels of similarities between them
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and the known ABCE floral gene orthologs in other plants.
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has been repeatedly confirmed to fall phylogenetically into the Malpighiales, a relatively
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recently recognized angiosperm order including many families.
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and multi-gene investigations (Tokuoka and Hiroshi, 2006; Savolainen et al., 2000), the
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Violaceae have been placed in that order with Passifloraceae and Salicaceae as close
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relatives.
The violet family, Violaceae,
In previous single gene
In our phylogenetic analyses of API, PI and SEP3 genes, the Viola orthologs
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orthologs grouped with Salicaceae orthologs represented by genera Populus and Salix,
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suggesting high sequence identity of the genes in these families and providing further
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evidence for considering the Viola sequences as orthologs.
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Expression of ABCE orthologs in both chasmogamous and cleistogamous flowers
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(both types of flowers have sepals, petals, stamens and carpels) in V. pubescens
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demonstrated their roles in flower development(Fig. 3).
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patterns of VAP1, VAP2, VAP3, VPI, VAG and VSEP3 in different plant organs (flowers,
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leaves and stems) were similar to those of their counterparts in Arabidopsis
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(https://www.genevestigator.ethz.ch/at/). For example, the AP2 gene of Arabidopsis is
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expressed in leaves while AP3 is not (https://www.genevestigator.ethz.ch/at/). Similarly,
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VAP2 but not VAP3 had expression in leaves (Fig. 3).
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VPI, VAG and VSEP3 are likely to be the ABCE floral gene orthologs in V. pubescens with
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evolutionarily conserved functions, just as these gene systems have been documented in
In addition, the expression
Taken together, VAP1, VAP2, VAP3,
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other species.
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Differential expression patterns of the ABCE floral gene ortholog contribute to the mixed
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breeding system in V. pubescens
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Generally, cleistogamous flowers have all four whorls of floral organs, but these are
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substantially reduced or even rudimentary in some instances. Accelerated sexual organ
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maturation is also typical in cleistogamous flowers (Uphof, 1938; Lord, 1981; Campbell et
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al., 1983), such that perianth parts are arrested in development at a much earlier
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chronological stage when the androecium and gynoecium mature to become functional.
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According to the ABCDE model, sepals are controlled by the A class genes (Coen and
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Meyerowitz, 1991; Theissen and Saedler, 2001).
The two A class genes in V. pubescens,
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VAP1 and VAP2, exhibited similar expression in both types of flowers (Fig. 3).
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pubescens, sepals of both types of flowers were well developed, surrounding and protecting
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the inner floral organs (Fig. 1a and 1b). The equivalent expression of VAP1 and VAP2
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was thought to ensure the development of sepals in both flower types.
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function in development of the petals and stamens (Coen and Meyerowitz, 1991; Theissen
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and Saedler, 2001). Underdevelopment of petals and stamens was apparent in the
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cleistogamous flowers of V. pubescens (Fig. 1c, 1d and 1e), as is typical of many other
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plants with the chasmogamous-cleistogamous mixed breeding system (Uphof, 1938; Lord,
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1981; Campbell et al, 1983). Corresponding with the rudimentary state of petals and
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stamens, distinctly less expression of VAP3 and VPI was detected in cleistogamous flowers
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of V. pubescens (Fig. 3).
In V.
B class genes
Because the E class genes are involved in the development of all
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floral organs, the less expression of VSEP3 in the morphologically rudimentary
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cleistogamous flowers is also not surprising (Fig. 3).
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Expression of the C class gene VAG was dramatically greater in cleistogamous flowers
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than in chasmogamous flowers (Fig. 3).
In Arabidopsis, AG specifies reproductive floral
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organs and later plays essential roles in their development and maturation (Bowman et al.
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1991; Ito et al., 2004; Skinner et al. 2004; Gomez-mena et al., 2005).
For example, SPL, a
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gene controlling microgenesis, is upregulated by AG (Ito et al., 2004).
High expression of
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AG in ovule primordia and integuments indicates the contribution of AG to the
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development of ovules (Bowman et al. 1991; Skinner et al. 2004). Maturation of
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reproductive organs is much earlier and faster in cleistogamous flowers than in
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chasmogamous flowers (Uphof 1938; Lord, 1981; Campbell et al., 1983).
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pollen meiosis occurs about eight days later in chasmogamous flowers than in
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cleistogamous flowers, and it takes 15 more days for chasmogamous flowers to mature
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(Mayers and Lord, 1983a, 1983b). At the same time, chasmogamous flowers of V.
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odorata are about four times bigger than cleistogamous flowers, but their ovaries are only
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1.25 times bigger than those of cleistogamous flowers (Mayers and Lord, 1983b).
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Similarly, in V. pubescens, cleistogamous flowers have a size about 1/3 that of
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chasmogamous flowers (Fig. 1a and 1b), but their ovaries were only slightly smaller than
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those of chasmogamous flowers (Fig. 1f and 1g).
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actually occupy a larger proportion of cleistogamous flowers than those of chasmogamous
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flowers. Despite all the differences between the two flower types, their seeds exhibit no
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significant differences in number per capsule, mean seed mass or seedling performance in V.
In V. ordorata,
Thus, in both violet species, ovaries
Wang, Ballard, McNally and Wyatt. - 14 -
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pubescens (Culley, 2002). Taken together, we think more expression of VAG in the
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cleistogamous flowers of V. pubescens accounts for their faster maturation and competent
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ability to produce seeds.
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ABCE floral genes in plants with different forms of flowers
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Many plants produce more than one type of flower for special reproductive advantages.
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Homeotic floral genes, especially those in the B and C classes, have been studied in several
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dioecious and monoecious species to gain insights into the molecular basis of unisexual
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flowers. Different spatial and temporal performances of floral homeotic genes in male
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and female flowers had been observed, suggesting important roles in the floral-sex
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determination in these plants (Hardennack et al., 1994; Ainsworth et al., 1995; Ambrose et
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al. 2000; Kater et al. 2001; Park et al., 2003; Di Stillo, et al., 2005; Pfent, et al., 2005).
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The terminal inflorescence of sunflower (Helianthus annuus L.) is composed of two flower
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forms: peripheral ray (sterile) flowers and central disc (fertile) flowers, and the B and C
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classes of floral genes were expressed at different levels in the two types of flowers (Dezar,
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et al., 2003). Our study also detected differential expression of B, C and E classes of
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genes in chasmogamous and cleistogamous flowers of V. pubescens.
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Altogether, these studies indicate the involvement of ABCE floral homeotic genes in
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the production of different flowers by the same species. These results urge further
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investigations into the details of molecular regulations of the floral homeotic gene systems
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in these and other plants.
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breeding system, the two types of flowers do not occur simultaneously.
In most cases of the chasmogamous-cleistogamous mixed
Different
Wang, Ballard, McNally and Wyatt. - 15 -
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environmental conditions, including photoperiod, light availability, temperature, and
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nutritional conditions, as well as different plant growth hormone levels, have been
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proposed to influence the production of specific flower types (Uphof, 1938; Lord, 1981;
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Campbell et al., 1983).
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be subjected to the regulation of exogenous environmental and endogenous hormonal
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conditions, resulting in different expression patterns and eventually the production of two
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types of flowers. Studying the regulation of ABC(D)E floral gene orthologs, especially
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with regard to potential hormone gradients and environmental signaling cues, will provide
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more insights into the basis of the chasmogamous-cleistogamous mixed breeding system.
Thus, the ABC(D)E floral genes of cleistogamous flowers might
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In summary, we have identified the ABCE floral gene orthologs in a cleistogamous
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violet V. pubescens and compared its expression in both chasmogamous and cleistogamous
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flowers. The results indicated that these floral genes function in the two flower types.
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The different expression patterns in chasmogamous and cleistogamous flowers, especially
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the heightened expression of the C class gene in cleistogamous flowers, suggested
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contributions of the ABCE floral genes in the differentiation of the two types of flowers in
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violets.
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a broader research program on the evolution of the chasmogamous-cleistogamous mixed
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breeding system and its underlying molecular basis.
Future studies will probe the details of floral homeotic gene regulation as part of
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Acknowledgements
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The authors thank Vijay Nadella and Matthew Shipp for technical assistance.
This
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research was funded by a Baker Fund award from Ohio University to Drs. Wyatt and
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Ballard.
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4
Wang, Ballard, McNally and Wyatt. - 17 -
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Tables
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Table 1. Primer sequences* and annealing temperatures for the amplification of ABCE
floral gene orthologs and the 26s rRNA gene in Viola pubescens
Primer name
VAP1-F2
VAP1-R1
VAP2-F2
VAP2-R1
VAP3-F1
VAP3-R1
VPI-F3
VPI-R1
VAG-F1
VAG-R1
VSEP3-F2
VSEP3-R2
V26S-F
V26S-R
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24
25
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Primer sequence
5’ GGTTGCYKTKATTRTCTTCTC 3’
5’ GARTCNARATCYTCYCCCA 3’
5’ GTTTAYYTAGGTGGATTTGACAC 3’
5’ TTATCRTAAGCYCTRGCAGCTTC 3’
5’ CTYACYGTTCTYTGTGATGC 3’
5’ TACYAAHCCAHMVTGTGGATC 3’
5’GAGATAAAGAGGATAGAGAAC 3’
5’ CACAACACAYCCATATARATA 3’
5’ GGMAARATWGARATMAAGAGGA 3’
5’ TTYTCMAVYTGYTTBAGYTCC 3’
5’ GARGTWGCTCTSATCATCTTCTC 3’
5’ CNAGATCYTCBCCVADVAGRTTCC 3’
5’ GCACCTGAGTCCGACGTC 3’
5’ GCGGCGAAGCAGCCAAGG 3’
Annealing Tm (°C)
50
60
50
50
58
60
58
* Degenerate nucleotide codes are used, with W=A or T; S=C or G; R=A or G; Y=C or T; K=G or
T; M=A or C; B=C, G or T; D=A, G or T; H=A, C or T; V=A, C or G; N=A, C, G, or T.
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Figure legends
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Fig. 1. Chasmogamous and cleistogamous flowers of Viola. pubescens. Chasmogamous
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flowers (a, c, d, f) at full anthesis and chasmogamous flowers (b, e, g) right before
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seed-setting were dissected to show their floral organs.
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flower (a). An intact cleistogamous flower (b).
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without sepals (c).
A chasmogamous flower without sepals and petals (Nec:
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nectary gland) (d).
A cleistogamous flower without sepals (Pe: petal; St: stamen)
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(e). Pistil of a chasmogamous flower (f). Pistil of a cleistogamous flower (g).
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The scale bar equals 1mm.
An intact chasmogamous
A chasmogamous flower
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Fig. 2. Phylogenetic analysis of nucleotide sequences of ABCE floral gene orthologs.
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Neighbor-joining trees for AP1 (a), AP2 (b), AP3 (c), PI (d), AG (e) and SEP3 (f)
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orthologs were generated by ClustalW2. The generic names are labeled on the
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trees. For genera with duplicated orthologs, the gene names are also labeled.
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Fig. 3. Expression of the ABCE floral gene orthologs in V. pubescens.
Expression of the
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ABCE floral gene orthologs was compared in chasmogamous flowers (CH fls),
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cleistogamous flowers (CL fls), chasmogamous leaves (CH lvs, leaves subtending
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chasmogamous flowers), cleistogamous leaves (CL lvs, leaves subtending
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cleistogamous flowers) and stems (Stem) by semi-quantitative RT-PCR with
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specific degenerate primers. The 26S rRNA gene of V. pubescens (V26S) was used
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as a control gene.
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VAG and VSEP3 in V. pubescens (a). Relative abundances of the mRNAs of VAP1,
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VAP2, VAP3, VPI, VAG and VSEP3 to that of V26S in V. pubescens (b).
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5
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Gel image showing the expression of VAP1, VAP2, VAP3, VPI,
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Fig. 1
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Fig. 2.
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Fig. 3