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Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants.

Skinner DJ, Gasser CS - BMC Plant Biol. (2009)

Bottom Line: Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes.Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization.The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA. dskinnr@illinois.edu

ABSTRACT

Background: Arabidopsis ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expression-based gene discovery can be used access such obscured genes.

Results: Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; inner no outer (ino, which lacks the outer integument), and aintegumenta (ant, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization.

Conclusion: The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and these form a set of candidate genes for evaluation of roles in ovule development using reverse genetics.

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Stages of ovule development collected in the pistil pools. Differential interference contrast images of wild-type ovules representing stages included in the pools. The FULL pools of pistils contained ovules from stage 1-II, through stage 3-IV ("maturity"). The EARLY pools of pistils contained ovules from stage 1-II through stage 3-I, when the integuments just cover the nucellus. The LATE pool included ovule stages 3-II to 3-IV during which here is little change in ovule shape. The genotypes that were collected for each pool are indicated in grey. Ovules stages are based on Schneitz et al. [2]. f, funiculus; ii, inner integument; oi, outer integument; n, nucellus.
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Figure 2: Stages of ovule development collected in the pistil pools. Differential interference contrast images of wild-type ovules representing stages included in the pools. The FULL pools of pistils contained ovules from stage 1-II, through stage 3-IV ("maturity"). The EARLY pools of pistils contained ovules from stage 1-II through stage 3-I, when the integuments just cover the nucellus. The LATE pool included ovule stages 3-II to 3-IV during which here is little change in ovule shape. The genotypes that were collected for each pool are indicated in grey. Ovules stages are based on Schneitz et al. [2]. f, funiculus; ii, inner integument; oi, outer integument; n, nucellus.

Mentions: Three different developmental classes of pooled pistils were used. The FULL (F) pool, collected from wild type (WT) and ino, contained pistils from the stage at which ovule primordia are emerging (floral stage 9, ovule stage 1-II), up to mature ovules, just prior to anthesis (Figure 2; floral stage 12, ovule stage 3-IV). Samples containing fewer stages were collected to decrease the complexity of the samples to provide better resolution of expression differences and to evaluate the temporal expression patterns of selected genes. The EARLY (E) pool, collected from all three genotypes, included the youngest stages described above up to the point when the integuments first enclose the nucellus (floral stage 11, ovule stage 3-I), while the LATE (L) pool, collected from WT, captured the remaining stages after the integuments enclose the nucellus up to anthesis. Three biological replicates of each sample were used, with the exception of the WT L arrays, which had two replicates.


Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants.

Skinner DJ, Gasser CS - BMC Plant Biol. (2009)

Stages of ovule development collected in the pistil pools. Differential interference contrast images of wild-type ovules representing stages included in the pools. The FULL pools of pistils contained ovules from stage 1-II, through stage 3-IV ("maturity"). The EARLY pools of pistils contained ovules from stage 1-II through stage 3-I, when the integuments just cover the nucellus. The LATE pool included ovule stages 3-II to 3-IV during which here is little change in ovule shape. The genotypes that were collected for each pool are indicated in grey. Ovules stages are based on Schneitz et al. [2]. f, funiculus; ii, inner integument; oi, outer integument; n, nucellus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664812&req=5

Figure 2: Stages of ovule development collected in the pistil pools. Differential interference contrast images of wild-type ovules representing stages included in the pools. The FULL pools of pistils contained ovules from stage 1-II, through stage 3-IV ("maturity"). The EARLY pools of pistils contained ovules from stage 1-II through stage 3-I, when the integuments just cover the nucellus. The LATE pool included ovule stages 3-II to 3-IV during which here is little change in ovule shape. The genotypes that were collected for each pool are indicated in grey. Ovules stages are based on Schneitz et al. [2]. f, funiculus; ii, inner integument; oi, outer integument; n, nucellus.
Mentions: Three different developmental classes of pooled pistils were used. The FULL (F) pool, collected from wild type (WT) and ino, contained pistils from the stage at which ovule primordia are emerging (floral stage 9, ovule stage 1-II), up to mature ovules, just prior to anthesis (Figure 2; floral stage 12, ovule stage 3-IV). Samples containing fewer stages were collected to decrease the complexity of the samples to provide better resolution of expression differences and to evaluate the temporal expression patterns of selected genes. The EARLY (E) pool, collected from all three genotypes, included the youngest stages described above up to the point when the integuments first enclose the nucellus (floral stage 11, ovule stage 3-I), while the LATE (L) pool, collected from WT, captured the remaining stages after the integuments enclose the nucellus up to anthesis. Three biological replicates of each sample were used, with the exception of the WT L arrays, which had two replicates.

Bottom Line: Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes.Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization.The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA. dskinnr@illinois.edu

ABSTRACT

Background: Arabidopsis ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expression-based gene discovery can be used access such obscured genes.

Results: Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; inner no outer (ino, which lacks the outer integument), and aintegumenta (ant, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization.

Conclusion: The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and these form a set of candidate genes for evaluation of roles in ovule development using reverse genetics.

Show MeSH