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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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Classification of identified genes by protein type and function. Proteins were classified into categories using GO annotations and published information and the percentages of each category encoded by the genes in each integument group are shown. 'Unknown biological function' includes those proteins with no recognized domains, as well as proteins with recognized, conserved domains of unknown function. The category 'transcriptional regulators and DNA binding proteins' includes recognized transcription factor families and chromatin binding proteins, that may or may not be involved in regulation.
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Figure 4: Classification of identified genes by protein type and function. Proteins were classified into categories using GO annotations and published information and the percentages of each category encoded by the genes in each integument group are shown. 'Unknown biological function' includes those proteins with no recognized domains, as well as proteins with recognized, conserved domains of unknown function. The category 'transcriptional regulators and DNA binding proteins' includes recognized transcription factor families and chromatin binding proteins, that may or may not be involved in regulation.

Mentions: The sets of genes described above were analyzed for their putative functions, as listed at The Arabidopsis Information Resource , using gene ontology searches and published literature. Divisions into broad functional classes are shown in Figure 4. The proportions of the different categories vary little between the putative expression groups. The most prominent categories are proteins with unknown function and proteins involved in metabolism. There are also many putative transcription factors and DNA binding proteins (Table 2), which are good candidates for regulators of ovule development. The proportion of transcription factors is approximately 20%, which is higher than estimates for the proportion of transcription factors found in the genome (6–7%) [84-86].


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

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

Classification of identified genes by protein type and function. Proteins were classified into categories using GO annotations and published information and the percentages of each category encoded by the genes in each integument group are shown. 'Unknown biological function' includes those proteins with no recognized domains, as well as proteins with recognized, conserved domains of unknown function. The category 'transcriptional regulators and DNA binding proteins' includes recognized transcription factor families and chromatin binding proteins, that may or may not be involved in regulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Classification of identified genes by protein type and function. Proteins were classified into categories using GO annotations and published information and the percentages of each category encoded by the genes in each integument group are shown. 'Unknown biological function' includes those proteins with no recognized domains, as well as proteins with recognized, conserved domains of unknown function. The category 'transcriptional regulators and DNA binding proteins' includes recognized transcription factor families and chromatin binding proteins, that may or may not be involved in regulation.
Mentions: The sets of genes described above were analyzed for their putative functions, as listed at The Arabidopsis Information Resource , using gene ontology searches and published literature. Divisions into broad functional classes are shown in Figure 4. The proportions of the different categories vary little between the putative expression groups. The most prominent categories are proteins with unknown function and proteins involved in metabolism. There are also many putative transcription factors and DNA binding proteins (Table 2), which are good candidates for regulators of ovule development. The proportion of transcription factors is approximately 20%, which is higher than estimates for the proportion of transcription factors found in the genome (6–7%) [84-86].

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