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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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Groups of inner and outer integument expressed genes identified by SOM clustering and significant differences in expression. The expression profiles of genes that showed significant changes and were more than two-fold changed in the mutants are shown grouped by predicted location of expression and cluster. The mean values for each gene were standardized to a mean of 0 and standard deviation of 1 (z-transformation), in order to focus on expression changes and not magnitude of expression. SOM cluster numbers (Additional file 5) are indicated at the bottom left of the graphs where applicable. (A) Genes likely to be expressed in the inner integument or other regions affected by the ant mutant are separated into 3 groups with different patterns. (B) Genes likely to be expressed in both integuments show a steady decrease in expression from WT E through ino E to ant E. (C) Genes likely to be expressed in the outer integument or at late stages in the embryo sac. The EARLY ("E") group is defined by lower expression levels in ino E, with similar levels in ant E while the FULL ("F") group contains those genes that only showed significant differences between WT F and ino F, and not at the early stages.
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Figure 3: Groups of inner and outer integument expressed genes identified by SOM clustering and significant differences in expression. The expression profiles of genes that showed significant changes and were more than two-fold changed in the mutants are shown grouped by predicted location of expression and cluster. The mean values for each gene were standardized to a mean of 0 and standard deviation of 1 (z-transformation), in order to focus on expression changes and not magnitude of expression. SOM cluster numbers (Additional file 5) are indicated at the bottom left of the graphs where applicable. (A) Genes likely to be expressed in the inner integument or other regions affected by the ant mutant are separated into 3 groups with different patterns. (B) Genes likely to be expressed in both integuments show a steady decrease in expression from WT E through ino E to ant E. (C) Genes likely to be expressed in the outer integument or at late stages in the embryo sac. The EARLY ("E") group is defined by lower expression levels in ino E, with similar levels in ant E while the FULL ("F") group contains those genes that only showed significant differences between WT F and ino F, and not at the early stages.

Mentions: SOM clusters 4, 5 and 8 – 10 (Figure 3A) contain a total of 310 genes that show little or no change between WT and ino arrays even at later stages and that have higher WT E than WT L levels (except cluster 10), indicating little or no outer integument or embryo sac expression later in development, and more expression early in development. Known genes in this group are expressed in medial regions, placenta and ovule primordia, for example CUC2, PERIANTHIA (PAN) and NUBBIN [19,63,64], while others also show some expression in integument primordia, such as BEL1, SPATULA (SPT) and PIN-FORMED 1 (PIN1) [10,65,66]. For these genes, outer integument expression is too low to be discerned in the ino arrays relative to the overall expression levels in the pistil. The patterns can be roughly separated on the basis of fold change: expression in ovule primordia regions results in smaller changes (approximately -1.4) and ovule and integument expression results in slightly higher fold changes.


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

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

Groups of inner and outer integument expressed genes identified by SOM clustering and significant differences in expression. The expression profiles of genes that showed significant changes and were more than two-fold changed in the mutants are shown grouped by predicted location of expression and cluster. The mean values for each gene were standardized to a mean of 0 and standard deviation of 1 (z-transformation), in order to focus on expression changes and not magnitude of expression. SOM cluster numbers (Additional file 5) are indicated at the bottom left of the graphs where applicable. (A) Genes likely to be expressed in the inner integument or other regions affected by the ant mutant are separated into 3 groups with different patterns. (B) Genes likely to be expressed in both integuments show a steady decrease in expression from WT E through ino E to ant E. (C) Genes likely to be expressed in the outer integument or at late stages in the embryo sac. The EARLY ("E") group is defined by lower expression levels in ino E, with similar levels in ant E while the FULL ("F") group contains those genes that only showed significant differences between WT F and ino F, and not at the early stages.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Groups of inner and outer integument expressed genes identified by SOM clustering and significant differences in expression. The expression profiles of genes that showed significant changes and were more than two-fold changed in the mutants are shown grouped by predicted location of expression and cluster. The mean values for each gene were standardized to a mean of 0 and standard deviation of 1 (z-transformation), in order to focus on expression changes and not magnitude of expression. SOM cluster numbers (Additional file 5) are indicated at the bottom left of the graphs where applicable. (A) Genes likely to be expressed in the inner integument or other regions affected by the ant mutant are separated into 3 groups with different patterns. (B) Genes likely to be expressed in both integuments show a steady decrease in expression from WT E through ino E to ant E. (C) Genes likely to be expressed in the outer integument or at late stages in the embryo sac. The EARLY ("E") group is defined by lower expression levels in ino E, with similar levels in ant E while the FULL ("F") group contains those genes that only showed significant differences between WT F and ino F, and not at the early stages.
Mentions: SOM clusters 4, 5 and 8 – 10 (Figure 3A) contain a total of 310 genes that show little or no change between WT and ino arrays even at later stages and that have higher WT E than WT L levels (except cluster 10), indicating little or no outer integument or embryo sac expression later in development, and more expression early in development. Known genes in this group are expressed in medial regions, placenta and ovule primordia, for example CUC2, PERIANTHIA (PAN) and NUBBIN [19,63,64], while others also show some expression in integument primordia, such as BEL1, SPATULA (SPT) and PIN-FORMED 1 (PIN1) [10,65,66]. For these genes, outer integument expression is too low to be discerned in the ino arrays relative to the overall expression levels in the pistil. The patterns can be roughly separated on the basis of fold change: expression in ovule primordia regions results in smaller changes (approximately -1.4) and ovule and integument expression results in slightly higher fold changes.

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