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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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In situ hybridization pattern of At3g55560 in inflorescences. (A – F): anti-sense probe; (G, H): sense probe. (A) Transcripts of At3g55560 were detected in the outer cell layers of floral primordia and floral organ primordia, and were maintained in the medial region of the elongating carpel (B). (C, D) Expression specific to the placenta was observed prior to fusion of the septum. (E) Emerging ovule primordia showed specific expression, that was maintained most strongly in the outer cell layers of the chalaza as the ovules developed (F). Expression was maintained at low levels in the distal funiculus and chalaza as the integuments initiated. (G) The sense probe detected RNA in the megaspore mother cell and developing embryo sac, as well as in the tapetum and pollen at maturity (H), but not in the structures that hybridized to the anti-sense probe. Bar = 20 μm in A, B, and F (bottom); 5 μm in C, and G; 10 μm in D, E, and F (top); 40 μm in H. c, carpel; ch, chalaza; es, embryo sac; f, funiculus; fm, floral meristem; fp, flower primordium; im, inflorescence meristem; n, nucellus; oi, outer integument; op, ovule primordium; p, pollen; pl, placenta; se, septum; st, stamen.
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Figure 6: In situ hybridization pattern of At3g55560 in inflorescences. (A – F): anti-sense probe; (G, H): sense probe. (A) Transcripts of At3g55560 were detected in the outer cell layers of floral primordia and floral organ primordia, and were maintained in the medial region of the elongating carpel (B). (C, D) Expression specific to the placenta was observed prior to fusion of the septum. (E) Emerging ovule primordia showed specific expression, that was maintained most strongly in the outer cell layers of the chalaza as the ovules developed (F). Expression was maintained at low levels in the distal funiculus and chalaza as the integuments initiated. (G) The sense probe detected RNA in the megaspore mother cell and developing embryo sac, as well as in the tapetum and pollen at maturity (H), but not in the structures that hybridized to the anti-sense probe. Bar = 20 μm in A, B, and F (bottom); 5 μm in C, and G; 10 μm in D, E, and F (top); 40 μm in H. c, carpel; ch, chalaza; es, embryo sac; f, funiculus; fm, floral meristem; fp, flower primordium; im, inflorescence meristem; n, nucellus; oi, outer integument; op, ovule primordium; p, pollen; pl, placenta; se, septum; st, stamen.

Mentions: Analysis of mRNA expression patterns with in situ hybridization tests the predictive value of the expression profiling groups and provides important information for understanding gene function. In situ hybridizations were performed for At3g55560 (AT-HOOK PROTEIN OF GA FEEDBACK 2, AGF2), that encodes an At-hook DNA binding protein [102]. This gene showed a 2.7 fold decrease in ant relative to wild type, and a slight decrease (1.5 fold) in ino in the FULL arrays, and was in cluster 2, predicting expression in primordia, medial regions or inner integument with later embryo sac or outer integument expression. In confirmation of this prediction, early expression was seen in the placenta and ovule primordia, as well as the inflorescence meristem and flower primordia, and in the outer integument and distal funiculus of the ovule later. Serial sections indicated that expression was highest in the anlagen and primordia in the outermost 2 to 3 cell layers of flower primordia (Figure 6A). Expression was observed in the floral organ primordia, and persisted in growing carpels, stamens and petals (Figure 6B). The petal expression was highest in the edges of the petals, and expression in the anthers was highest in the center of each locule, prior to pollen formation. After microsporogenesis, expression in the tapetum and pollen decreased and was undetectable at maturity (not shown). In carpels, expression was limited very early to the parietal placental regions, before fusion of the septum (Figure 6C). Expression remained high in the ovule primordia as they formed as protrusions from the placenta (Figure 6D, E), and localized to the distal funiculus and outer integument after integument initiation (Figure 6F). By maturity, expression could not be detected in any part of the ovule (data not shown). A sense probe made from the same construct showed a distinct pattern confined to sporogenous cells, with a high level of expression seen in the tapetum and pollen and in the developing embryo sac (Figure 6G). In addition, MPSS signatures exist in this genomic region for this strand which have a different distribution pattern from the signatures for the coding strand [24].


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

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

In situ hybridization pattern of At3g55560 in inflorescences. (A – F): anti-sense probe; (G, H): sense probe. (A) Transcripts of At3g55560 were detected in the outer cell layers of floral primordia and floral organ primordia, and were maintained in the medial region of the elongating carpel (B). (C, D) Expression specific to the placenta was observed prior to fusion of the septum. (E) Emerging ovule primordia showed specific expression, that was maintained most strongly in the outer cell layers of the chalaza as the ovules developed (F). Expression was maintained at low levels in the distal funiculus and chalaza as the integuments initiated. (G) The sense probe detected RNA in the megaspore mother cell and developing embryo sac, as well as in the tapetum and pollen at maturity (H), but not in the structures that hybridized to the anti-sense probe. Bar = 20 μm in A, B, and F (bottom); 5 μm in C, and G; 10 μm in D, E, and F (top); 40 μm in H. c, carpel; ch, chalaza; es, embryo sac; f, funiculus; fm, floral meristem; fp, flower primordium; im, inflorescence meristem; n, nucellus; oi, outer integument; op, ovule primordium; p, pollen; pl, placenta; se, septum; st, stamen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 6: In situ hybridization pattern of At3g55560 in inflorescences. (A – F): anti-sense probe; (G, H): sense probe. (A) Transcripts of At3g55560 were detected in the outer cell layers of floral primordia and floral organ primordia, and were maintained in the medial region of the elongating carpel (B). (C, D) Expression specific to the placenta was observed prior to fusion of the septum. (E) Emerging ovule primordia showed specific expression, that was maintained most strongly in the outer cell layers of the chalaza as the ovules developed (F). Expression was maintained at low levels in the distal funiculus and chalaza as the integuments initiated. (G) The sense probe detected RNA in the megaspore mother cell and developing embryo sac, as well as in the tapetum and pollen at maturity (H), but not in the structures that hybridized to the anti-sense probe. Bar = 20 μm in A, B, and F (bottom); 5 μm in C, and G; 10 μm in D, E, and F (top); 40 μm in H. c, carpel; ch, chalaza; es, embryo sac; f, funiculus; fm, floral meristem; fp, flower primordium; im, inflorescence meristem; n, nucellus; oi, outer integument; op, ovule primordium; p, pollen; pl, placenta; se, septum; st, stamen.
Mentions: Analysis of mRNA expression patterns with in situ hybridization tests the predictive value of the expression profiling groups and provides important information for understanding gene function. In situ hybridizations were performed for At3g55560 (AT-HOOK PROTEIN OF GA FEEDBACK 2, AGF2), that encodes an At-hook DNA binding protein [102]. This gene showed a 2.7 fold decrease in ant relative to wild type, and a slight decrease (1.5 fold) in ino in the FULL arrays, and was in cluster 2, predicting expression in primordia, medial regions or inner integument with later embryo sac or outer integument expression. In confirmation of this prediction, early expression was seen in the placenta and ovule primordia, as well as the inflorescence meristem and flower primordia, and in the outer integument and distal funiculus of the ovule later. Serial sections indicated that expression was highest in the anlagen and primordia in the outermost 2 to 3 cell layers of flower primordia (Figure 6A). Expression was observed in the floral organ primordia, and persisted in growing carpels, stamens and petals (Figure 6B). The petal expression was highest in the edges of the petals, and expression in the anthers was highest in the center of each locule, prior to pollen formation. After microsporogenesis, expression in the tapetum and pollen decreased and was undetectable at maturity (not shown). In carpels, expression was limited very early to the parietal placental regions, before fusion of the septum (Figure 6C). Expression remained high in the ovule primordia as they formed as protrusions from the placenta (Figure 6D, E), and localized to the distal funiculus and outer integument after integument initiation (Figure 6F). By maturity, expression could not be detected in any part of the ovule (data not shown). A sense probe made from the same construct showed a distinct pattern confined to sporogenous cells, with a high level of expression seen in the tapetum and pollen and in the developing embryo sac (Figure 6G). In addition, MPSS signatures exist in this genomic region for this strand which have a different distribution pattern from the signatures for the coding strand [24].

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