Molecular and phenotypic analysis of a new allele of ABSCISIC ACID-INSENSITIVE3 (ABI3)

Plants have the ability to enter a quiescent stage in their lifecycle by forming seeds. During maturation, the embryo acquires dormancy, storage reserves, and products related to desiccation tolerance. The best characterized regulator of these processes is the plant hormone abscisic acid (ABA). Genes involved in these responses have been identified, but little is known about how they contribute to maturation. This study analyzed an ABA-insensitive mutant isolated from a population of Arabidopsis seeds transformed with a T-DNA library. This mutant was named helmethead ( helm) because cotyledons remained white and unexpanded so that only the root and hypocotyl emerge from the seed coat after germination. Because this defect has not been reported for other ABA-insensitive mutants, helm was the subject of characterization. Many lines of evidence support that the helm mutation is a new allele of ABA-INSENSITIVE3 (ABI3). Although the ABI3 coding region is unaffected, the majority of its promoter is not in tandem to this sequence. Defects similar to helm were observed in lines predicted to alter ABI3 expression including those with T-DNA insertions in the ABI3 promoter, ABI3 antisense lines and the severe abi3-6 mutant. Mutant abi3 alleles failed to genetically complement helm defects but over-expression of ABI3 was sufficient to confer normal ABA responsiveness and cotyledon growth. Relative to weak and severe abi3 mutants, helm defects are moderate. helm tissues were analyzed to determine the properties of the cotyledons that were defective. These observations support that embryonic storage functions are normal while post-germination functions likely prevent seedling growth of helm. Finally, as ABI3 is a transcriptional regulator, microarray analysis was performed. In addition to previously-reported ABI3- and ABA-regulated genes, those associated with desiccation tolerance and stress responses showed reduced expression in helm. Together, these results suggest that ABI3 regulates processes differently across embryonic tissues and that seed maturation is sensitive to ABI3 levels. As such, this study has contributed to our understanding of ABI3 function and extended the ABI3 allelic series to provide a more complete set of tools for studying seed development.