| Abscisic acid (ABA) regulates seed dormancy and germination, and gene expression in response to various environmental stresses. In plants, ABA is synthesized from epoxycarotenoid precursors that are localized exclusively in the membranes of plastids. Oxidative cleavage of epoxycarotenoids catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED) is the key regulatory step in the ABA biosynthetic pathway. Previous work in maize and other species has shown that NCED is encoded by a complex gene family. The Arabidopsis genome sequence revealed nine putative genes showing sequence homology to NCED. These are designated 9-cis epoxycarotenoid dioxygenase (AtNCED). The AtNCED2, 3, 5, and 9 genes show a strong sequence identity to the Vp14 from maize as well as NCEDs from other species, and are thus potentially the NCED homologs involved in ABA biosynthesis. The AtNCED1, 4, and 6 genes have moderate homology to NCED and have unknown functions. The AtNCED7 and AtNCED8 are more closely related to beta-carotene dioxygenases of animals that catalyze retinal biosynthesis. Northern analysis revealed that the AtNCEDs are differentially expressed in leaves and roots, and in some cases induced by water stress. In vitro chloroplast import experiments showed that AtNCED2, 4, and 5 proteins are differentially localized within chloroplasts. The AtNCED2 is partially localized in the stroma, and partially associated with the thylakoid membranes. In contrast, AtNCED4 is localized only in the stroma, whereas AtNCED5 is associated exclusively with thylakoid membranes. The imported mature AtNCED5 was larger than its translation precursor suggesting modification post-import. The differential expression, localization, and post-import modification of the AtNCEDs suggest that ABA biosynthesis is regulated both at the transcriptional and posttranslational levels.; AtNCED2 is 90% similar to VP14 of maize. To study the regulation of ABA biosynthesis and the AtNCED2 gene, AtNCED2 promoter-GUS, sense overexpression, and antisense expression transgenic plants were constructed. The AtNCED2 promoter-GUS transgenic lines showed GUS staining at the base of mature lateral roots, a narrow ring of cells in the root cap, and pericycle and cortex cells surrounding the lateral root initials. Auxin treatment induced lateral root formation and also strongly induced GUS staining, suggesting that ABA may be involved in lateral root initiation. Transgenic plants overexpressing the AtNCED2 showed phenotypes of inhibited growth, fewer lateral roots, male sterility, and anthocyanin accumulation. No obvious phenotype was observed in antisense lines.; We also screened knockout mutants of the AtNCED gene family. Two knockout mutants of AtNCED2 and one mutant of AtNCED5 have been identified and narrowed down to individual plants. Positive candidates have been identified for all the other family members, and further screening is in progress. |
