Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase.

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.

Plants with increased expression of ent-kaurene oxidase are resistant to chemical inhibitors of this gibberellin biosynthesis enzyme.

The gibberellin (GA) biosynthetic pathway includes the three-step oxidation of ent-kaurene to ent-kaurenoic acid, catalyzed by the enzyme ent-kaurene oxidase (KO). Arabidopsis plants overexpressing the KO cDNA under the control of the cauliflower mosaic virus 35S promoter, with or without a translational fusion to a modified green fluorescent protein (GFP), are very similar to wild-type (WT) plants under normal growth conditions. In contrast, when WT and 35S:KO (or 35S:KO-GFP) seeds, seedlings or pollen tubes are grown in the presence of chemical inhibitors of KO, such as paclobutrazol and uniconazole, plants with increased KO expression are partially resistant to the effects of these inhibitors. In combination with the observation that decreased KO levels increase the sensitivity to KO inhibitors, the 35S:KO phenotypes demonstrate that the modification of KO enzyme levels could be used to create transgenic crop plants with altered KO inhibitor response. These results also suggest that the KO gene could be used as a selectable marker for plant regeneration based on resistance to KO inhibitors. Finally, the observation that pollen tubes expressing 35S:KO or 35S:KO-GFP have decreased sensitivity to KO inhibitors provides further evidence for a physiological role for GAs in pollen tube elongation.

The pea gene LH encodes ent-kaurene oxidase.

The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants.

The pea gene NA encodes ent-kaurenoic acid oxidase.

The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

Abscisic acid, phaseic acid and gibberellin contents associated with dormancy and germination in barley.

Analyses of abscisic acid (ABA), ent-kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley (Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent-kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent-kaurenoids and GAs, including GA1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent-kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent-kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.