Contribution of gibberellins to the formation of Arabidopsis seed coat through starch degradation.

To clarify the role of gibberellins in the seed development of Arabidopsis, we investigated the sites where gibberellins are synthesized and induce alpha-amylase genes. The spatial and temporal expression of the genes encoding gibberellin biosynthetic enzymes and alpha-amylases was examined by reverse transcription-PCR (RT-PCR) and in situ hybridization. The mRNAs of AtGA20ox2, AtGA20ox3 and AtGA3ox4 began to be detectable 5-7 d after pollination. In situ hybridization showed that these genes were expressed almost simultaneously around starch granules in the outer integument, preceding the disappearance of those granules. AtGA20ox2 and AtGA3ox4 but not AtGA20ox3 also showed their signals at the rim of the developing embryo. The alpha-amylase gene, Amy3, which responded to gibberellin, was mainly expressed in the developing seed, spatially overlapping with the expression of AtGA20ox2 and AtGA3ox4. These results suggest that gibberellins function in at least two sites of the seed: the outer integument and part of the embryo. We examined the phenotypes of a T-DNA insertion line of AtGA3ox4 and observed the following: (i) a decrease of alpha-amylase gene transcripts in young siliques; (ii) delay of starch degradation in the outer integument; (iii) disarrangement of the seed surface structure; and (iv) abnormal swelling pattern of polysaccharides after imbibition by the mature seed. These characteristics are phenotypes of plants under gibberellin starvation, because the abnormalities could be almost overcome with applied gibberellin, and the gibberellin-treated mutant was indistinguishable from the wild type. These results strongly suggest that gibberellins in the outer integument would be required for the normal formation of the Arabidopsis seed coat.