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AUXIN RESPONSE FACTORS (ARFs) are transcription factors involved in auxin signal transduction during many stages of plant growth development. ARF10, ARF16 and ARF17 are targeted by microRNA160 (miR160) in Arabidopsis thaliana. Here, we show that negative regulation of ARF10 by miR160 plays important roles in seed germination and post-germination. Transgenic plants expressing an miR160-resistant form of ARF10, which has silent mutations in the miRNA target site (termed mARF10), exhibited developmental defects such as serrated leaves, curled stems, contorted flowers and twisted siliques. These phenotypes were not observed in wild-type plants or plants transformed with the targeted ARF10 gene. During sensu stricto germination and post-germination, mARF10 mutant seeds and plants were hypersensitive to ABA in a dose-dependent manner. ABA hypersensitivity was mimicked in wild-type plants by exogenous auxin. In contrast, overexpression of MIR160 (35S:MIR160) resulted in reduced sensitivity to ABA during germination. Transcriptome analysis of germinating ARF10 and mARF10 seeds indicated that typical ABA-responsive genes expressed during seed maturation were overexpressed in germinating mARF10 seeds. These results indicate that negative regulation of ARF10 by miR160 plays a critical role in seed germination and post-embryonic developmental programs, at least in part by mechanisms involving interactions between ARF10-dependent auxin and ABA pathways.

In many plant species, seed dormancy is broken by cold stratification, a pre-chilling treatment of fully imbibed seeds. Although the ecological importance of seed response to cold temperature is well appreciated, the mechanisms underlying the physiological changes during cold stratification is unknown. Here we show that the GATA zinc finger protein expressed in Arabidopsis seeds during cold stratification plays a critical role in germination. Characterization of an enhancer-trap population identified multiple lines that exhibited beta-glucuronidase (GUS) expression in the micropylar end of the seed (named Blue Micropylar End, BME lines). One of these lines, BME3, had a T-DNA insertion site in the 5' upstream region of a GATA-type zinc finger transcription factor gene (termed BME3-ZF). The BME3-ZF mRNA accumulated in seeds during cold stratification. Characterization of the BME3-ZF promoter indicated that this gene was activated specifically in the embryonic axis, which was still enclosed by the endosperm. The zinc finger gene knockout plants produced seeds exhibiting deeper dormancy, which showed reduced response to cold stratification. The ungerminated knockout seeds exhibited testa rupture, but failed to penetrate the endosperm layer. Application of gibberellic acid (GA3) rescued impaired germination of knockout seeds without cold stratification, indicating that the normal GA signal transduction pathway is present in the knockout mutants. Expression of GA20-oxidase and GA3-oxidase genes was greatly reduced in the knockout seeds, suggesting the potential involvement of the zinc finger protein in GA biosynthesis. These results suggest that the GATA zinc finger protein is a positive regulator of seed germination.

Enhancer trap is a powerful approach for identifying tissue- and stage-specific gene expression in plants and animals. For Arabidopsis research, beta-glucuronidase (GUS) enhancer-trap lines have been created and successfully used to identify tissue-specific gene expression in many plant organs. However, limited applications of these lines for seed germination research have been reported. This is probably due to the impermeability of the testa to the GUS substrate. By focusing on the stages between testa and endosperm rupture, we were able to circumvent the testa barrier to the GUS substrate and observe diverse tissue-specific gene expression during germination sensu stricto. One hundred and twenty-one positive subpools of 10 lines out of 1130 were isolated. Approximately 4500 plants from these subpools were grown in a greenhouse and one to seven individual plants exhibiting GUS expression in seeds were isolated for each subpool. This library of the Arabidopsis seed enhancer-trap lines is an efficient tool for identifying seed germination-associated genes. The individual lines from this library will be provided to the international seed biology research community. International collaboration to identify the trapped genes using genome-walking PCR and to characterize the gene functions using knockout plants will significantly enhance our understanding of the molecular mechanisms of seed germination.