Movement of the plant hormone abscisic acid (ABA) within plants has been documented; however, the molecular mechanisms that regulate ABA transport are not fully understood. By using a modified yeast two-hybrid system, we screened Arabidopsis cDNAs capable of inducing interactions between the ABA receptor PYR/PYL/RCAR and PP2C protein phosphatase under low ABA concentrations. By using this approach, we identified four members of the NRT1/PTR family as candidates for ABA importers. Transport assays in yeast and insect cells demonstrated that at least one of the candidates ABA-IMPORTING TRANSPORTER (AIT) 1, which had been characterized as the low-affinity nitrate transporter NRT1.2, mediates cellular ABA uptake. Compared with WT, the ait1 / nrt1 . 2 mutants were less sensitive to exogenously applied ABA during seed germination and/or postgermination growth, whereas overexpression of AIT1 / NRT1 . 2 resulted in ABA hypersensitivity in the same conditions. Interestingly, the inflorescence stems of ait1 / nrt1 . 2 had a lower surface temperature than those of the WT because of excess water loss from open stomata. We detected promoter activities of AIT1 / NRT1 . 2 around vascular tissues in inflorescence stems, leaves, and roots. These data suggest that the function of AIT1 / NRT1 . 2 as an ABA importer at the site of ABA biosynthesis is important for the regulation of stomatal aperture in inflorescence stems.

Summary: We have developed a novel gain‐of‐function system that we have named the FOX hunting system (Full‐length cDNA Over‐eXpressing gene hunting system). We used normalized full‐length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full‐length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T₁ mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T₂ progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full‐length cDNAs.

Summary: In a screen for morphological mutants from the T₁ generation of approximately 50 000 activation‐tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward‐pointing flowers and decreased apical dominance. We designated them isoginchaku (iso). The iso‐1D and iso‐2D are allelic mutants caused by activation of the AS2 gene. The T‐DNAs were inserted in the 3′ downstream region of AS2. Iso‐3D and iso‐4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation‐tagged T‐DNA flanking sequences, we found a dominant mutant that we designated peacock1‐D (pck1‐D) in which the ASL5/LBD12 gene was activated by a T‐DNA. The pck1‐D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant‐mining tool especially for genes that make up a gene family.