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8% agar under the same growth conditions. The plate-grown seedlings were transferred to soil after 1?week. For abo3 (Salk_007496) T-DNA insertion identification, total genomic DNA was extracted from two rosette leaves. PCR was performed using primer pairs: SALK_007496LP 5��-CTTCTTCTCGAGACATGGCAG-3�� and SALK_007496RP 5��-TTGTTCCATGTTGTGAGGTTG-3��; TF 5��-GCGTGGACCGCTTGCTGCAACT-3��. RT-PCR was performed on total RNA extracted from 2-week-old Arabidopsis thaliana Columbia seedlings with the use of TRIzol (Invitrogen, Cat. No. 15596-026). The first-strand cDNA was synthesized with an 18-mer oligo(dT) primer. Subsequent PCR amplification of ABO3 cDNA involved the primer pair ABO3F, http://www.selleckchem.com/products/bmn-673.html 5��-ATGTTTTCAAACATCGATCACAAGGCTGTGG-3��, and ABO3R, 5��-CAACATCAGGTCTTCCGATGAAAATAGAGGAAATTCATTCC-3��. The PCR products were cloned into the CaMV 35S promoter driving the binary vector pMDC32 (for complementation and overexpression analysis) or pMDC85 (for protein targeting; Curtis and Grossniklaus, 2003) with Gateway Technology (Invitrogen). A promoter fragment, 1544?bp of the ABO3 gene, pABO3, was amplified from Columbia genomic DNA by PCR with the primer pair 5��-TGAGCCCTCTGATCTCCTTACGACTTTACGTGTCTTTGTC-3�� and 5��-CATCGATCACAAGGCTGTGGCAGC-3��. The amplified fragment http://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html was cloned into the pCAMBIA 1391 for a transcriptional fusion of the ABO3 promoter with the GUS coding region. The constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed by floral infiltration into wild-type Arabidopsis (Columbia accession, for gene overexpression and GUS staining assays) and abo3 mutants (for gene complementation). Stomatal closing assays were conducted as described by Chen et?al. (2006). Rosette leaves were floated in solutions containing 50?��m CaCI, 10?mm KCl, 10?mm Mes-Tris, pH?6.15, and exposed to light (150?��mol?m?2?sec?l) for 2?h. Subsequently, ABA was added to the solution at 0.5 and 1?��m to assay for stomatal closing. After ABA treatment for 2?h, stomatal apertures were measured as described by Chen et?al. (2006). Values are http://www.selleck.cn/products/incb024360.html means?��?SE, n?=?60. Significance (P?