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-1. Subsequently, the indicated concentrations of ABA, or 2 μM HFPA or 5 μM manumycin, or ABA and 2 μM HFPA or 5 μM manumycin were added to the solutions to assay for stomatal closing. After treatments for 2 hours, stomatal apertures were observed with a digital video camera attached to an inverted microscope. Stomatal density was not affected in era1-2.
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ERA1-β-glucuronidase (GUS) fusion constructs were generated by inserting a 2.5-kb polymerase chain reaction (PCR)-amplified genomic fragment of the ERA1 promoter into a promoterless GUS T-DNA plasmid (pBI121). This construct was transformed into the Agrobacterium strain LB4404. Transgenic plants were generated by vacuum-infiltrating plants with Agrobacterium [N. Bechtold, J. Ellis, G. Pelletier, C. R. Acad. Sci. (Paris) 316, 1194 (1993)]. Kanamycin-resistant plants were selected in the next generation, and intact whole leaves were tested for GUS activity with the fluorescent GUS substrate Imagene Green (Molecular Probes, Oregon). Seedlings were incubated in GUS-buffer for 2 to 4 hours at room temperature and then directly viewed under a microscope (magnification, X25) by using blue excitation light. Positive fluorescent signal is yellow on a red chlorophyll autofluorescent background.
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data not shown (n = 480 stomata in three experiments)
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T. A. Theobald and Z. M. Pei, data not shown (n = 480 stomata in three experiments).
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Theobald, T.A.1
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3643069525
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note
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In addition, ABA activation of anion-channel currents was also analyzed at 1 and 50 μM ABA (n = 23 and 28 cells for WT and era1-2, respectively). Activation of anion currents was also potentiated in era1-2 compared with WT at 1 μM ABA, whereas at 50 μM ABA both WT and era1-2 responses were similar.
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2 seeds were screened for ABA insensitivity to select for abi1 or abi2. In the next generation seeds were screened for ABA supersensitivity (era1/era1). Supersensitive seeds were advanced to the next generation. Homozygous double mutants were identified by PCR amplification with primers of 5′-GATATCTCCGCCGGAGAT-3′ and 5′-CCATTCCACTGAATCACTTT-3′ for abi1-1, and 5′-CATCATCTGCTATGGCAGG-3′ and 5′-CCGGAGCATGAGCCACAG-3′ for abi2-1, as described (19). The era1-2 deletion was verified by Southern (DNA) blot with ERA1 cDNA as a probe. The era1/abi1 mutant was further verified by back crosses to both parents to confirm genetically that it was a double mutant.
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44
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3643140476
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data not shown (n = 960 stomata for era1/abi1 experiments; n = 220 stomata for drought experiments)
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C. M. Kwak, data not shown (n = 960 stomata for era1/abi1 experiments; n = 220 stomata for drought experiments).
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Kwak, C.M.1
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3643069524
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data not shown
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C. M. Kwak, data not shown. ABA inhibition of seed germination was analyzed as described (17). Germination of seeds was defined as positive when a radical tip had fully penetrated the seed coat (n = 50 per condition). Each experiment (n = 25) included conditions comparing the indicated lines at multiple ABA concentrations.
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Kwak, C.M.1
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-1) and watered by irrigation until just before the plants bolted (≈3 weeks). Because era1 affects growth, WT and era1-2 plants (n = 16 each) were selected that were at the same developmental stages and had similar numbers of leaves. At this point pots were removed from water and allowed to dry over time. Evaporation from soil was reduced by covering the soil surface with tinfoil so that water loss occurring primarily through plant transpiration could be quantified. Watered control plants were also analyzed. Pots were weighed every day at the same time. Pots containing no plants were subjected to the same treatments to determine the background rate of water loss.
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We thank T. A. Theobald, V. M. Baizabal-Aguirre, K. Kuchitsu, and X.-F. Cheng for assistance, and E. J. Kim, G. J. Allen, W. R. Schafer, N. M. Crawford, M. F. Yanofsky, and R. Y. Hampton for discussions and reading of the manuscript. Supported by National Science Foundation (MCB-9506191) and Department of Energy grants (94-ER20148) to J.I.S. and by a Natural Science and Engineering Research Council of Canada grant to P.M.
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