![Isolation of the atairp3/log2-2 T-DNA-inserted loss-of-function mutant line. A, Screening of an ABA-hyposensitive RING E3 Ub ligase mutant in the germination stage. Wild-type (WT) and knockout mutant seeds were germinated on MS growth medium in the absence (top panel) or presence (bottom panel) of 0.5 μm ABA. Germination rates with respect to cotyledon greening were determined after 5 d. The atairp1 and atairp2 mutants served as positive controls for ABA-insensitive phenotypes, while the #99 RING mutant served as a negative control to demonstrate the wild-type phenotype in response to ABA. Bars = 1 cm. B, Schematic structure of the atairp3/log2-2 mutant line (SAIL_729_A08). Gray bars represent the 5′ and 3′ untranslated regions, black bars show the coding regions, and solid lines indicate the introns of AtAIRP3/LOG2 (GenBank accession no. NC_003074). The atairp3/log2-2 mutant contains double T-DNA insertions in the first introns after nucleotides 873 and 887 in an antisense orientation. White boxes depict T-DNA insertions. Primers used for genotyping PCR and RT-PCR are shown with arrows. Nucleotide sequences of the primers are listed in Supplemental Table S1. C, Genotyping and RT-PCR analyses of the atairp3/log2-2 knockout mutant and 35S:AtAIRP3-RNAi knockdown transgenic plants. Left panel, genotyping PCR of wild-type and atairp3/log2-2 plants. Primers used for genomic PCR are shown on the right side of the agarose gel. Middle panel, RT-PCR of wild-type and atairp3/log2-2 plants. Primers used for RT-PCR are shown on the right side of the gel. The level of Arabidopsis AtUBC10 (E2 ubiquitin-conjugating enzyme) transcripts was used as a loading control. Right panel, RT-PCR of wild-type, atairp3/log2-2, and 35S:AtAIRP3-RNAi (independent transgenic T4 lines 1 and 2) plants. Primers used for RT-PCR are shown on the right. AtUBC10 was used as a loading control. D, Schematic structure of the AtAIRP3/LOG2 gene and its predicted protein. Gray bars represent 5′ and 3′ untranslated regions, black bars show coding regions, and solid lines indicate introns. A putative N-terminal myristoylation site, a DAR2 domain, and a single C3HC4-type RING motif are indicated. Primers used for RT-PCR are shown with arrows. [See online article for color version of this figure.]](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/plphys/162/3/10.1104_pp.113.220103/3/plphys_v162_3_1733_f1.jpeg?Expires=1749873861&Signature=pGvPElTQFWZqgFEuiaQDWy5WPS3u4v1jZ4bldMM3y7~ppUvcmRk6YakL7usj-yGgwNVzTLyo02IrWq07DCnxG3WsNesZUOCD~9TmNdQVG4a8eZcg9vjE5iADF-05zuh5MhWVqpI3o~~BjKBORgI4damt5DDMiqsOIjdcf8Rd6qU9vuUtNGX5ldkf5~vEtpPBqDd~3tApySUyXk2NeTvEuCyOVvJZUpRW-ZxT1CshKn1yRJ~lXXm45v1DBYSVGO9zlSIotrYiquP9pZ9U4huFbA4Se~F-~eSR95EEb50ZQpiU-V5A3LOM0ks4hLN~OeejtVeOMQUn2c~p2fU4S5Cz4w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Isolation of the atairp3/log2-2 T-DNA-inserted loss-of-function mutant line. A, Screening of an ABA-hyposensitive RING E3 Ub ligase mutant in the germination stage. Wild-type (WT) and knockout mutant seeds were germinated on MS growth medium in the absence (top panel) or presence (bottom panel) of 0.5 μm ABA. Germination rates with respect to cotyledon greening were determined after 5 d. The atairp1 and atairp2 mutants served as positive controls for ABA-insensitive phenotypes, while the #99 RING mutant served as a negative control to demonstrate the wild-type phenotype in response to ABA. Bars = 1 cm. B, Schematic structure of the atairp3/log2-2 mutant line (SAIL_729_A08). Gray bars represent the 5′ and 3′ untranslated regions, black bars show the coding regions, and solid lines indicate the introns of AtAIRP3/LOG2 (GenBank accession no. NC_003074). The atairp3/log2-2 mutant contains double T-DNA insertions in the first introns after nucleotides 873 and 887 in an antisense orientation. White boxes depict T-DNA insertions. Primers used for genotyping PCR and RT-PCR are shown with arrows. Nucleotide sequences of the primers are listed in Supplemental Table S1. C, Genotyping and RT-PCR analyses of the atairp3/log2-2 knockout mutant and 35S:AtAIRP3-RNAi knockdown transgenic plants. Left panel, genotyping PCR of wild-type and atairp3/log2-2 plants. Primers used for genomic PCR are shown on the right side of the agarose gel. Middle panel, RT-PCR of wild-type and atairp3/log2-2 plants. Primers used for RT-PCR are shown on the right side of the gel. The level of Arabidopsis AtUBC10 (E2 ubiquitin-conjugating enzyme) transcripts was used as a loading control. Right panel, RT-PCR of wild-type, atairp3/log2-2, and 35S:AtAIRP3-RNAi (independent transgenic T4 lines 1 and 2) plants. Primers used for RT-PCR are shown on the right. AtUBC10 was used as a loading control. D, Schematic structure of the AtAIRP3/LOG2 gene and its predicted protein. Gray bars represent 5′ and 3′ untranslated regions, black bars show coding regions, and solid lines indicate introns. A putative N-terminal myristoylation site, a DAR2 domain, and a single C3HC4-type RING motif are indicated. Primers used for RT-PCR are shown with arrows. [See online article for color version of this figure.]
