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Vector Construction and Plant Materials.&l … Vector Construction and Plant Materials.<br />
For overexpression lines, full-length Myb28 cDNA was amplified by PCR using Arabidopsis leaf cDNA as a template. The cDNA was introduced into binary vector pGWB2 by TOPO and the Gateway system (Invitrogen, Carlsbad, CA), in which the expression of cDNA is under the control of the CaMV35S promoter. For the genetic complementation study, an ≈4-kb fragment spanning the upstream sequence and coding region of Myb28 was amplified by PCR using Arabidopsis leaf DNA as a template. This genomic fragment was introduced in pGWB1 by TOPO and the Gateway system. The resulting vectors were introduced into Agrobacterium tumefaciens EHA101 by the method of An et al..<br />
Wild-type Arabidopsis accession Columbia was transformed with full-length Myb28 cDNA by the floral dip method to obtain Myb28-overexpressing plants. The T-DNA insertion mutant myb28 (see below) was complemented with a genomic fragment containing an intact copy of Myb28. Arabidopsis T87 cultured suspension cells were transformed with the fusion construct of CaMV35S promoter linked to Myb28 cDNA to obtain Myb28 overexpressing suspension cell lines. Details of suspension cell culture and transformation are described in SI Methods.<br />
Myb28-knockout plants, in which T-DNA was inserted into the 5′ UTR of Myb28 (SALK_136312), was obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH). Homozygous lines of the T-DNA insertion mutant were selected and designated as myb28. A homozygous T-DNA-inserted line of Myb29, designated as myb29, in which T-DNA is inserted in the 5′ UTR of Myb29 (CS121027), was a kind gift from Mitsuhiro Aida (Nara Institute of Science and Technology, Ikoma, Japan).<br />
T2 and T3 generations of mutants and transgenic plants were used for analysis. Plants were grown for ≈3 weeks on soil [PRO-MIX BX (Premier Horticulture Inc., Quakertown, PA): vermiculite = 2:1, supplemented with fertilizer] in a greenhouse at 22°C under natural and fluorescent light (16 h light/8 h dark cycle). Rosette leaves were harvested, immediately frozen in liquid nitrogen, and stored at −80°C.<br />
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MeJA Treatment.<br />
Wild-type Arabidopsis plants were grown for 7 days in liquid culture . Plants were treated with MeJA for 3 h by direct addition to the liquid medium (final concentration of 10 μM). DMSO was used for mock-treatments (final concentration of 0.1%, vol/vol). The seedlings were harvested, immediately frozen in liquid nitrogen, and stored at −80°C until use. d nitrogen, and stored at −80°C until use.
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