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Plant materials<br />
A double knock … Plant materials<br />
A double knock-out mutant myb28myb29 was generated by crossing two single mutants, myb28 and myb29, possessing the T-DNA insertion and transposon element in At5g61420 (SALK_136312) and At5g07690 (SM_3_34316), respectively. Note that the combination of single mutants was different from that in the previous studies (Sønderby et al. 2007, Beekwilder et al. 2008). To construct the ProMYB:MYB lines, about 4,000 (for ProMYB28:MYB28 and ProMYB29:MYB29) or 3,000 (for ProMYB76:MYB76) bp fragments spanning the promoters (2,683, 2,674 and 2,000 bp for MYB28, MYB29 and MYB76, respectively) and the coding regions of MYB28, MYB29 and MYB76 were amplified by PCR by using Arabidopsis genomic DNA (Supplementary Fig. S2). Primers used for amplification of the ProMYB28:MYB28, ProMYB29:MYB29 and ProMYB76:MYB76 fragments are shown in Supplementary Table S2. The 3′-UTRs of MYB genes were not included in the amplified fragments. They were then cloned into a binary vector pGWB1 (Nakagawa et al. 2007). The resulting vectors were transformed into Agrobacterium tumefaciens EHA101 and then used for transformation of Arabidopsis myb28myb29. Transgenic lines were selected on agar-solidified 1/2 Murashige and Skoog medium with 50 mg l−1 kanamycin or 20 mg l−1 hygromycin. The homozygous lines were used for subsequent analyses.<br /><br />
Plant growth conditions<br />
The wild type (Columbia), myb28myb29 and seven lines of transgenic Arabidopsis (ProMYB28:MYB28_1, ProMYB28:MYB28_2, ProMYB28:MYB28_3, ProMYB29:MYB29_1, ProMYB29:MYB29_2, ProMYB76:MYB76_1 and ProMYB76:MYB76_2) were grown for 3 weeks in a growth chamber at 22°C under fluorescent light with a light/dark cycle of 16 h/8 h. Agar-solidified 1/2 Murashige and Skoog medium with 1% sucrose was utilized for plant cultivation. For the sulfur stress experiment, the medium containing 1.5 mM sulfate (Full S) was adjusted to 0.15 mM (1/10 S) and 0 mM (0 S) sulfate by substitution of MgSO4 by MgCl2. sulfate by substitution of MgSO4 by MgCl2.
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