Expression of Cytokinin Receptor Genes. The following primers were used for amplification of CRE-family genes from WT (Col) total RNA: CRE1, 5'-aatgcaatgttcaatcctctcacaactc-3' and 5'-caatgatgatccacagaatcaaagct-3'; AHK2, 5'-gtggatggtttcaagaactcaactatgag-3' and 5'-gagaaggatattcttcctccatcttccag-3'; and AHK3, 5'-tgggaagcctacaaggatt-3' and 5'-aaaccaccacaagcttcctccac-3'. Amplified fragments were used as templates for riboprobe synthesis. For expression patterns of reporter genes, all nucleotide positions are reported relative to the translational start codon. The genomic region between the –2682 nucleotide position and the +11 position of CRE1 was cloned in-frame upstream of the b -glucuronidase (GUS) gene in the binary vector pBI101 (1). This fusion gene was designated CRE1::GUS. The genomic region between the –2824 position and the +4599 position of the AHK2 gene was cloned in-frame upstream of the GUS gene in the binary vector pGPTVBar (2), and the terminator region (+4607 to +5252) of AHK2 was cloned downstream of the GUS gene. This GUS-fusion gene was designated AHK2::GUS. The genomic region between the -2061 nucleotide position and the +4246 position of AHK3 was cloned in-frame upstream of the GUS gene in the binary vector pBI101. This GUS fusion gene was designated AHK3::GUS.

Screening for T-DNA Insertion Mutants. Plants containing the T-DNA insertion alleles cre1-10, cre1-11, ahk2-1, ahk3-1, and ahk3-2 were obtained in a homozygous state according to Pischke et al. (3). The CRE1 gene-specific primers used were 5'-ccggaagattggattacgacgaaggtgag-3' and 5'-gtaccagagatagtcaaaggagattcagg-3'. The AHK2 gene-specific primers used were 5'-atgtctataacttgtgagctcttgaatct-3' and 5'-tggatgttcaagatatactacccgatgtc-3'. The AHK3 gene-specific primers used were 5'-atgctatgctgctggtttgtttcttggtt-3' and 5'-ttccagctgctactctcatctacatacca-3'. The T-DNA-specific primers used were 5'-cattttataataacgctgcggacatctac-3' and 5'-tgggaaaacctggcgttacccaacttaat-3'. T-DNA insertion sites were determined by sequencing PCR products containing the T-DNA/plant genomic DNA junction. The above primers were used for routine genotyping using PCR.

Plants containing the T-DNA insertion mutants cre1-12 and ahk3-3 were obtained from the Salk T-DNA collection (4). The T-DNA insertion mutant ahk2-2 was obtained from the Kazusa DNA Research Institute (www.kazusa.or.jp/arabi). T-DNA insertion sites were determined by sequencing PCR products containing the T-DNA/plant genomic DNA junction. For cre1-12, the gene-specific primers used were 5'-ggagagccttcaccggttaggg-3' and 5'-aagctcttgcatttcatggaaatc-3'. The T-DNA-specific primer used was 5'-tggttcacgtagtgggccatcg-3'. For ahk3-3, the gene-specific primers used were 5'-cttgtgattgcgttacttgttgcac-3' and 5'-gcaggcctatggtccacaaccacag-3'. The T-DNA-specific primers used were 5'-tggttcacgtagtgggccatcg-3' and 5'-gtgactcccttaattctccgctcatg-3'. For ahk2-2, the gene-specific primers used were 5'-gtctataacttgtgagctcttgaatc-3' and 5'-gctcgtgtcatagacagcaaaggtc-3'. The T-DNA-specific primer used was 5'-ataacgctgcggacatctac-3'. These primers were also used for routine genotyping using PCR.

Expression of Cytokinin Receptor Genes in Mutant Backgrounds. Sterilized seeds from plants homozygous for the cre1-12, ahk2-2, or ahk3-3 mutation were incubated in germination medium (GM) lacking Phytagel (Sigma-Aldrich) for 1 day at 4°C and subsequently incubated for 4 days at 22°C. One hundred fifty mg of seedling tissue was pooled, per genotype, for RNA preparation. RNA extraction, reverse transcription, and PCR were carried out as described in Miyawaki et al. (5), except that DNase treatment was omitted. cDNA prepared from 10 ng of total RNA was used for a PCR. PCR cycle numbers were 35 for CRE1 and AHK2, 30 for AHK3, and 28 for AtIPT9. These numbers of PCR cycles gave saturated amplification of CRE-family transcripts and unsaturated amplification of the AtIPT9 transcript, which was used as a control for cDNA amplification. Primers used for PCR were: CRE1, 5'-gaacccttctgcaattgatcaggag-3' and 5'-ctaggagcatagcaagcattccgag-3'; AHK2, 5'-catctgccattgatcagagaac-3' and 5'-catatgatgcaccaaggtaccca-3'; AHK3, 5'-gagagaattgaggctactaacgggtatc-3' and 5'-cagaccacatcagctccgtac-3'; and AtIPT9, 5'-catcagatagcgatagaaaggtggtg-3' and 5'-ttatgctattgcgctttccacgcatgaagc-3'. The sequence of at least one primer from each pair was interrupted by an intron.

Expression of Cytokinin Primary-Response Genes. Primer and probe sequences used were as follows. For SHR, primers used were 5'-gacataagctccacgttttgca-3' and 5'-gtctgatcttgtggctaaagcttct-3', and probe sequence was 5'-caatggccgactcttc-3'. For ARR5, primers used were 5'-agtgcgacaagagctttacaatatctt-3' and 5'-ccaggcatagagtaatccgtcatt-3', and probe sequence was 5'-ctcaaatccaaccgaactat-3'. For ARR15, primers used were 5'-cactcagagaaatcccagtagtgat-3' and 5'-gcaaaaactcctctgctccttctat-3', and probe sequence was 5'-caacctcgtatagaacaatg-3'.

1. Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. (1987) EMBO J. 6, 3901-3907.

2. Becker, D., Kemper, E., Schell, J. & Masterson, R. (1992) Plant Mol. Biol. 20, 1195-1197.

3. Pischke, M. S., Jones, L. G., Otsuga, D., Fernandez, D. E., Drews, G. N. & Sussman, M. R. (2002) Proc. Natl. Acad. Sci. USA 99, 15800-15805.

4. Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R., et al.. (2003) Science 301, 653-657.

5. Miyawaki, K., Matsumoto-Kitano, M. & Kakimoto, T. (2004) Plant J. 37, 128-138.