CKI1, a Histidine Kinase Homolog Implicated in Cytokinin Signal Transduction

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Science  08 Nov 1996:
Vol. 274, Issue 5289, pp. 982-985
DOI: 10.1126/science.274.5289.982


  • Fig. 1.

    (top). Screen for cytokinin-independent mutants. Wild-type Arabidopsis calli were transformed with pPCVICEn4HPT and cultured in the absence of phytohormones for 3 weeks. Note that a portion of one callus is green, whereas most of the transformed calli are a yellowish color. This green region proliferated rapidly and regenerated shoots in the absence of exogenous cytokinin and auxin. Bar, 1 cm. Fig. 2 (bottom). Genomic structure of the CKI1 region. Sites of the T-DNA insert in cki1-1, -2, and -3 are indicated by arrows. Structure of the T-DNA insert in cki1-1 is indicated, with hatched boxes representing the tetramer of CaMV 35S RNA enhancers. Orientation of the T-DNA inserts in cki1-1, -2, and -3 is the same. The position of the insert in cki1-4 could not be defined because, in this strain, there appeared to have been a rearrangement in the upstream region of the CKI1 gene. pC1S1 corresponds to the region between two Spe I sites with the T-DNA insert. Sequences that are common between cCKI1-16 and pC1S1 are shown below the diagram of the genomic structure as solid boxes, together with the presumed ATG initiation codon and TAG stop codon for the longest open reading frame. A region of 143 bp of the 3′ noncoding part of cCKI1-16, which was not carried by pC1S1, is not shown. The stop codon is at the Spe I recognition site. B, Bam HI; P, Pst I; S, Spe I; X, Xho I; LB, left border of T-DNA; RB, right border of T-DNA.

  • Fig. 3.

    Effects of transformation with the rescued DNA or with the cDNA of the CKI1 gene fused under the CaMV 35S RNA promoter. Calli were transformed with pPCVICEn4HPT (A and B), pC1S1Ti (C and D), pMON530 (E and F), or p35ScCKI1 (G and H) and cultured for 18 days in the presence [(A), (C), (E), and (G)] or absence [(B), (D), (F), and (H)] of cytokinin [trans-zeatin (2 mg/liter)]. All plates contained indolebutyric acid (0.5 mg/liter) and cefotaxime (150 mg/liter). Plates in (A) through (D) contained hygromycin (20 mg/liter) and plates in (E) through (H) contained kanamycin (50 mg/liter).

  • Fig. 4.

    Southern blot analysis of the wild-type and cki1-1 lines. DNA from the wild type (lanes 1 and 3) and from cki1-1 (lanes 2 and 4) was digested with Pst I (lanes 1 and 2) or Spe I (lanes 3 and 4), and fragments were allowed to hybridize with the 5′ half of cCKI1-16 (nucleotides 1 to 2652).

  • Fig. 5.

    (A) Deduced amino acid sequence of the CKI1 gene product (26). Potential transmembrane regions are indicated with double underlining. Eleven potential sites of N-glycosylation between the putative transmembrane regions are indicated by single underlining. (B and C) Deduced amino acid sequence of CKI1, aligned with conserved regions of several histidine kinase and receiver domains of LemA of Pseudomonas syringae (17), BarA of E. coli (18), DhkA of Dictyostelium (22), Sln1 of Saccharomyces cerevisiae (19), and ETR1 of A. thaliana (21). Alignment was achieved with the ClustalW program (Human Genome Center, Baylor College of Medicine). Amino acids shown in boldface indicate residues that are identical to those found at the same positions in at least three of the other sequences. (B) shows the deduced amino acid sequence of CKI1 aligned with the histidine kinase domains. Boxes surround the five consensus motifs that are characteristic of histidine kinase domains, as compiled by Perkinson and Kofoid (15). Asterisk at His405 indicates a putative phosphoryl group acceptor. (C) shows the deduced amino acid sequence of CKI1 aligned with the receiver domains. Asterisk at Asp1050 indicates a putative phosphoryl group acceptor. Boxes surround the highly conserved regions in the receiver domains (16). (D) Schematic representation of predicted structure of the CKI1 protein in comparison with the ETR1 protein. Open boxes represent histidine kinase domains, open ovals represent receiver domains, and filled boxes represent stretches of hydrophobic residues characteristic of membrane-spanning sequences. Putative histidine and aspartate phosphorylation sites are indicated.

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