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. 2004 Jun;16(6):1365-77.
doi: 10.1105/tpc.021477. Epub 2004 May 21.

Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis

Chika Nishimura  1 , Yoshi Ohashi, Shusei Sato, Tomohiko Kato, Satoshi Tabata, Chiharu Ueguchi
Affiliations

Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis

Chika Nishimura et al. Plant Cell. 2004 Jun.

Abstract

Cytokinins are plant hormones that may play essential and crucial roles in various aspects of plant growth and development. Although the functional significance of exogenous cytokinins as to the proliferation and differentiation of cells has been well documented, the biological roles of endogenous cytokinins have remained largely unknown. The recent discovery of the Arabidopsis Histidine Kinase 4 (AHK4)/CRE1/WOL cytokinin receptor in Arabidopsis thaliana strongly suggested that the cellular response to cytokinins involves a two-component signal transduction system. However, the lack of an apparent phenotype in the mutant, presumably because of genetic redundancy, prevented us from determining the in planta roles of the cytokinin receptor. To gain insight into the molecular functions of the three AHK genes AHK2, AHK3, and AHK4 in this study, we identified mutational alleles of the AHK2 and AHK3 genes, both of which encode sensor histidine kinases closely related to AHK4, and constructed a set of multiple ahk mutants. Application of exogenous cytokinins to the resultant strains revealed that both AHK2 and AHK3 function as positive regulators for cytokinin signaling similar to AHK4. The ahk2 ahk4 and ahk3 ahk4 double mutants and the ahk single mutants grew normally, whereas the ahk2 ahk3 double mutants exhibited a semidwarf phenotype as to shoots, such as a reduced leaf size and a reduced influorescence stem length. The growth and development of the ahk2 ahk3 ahk4 triple mutant were markedly inhibited in various tissues and organs, including the roots and leaves in the vegetative growth phase and the influorescence meristem in the reproductive phase. We showed that the inhibition of growth is associated with reduced meristematic activity of cells. Expression analysis involving AHK:beta-glucuronidase fusion genes suggested that the AHK genes are expressed ubiquitously in various tissues during postembryonic growth and development. Our results thus strongly suggest that the primary functions of AHK genes, and those of endogenous cytokinins, are triggering of the cell division and maintenance of the meristematic competence of cells to prevent subsequent differentiation until a sufficient number of cells has accumulated during organogenesis.

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Figures

Figure 1.
Figure 1.
Structure of the AHK Genes and Expression of AHK2 in ahk2 Mutants. (A) The structure of the chromosomal region encompassing each AHK gene is schematically presented. The closed rectangles represent the exons. The arrows above the genomic structures indicate the T-DNA insertion points in the ahk mutants. (B) AHK2 transcripts in the ahk2 mutants. Total RNA was isolated from the indicated 2-week-old rosette plants and then subjected to RT-PCR analysis to amplify the cDNA region encoding the receiver domain as well as the TUB2 transcript, as indicated above.
Figure 2.
Figure 2.
In Vitro and in Vivo Responses to Exogenous Cytokinins. (A) Effect of a cytokinin (t-zeatin) on rapid proliferation, greening, and shoot induction of calli. From the hypocotyl tissues of the indicated strains, calli were prepared. For the ahk4-1 single mutant and ahk2-1 ahk3-1 double mutants, root tissues (R) as well as hypocotyl tissues (H) were used. The resultant calli were incubated with continuous fluorescent illumination at 22°C for 10 d on shoot induction medium comprising MS medium supplemented with indolebutyric acid (0.2 μg/mL) and the indicated concentrations of t-zeatin. (B) Root inhibition by an exogenous cytokinin. The wild types (Col and Ws, open and closed circles, respectively) and the ahk2-1 (open triangles), ahk3-1 (closed triangles), ahk4-1 (open squares), and ahk2-1 ahk3-1 (closed squares) mutants were grown on the surface of MS agar plates supplemented with the indicated concentrations of BA with continuous fluorescent illumination at 22°C for 10 d. The length of each main root was measured and expressed relative to the average value for the same strain on the medium without BA. Each value represents the average with standard deviation for 20 plants. (C) Induction of ARR5 transcription by a cytokinin. Two-week-old rosette plants of the indicated strains grown on MS agar plates were sprayed with 100 μM t-zeatin (Z) or 0.36% methanol (the solvent for t-zeatin; M). After 3 h, total RNA from the resultant plants as well as untreated ones (−) was prepared, treated with DNase I, and then subjected to RT-PCR analysis to amplify ARR5 or TUB2 cDNAs. PCR products were separated by 2% agarose gel electrophoresis and then visualized with ethidium bromide.
Figure 3.
Figure 3.
Seedling Phenotypes of the Multiple ahk Mutants. Plants were grown on MS gellan gum plates with continuous fluorescent illumination at 22°C. (A) Five-day-old seedlings. From left to right are Col, Ws, ahk2-1 ahk4-1, ahk3-1 ahk4-1, ahk2-1 ahk3-1, and ahk2-1 ahk3-1 ahk4-1 seedlings. Bar = 1 mm. (B), (C), and (D) The hypocotyl length is shortened in the ahk multiple mutants. The length (B), cell number (C), and average cell length (D) of the hypocotyls of the 5-d-old seedlings were measured. Each value represents the average with standard deviation for 20 plants.
Figure 4.
Figure 4.
Root Growth Is Strongly Retarded in the ahk2-1 ahk3-1 ahk4-1 Triple Mutant. (A) The kinetics of root growth. Plants were germinated and grown on the surface of MS agar plates with continuous fluorescent illumination at 22°C. The length of main roots was measured every day. Each value represents the average with standard deviation for 20 plants. The strains used were as follows: Col (open circles), Ws (closed circles), ahk2-1 ahk4-1 (open triangles), ahk3-1 ahk4-1 (closed triangles), ahk2-1 ahk3-1 (open squares), and ahk2-1 ahk3-1 ahk4-1 (closed squares). (B) Longitudinal section of the hypocotyl of the ahk2-1 ahk3-1 ahk4-1 triple mutant (12-d-old seedling). The main root and adventitious roots are indicated by an arrow and arrowheads, respectively. (C) to (F) Propidium iodine–stained root tips of 5-d-old seedlings of Col (C), Ws (D), ahk2-1 ahk3-1 (E), and ahk2-1 ahk3-1 ahk4-1 (F) plants. Bars = 50 μm. (G) to (J) DAPI-stained root tips of 5-d-old seedlings of Col (G), Ws (H), ahk2-1 ahk3-1 (I), and ahk2-1 ahk3-1 ahk4-1 (J) plants. Bars = 50 μm.
Figure 5.
Figure 5.
Rosette Phenotypes of the ahk Multiple Mutants. Plants were grown on MS gellan gum plates with 16-h-light/8-h-dark fluorescent illumination at 22°C. (A) to (G) Morphology of 3-week-old rosettes. The strains used were Col (A), Ws (B), ahk2-1 ahk4-1 (C), ahk3-1 ahk4-1 (D), ahk2-1 ahk3-1 (E), ahk2-2 ahk3-2 (F), and ahk2-1 ahk3-1 ahk4-1 (G). Bars = 1 cm. (H) to (K) Vascular patterns of the fully expanded fourth mature leaves of 3-week-old Col (H), Ws (I), ahk2-1 ahk3-1 (J), and ahk2-1 ahk3-1 ahk4-1 (K) plants. Leaves were fixed and cleared, followed by microscopic observation. The entire morphology (left) and a close-up view (right) are shown. Bars = 1 mm. (L) to (N) Abaxial side of the fully expanded second mature leaves of 17-d-old Col (L), Ws (M), and ahk2-1 ahk3-1 ahk4-1 (N) plants. Leaves were harvested, fixed, and cleared, followed by microscopic observation. (O) Trichomes of the ahk2-1 ahk3-1 ahk4-1 triple mutant. (P) to (S) The number and morphology of leaves of 3-week-old Col (P), Ws (Q), ahk2-1 ahk3-1 (R), and ahk2-1 ahk3-1 ahk4-1 (S) plants. Two cotyledons and mature leaves of each plant were collected and are presented according to age from left to right. Bars = 1 cm. (T) and (U) Longitudinal sections of the SAM of Col (T) and ahk2-1 ahk3-1 ahk4-1 (U) plants. Bars = 50 μm.
Figure 6.
Figure 6.
Kinematic Analysis of Leaf Development. Plants were grown on MS gellan gum plates with continuous fluorescent illumination at 22°C. Second mature leaves were harvested every day and subjected to determination of leaf size and number and size of abaxial epidermal cells. The strains used were as follows: Col (open circles), Ws (closed circles), ahk2-1 ahk3-1 (open triangles), and ahk2-1 ahk3-1 ahk4-1 (open squares). (A) Leaf blade area. (B) Number of abaxial epidermal cells. (C) Size of abaxial epidermal cells. Each value represents the average with standard deviation for six plants.
Figure 7.
Figure 7.
Mutational Phenotypes of the ahk Multiple Mutants in the Reproductive Growth Phase. (A) Morphology of 5-week-old plants. From left to right are Col, Ws, ahk2-1 ahk4-1, ahk3-1 ahk4-1, ahk2-1 ahk3-1, and ahk2-1 ahk3-1 ahk4-1 plants. Bar = 10 cm. (B) Close-up view of the 7-week-old ahk2-1 ahk3-1 ahk4-1 triple mutant. Bar = 1 cm. (C) to (E) Close-up views of Col (C), Ws (D), and ahk2-1 ahk3-1 ahk4-1 (E) flowers. Bars = 1 mm.
Figure 8.
Figure 8.
Expression of AHK:GUS Fusion Genes. GUS staining of AHK2:GUS (left column), AHK3:GUS (middle column), and AHK4:GUS (right column) is shown. Plants were grown on MS gellan gum plates or on soil with 16-h-light/8-h-dark fluorescent illumination at 22°C. (A), (B), and (C) Five-day-old seedlings and close-up views of SAM and young leaf primordia (insets). (D), (E), and (F) Growing lateral root primordia of 5-d-old seedlings. (G), (H), and (I) Root tips of 5-d-old seedlings. (J), (K), and (L) Close-up views of the surface of first mature leaves. Ten-day-old rosette plants were stained. (M), (N), and (O) Cross-sections of influorescences of 4-week-old plants. (P), (Q), and (R) Floral tissues of 4-week-old plants.
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