Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism

Abstract

We used loss-of-function mutants to study three Arabidopsis thaliana sensor histidine kinases, AHK2, AHK3, and CRE1/AHK4, known to be cytokinin receptors. Mutant seeds had more rapid germination, reduced requirement for light, and decreased far-red light sensitivity, unraveling cytokinin functions in seed germination control. Triple mutant seeds were more than twice as large as wild-type seeds. Genetic analysis indicated a cytokinin-dependent endospermal and/or maternal control of embryo size. Unchanged red light sensitivity of mutant hypocotyl elongation suggests that previously reported modulation of red light signaling by A-type response regulators may not depend on cytokinin. Combined loss of AHK2 and AHK3 led to the most prominent changes during vegetative development. Leaves of ahk2 ahk3 mutants formed fewer cells, had reduced chlorophyll content, and lacked the cytokinin-dependent inhibition of dark-induced chlorophyll loss, indicating a prominent role of AHK2 and, particularly, AHK3 in the control of leaf development. ahk2 ahk3 double mutants developed a strongly enhanced root system through faster growth of the primary root and, more importantly, increased branching. This result supports a negative regulatory role for cytokinin in root growth regulation. Increased cytokinin content of receptor mutants indicates a homeostatic control of steady state cytokinin levels through signaling. Together, the analyses reveal partially redundant functions of the cytokinin receptors and prominent roles for the AHK2/AHK3 receptor combination in quantitative control of organ growth in plants, with opposite regulatory functions in roots and shoots.

Figure 1.

Shoot Development of ahk Mutant Plants. (A) Rosette sizes of wild-type and cytokinin receptor mutants 25 d after germination (DAG). The mutant alleles were ahk2-5, ahk3-7, and cre1-2. Error bars represent se (n = 30). (B) Rosettes of plants grown on soil 25 DAG. Bar = 10 mm for the close-up of the ahk2-5 ahk3-7 cre1-2 triple mutant shown in the bottom right. (C) A triple ahk2-5 ahk3-7 cre1-2 mutant plant (right) 70 DAG compared with the wild type. (D) Downward bending of cotyledons of ahk2-5 ahk3-7 (middle) and ahk2-5 ahk3-7 cre1-2 (bottom) seedlings compared with the wild type (top) (3 DAG). (E) Number of epidermal cells per mm² on the adaxial (white bars) and abaxial sides (black bars) of the seventh leaf of the wild type and ahk2-5 ahk3-7 (21 DAG) and of the full expanded seventh leaf of ahk2-5 ahk3-7 cre1-2 (28 DAG). Cell size was measured at three different positions on the seventh leaf in the middle of the leaf blade. Error bars represent se (n = 5). (F) Number of epidermal cells on the adaxial side on the seventh leaf. Error bars represent se (n = 5).

Figure 2.

AHK2 and AHK3 Are Required to Mediate Cytokinin-Dependent Chlorophyll Retention in the Dark. (A) Chlorophyll content of in vitro–grown plants 24 DAG. Wild type (1.92 ± 0.01 μg/g leaf fresh weight) was set at 100%. For each of five independent samples per clone, five seventh leaves from different plants were pooled and analyzed. Error bars represent se (n = 5). The mutant alleles used were ahk2-5, ahk3-7, and cre1-2. (B) Dark-induced senescence in a detached leaf assay and its inhibition by cytokinin. The leaf chlorophyll content before the start of dark incubation was set at 100% for each genotype tested. Bars: white, water plus DMSO; gray, 0.1 μM BA; black, 1 μM BA. Chlorophyll content at the beginning of the assay was for the wild type, 1.92 ± 0.01; ahk2-5, 1.64 ± 0.11; ahk3-7, 1.46 ± 0.11; cre1-2, 1.95 ± 0.14; ahk2-5 ahk3-7, 1.23 ± 0.03; ahk2-5 cre1-2, 2.23 ± 0.17; ahk3-7 cre1-2, 1.44 ± 0.09; ahk2-5 ahk3-7 cre1-2, 0.61 ± 0.31 μg/g leaf fresh weight. Asterisks represent significant changes to wild-type control at respective hormone concentrations. Error bars represent se (n = 5). (C) Leaves of different genotypes at the end of the chlorophyll retention assay described in (B). (D) Time course of dark-induced leaf senescence in wild-type leaves. The chlorophyll content at the beginning of the experiment was set at 100%. Three independent plates with five leaves per plate were examined at each time point and concentration. The graph shows pooled results from three independent experiments ±se (n = 3). (E) Time course of dark-induced leaf senescence in leaves of the ahk2-5 ahk3-7 mutant. Conditions are as described in (D).

Figure 3.

Seeds and Embryos of the Triple Mutant Are Increased in Size. (A) ahk2-5 ahk3-7 cre1-2 triple mutant seeds (top two seeds) compared with the wild type (bottom two seeds). (B) Embryos of the ahk2-5 ahk3-7 cre1-2 triple mutant (top) and the wild type (bottom). Bar = 3 mm. (C) Width (white bars) and length (black bars) of seeds from the wild type and the triple mutant. Error bars represent se (n = 60). (D) Calculated volume of wild-type and ahk2-5 ahk3-7 cre1-2 triple mutant seeds. Error bars represent se (n = 60).

Figure 4.

Seeds of Cytokinin Receptor Mutants Show Early Germination and Resistance to Far-Red Light. (A) Percentage of germinated seeds after transfer to white light (WL) and incubation under long-day conditions on sugar-free Murashige and Skoog (MS) medium. (B) Percentage of seeds germinated after 7 d of incubation in the dark on sugar-free MS medium. (C) Percentage of seeds germinated under constant red light (R; 660 nm/3.4 μE). (D) Percentage of germinated seeds after 3 d of incubation under constant far-red light (FR; 724 nm/3.4 μE). Germination rate was determined after an additional 3 d in the dark. Data are means of four to six replicates of two independent seed batches. The mutant alleles used in all assays were ahk2-5, ahk3-7, and cre1-2.

Figure 5.

Hypocotyl Elongation of Cytokinin Receptor Mutants Shows No Altered Red Light Sensitivity. (A) Hypocotyl elongation in the light and in the dark 7 DAG. Error bars represent se (n = 15). (B) Hypocotyl elongation of Arabidopsis plants grown in continuous red light (R; 660 nm/3.4 μE, 4 DAG) or far-red light (FR; 724 nm/3.4 μE, 4 DAG). Error bars represent se (n > 30). (C) Reduction of hypocotyl elongation in red light or far-red light shown in (B) compared with the elongation in the dark. The ahk2-5 ahk3-7 cre1-2 triple mutant was used in all assays.

Figure 6.

Cytokinin-Dependent Deetiolation of Dark-Grown Seedlings Is Altered in Receptor Mutants. (A) Hypocotyl elongation in the dark 7 DAG on MS medium containing no BA or 3 μM BA. Error bars represent se (n = 15). (B) Deetiolation of wild-type and mutants seedlings 14 DAG on MS medium containing 60 μM BA. Bar = 10 mm. The mutant alleles used in all assays were ahk2-5, ahk3-7, and cre1-2.

Figure 7.

ahk2 ahk3 Double Mutants Form an Enhanced Root System. (A) Elongation of primary roots 14 DAG on standard MS medium. Error bars represent se (n ≥ 10). (B) Number of lateral roots of first and second order 14 DAG on standard MS medium. Error bars represent se (n ≥ 15). (C) Root system of in vitro–grown wild-type (left) and ahk2-5 ahk3-7 mutant plants 14 DAG. (D) Enhanced root system of ahk2-5 ahk3-7 mutant plants grown in vitro for 28 d on vertical plates (bottom) compared with the wild type (top). (E) Dry weight of the root system 21 DAG. The root system was harvested 3 weeks after germination, the roots of eight plants were pooled, and the dry weight of three independent pools per genotype was determined. Error bars represent se (n = 3). The mutant alleles used in all assays were ahk2-5, ahk3-7, and cre1-2.

Figure 8.

Contributions of Different Cytokinin Receptors and Receptor Combinations to Cytokinin-Regulated Processes. Cytokinin receptors that make a major contribution to a given process are printed in bold letters. The figure is based on data from this article. Effects of receptor loss-of-function mutations on fertility, plastochrone, leaf cell formation, primary root elongation, the root response to exogenous cytokinin, and in vitro shoot regeneration were also reported by others (Inoue et al., 2001; Higuchi et al., 2004; Nishimura et al., 2004).