Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development

Abstract

The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.

Figure 1.

Characterization of the arr1-3, arr10-5, and arr12-1 T-DNA Insertion Alleles. (A) Positions of T-DNA insertions in genes encoding ARR1, ARR10, and ARR12. Black boxes represent exons of the coding sequence, lines represent introns, and triangles indicate positions of T-DNA insertions. (B) RNA gel blot analysis. Poly(A) RNA from the wild type, arr1-3, an arr1-3 arr10-5 double mutant, and an arr11-2 arr12-1 double mutant was analyzed by RNA gel blot analysis using ARR1, ARR10, ARR12, or a control ubiquitin (UBQ) probe as indicated.

Figure 2.

Primary Root Characteristics of T-DNA Insertion Mutants. (A) Root elongation responsiveness to cytokinin of arr mutants. Seedlings were grown on vertical plates supplemented with 1 μM BA or a DMSO vehicle control under constant light (100 μE·m⁻²·s⁻¹) at 22°C. Increase in root length from day 4 through day 7 was measured. The root elongation of each line receiving cytokinin treatment is expressed as a percentage of its DMSO control. Error bars represent se (n > 10). The triple mutant arr1-3 arr10-2 arr12-1 is abbreviated arr1-3,10-2,12-1; double mutants are similarly abbreviated. Genotypes were analyzed for significant differences in their responsiveness to cytokinin based on Duncan's multiple range test among the means on the analysis of variance (P < 0.05). Those genotypes having the same letter exhibit no significant difference in their responsiveness to cytokinin. (B) Dose response for the effect of BA on arr mutants grown under constant low light (25 μE·m⁻²·s⁻¹). Error bars represent se (n > 18). (C) CycB1;1:GUS expression in wild-type and arr1-3 arr12-1 primary roots grown for 8 d on the indicated concentrations of BA. Bar = 0.1 mm.

Figure 3.

The arr1-3 arr10-5 arr12-1 Triple Mutant Exhibits a Short Primary Root with Altered Vascular Development. (A) Wild-type and arr1-3 arr10-5 arr12-1 seedlings grown for 8 d in the absence (−) and presence (+) of 10 μM at 25 μE·m⁻²·s⁻¹ constant light. Squares on the plate are 13.3 mm per side. (B) Wild-type and arr1-3 arr10-5 arr12-1 seedlings grown for 18 d at 25 and 100 μE·m⁻²·s⁻¹ constant light (hv). A closeup of an arr1-3 arr10-5 arr12-1 seedling grown under 100 μE·m⁻²·s⁻¹ light is shown at right; note the adventitious root (arrowhead) and the purple curled cotyledons. Squares on the plate are 13.3 mm per side. (C) Time course for primary root growth of wild-type and ARR mutant seedlings (100 μE·m⁻²·s⁻¹ constant light; similar results were obtained for seedlings grown under 25 μE·m⁻²·s⁻¹ constant light). (D) CycB1;1:GUS expression in primary and adventitious arr1-3 arr10-5 arr12-1 roots from a 29-d-old seedling. Bar = 0.1 mm. (E) and (F) Fuschin-stained roots showing vasculature. The protoxylem (arrowheads) and metaxylem (asterisks) are indicated on the wild-type root images. Seedlings were grown under 100 μE·m⁻²·s⁻¹ constant light for 7 d in the absence (−) or presence (+) of 0.1 μM BA (E) or under 25 μE·m⁻²·s⁻¹ constant light for 21 d in the absence or presence of 10 μM BA to allow time for adventitious roots to develop (F). The ahk2 ahk3 ahk4 cytokinin receptor triple mutant is included for comparison. Bars = 5 μm.

Figure 4.

Hypocotyl Elongation and Deetiolation Response of arr Mutants to Cytokinin. (A) Dose response for the effect of BA on hypocotyl elongation of 5-d-old seedlings grown under 25 μE·m⁻²·s⁻¹ constant light. The hypocotyl length of each line receiving cytokinin treatment is expressed as a percentage of its DMSO control. The triple mutant arr1-3 arr10-5 arr12-1 is abbreviated arr1-3,10-5,12-1; other higher order mutants are similarly abbreviated. Error bars indicate se (n > 15). (B) Effect of 10 μM BA on hypocotyl elongation in 4-d-old dark-grown seedlings. The hypocotyl length of each line receiving cytokinin treatment is expressed as a percentage of its DMSO control. Error bars indicate se (n > 16). Those genotypes having the same letter exhibit no significant difference in their responsiveness to cytokinin. (C) Deetiolation response in 14-d-old dark-grown seedlings grown on 30 μM BA. Bar = 10 mm.

Figure 5.

Shoot Phenotypes of arr1-3 arr10-5 arr12-1 Triple Mutants. (A) Rosettes of wild type, arr1-3 arr10-5 arr12-1 (arr1,10,12), and ahk2 ahk3 ahk4 (ahk2,3,4) plants grown under short days (8-h daylength) at 60 d old. The ahk2 ahk3 ahk4 plant is circled. Mean rosette diameters are indicated, with se in parentheses (n > 3). Pot diameter is 62 mm. (B) Adaxial epidermal cells from fully expanded leaf 5 of wild-type and arr1-3 arr10-5 arr12-1 plants shown in (A). Mean cells/mm² and cells/leaf are indicated, with se in parentheses (n = 3). Bar = 0.1 mm. (C) Real-time RT-PCR analysis of CYCD3;1 expression in wild-type and arr1-3 arr10-5 arr12-1 14-d-old shoots. Expression level of the wild type is set to 1. (D) Primary inflorescence stems of wild-type (left) and arr1-3 arr10-5 arr12-1 (right) plants. Bar = 2 mm. (E) Altered spacing of siliques in arr1-3 arr10-5 arr12-1. Two examples of the mutant phenotype are shown, with arrows indicating positions with altered silique spacing. Bar = 10 mm. (F) Mean length of seeds from the wild type and arr mutants. Error bars indicate se (n = 60). The triple mutant arr1-3 arr10-5 arr12-1 is abbreviated arr1,10,12; other mutants are similarly abbreviated. Those genotypes having the same letter exhibit no significant difference in their seed length. The inset shows wild-type (top) and arr1-3 arr10-5 arr12-1 (bottom) seeds. (G) Effect of cytokinin treatment on the growth of the wild type and arr1-3 arr10-5 arr12-1 triple mutants. Images show 26-d-old plants grown under constant light (25 μE·m⁻²·s⁻¹) treated with a DMSO vehicle control or with 0.1 μM BA. Fresh weight (FW) and total chlorophyll (Chl) levels are indicated, with sd in parentheses, based on three replicates of five seedlings each. Bar = 5 mm.

Figure 6.

Altered Levels of Anthocyanin and Chlorophyll in arr1-3 arr10-5 arr12-1 and ahk2 ahk3 ahk4 Compared with the Wild Type. Fourteen-day-old seedlings were grown under either 25 or 100 μE·m⁻²·s⁻¹ constant light. Seedlings of the wild type, the arr1-3 arr10-5 arr12-1 triple mutant, the ahk2 ahk3 ahk4 triple receptor mutant, and a mixed population of ahk2 ahk4 and ahk2 ahk3(+/−) ahk4 seedlings were examined for both anthocyanin and chlorophyll levels. Each sample represents the mean of two replicates of 10 seedlings each.

Figure 7.

Reduced Transcriptional Response of Cytokinin-Regulated Genes in Shoots of arr1-3 arr10-5 arr12-1 Triple Mutants Compared with the Wild Type. Fourteen-day-old wild-type and arr1-3 arr10-5 arr12-1 (arr1,10,12) seedlings were treated with either 10 μM BA or a DMSO control for 1 h, and the gene expression was analyzed in shoots by either microarray ([A] to [C]) or real-time RT-PCR (D). For microarray analysis, absolute expression levels are given relative to the normalized expression value of 1 based on all genes represented on the array. (A) Box-plot analysis for the expression levels of 71 genes induced (Up) and 95 genes repressed (Down) threefold or more based on microarray analysis. Whiskers are drawn to 1.5× the interquartile distance, with genes whose expression lies outside this range indicated by open circles. (B) Expression levels of 71 genes induced threefold or more by BA treatment in the wild type compared with the arr1-3 arr10-5 arr12-1 mutant. Expression levels are indicated by color according to the scale. (C) Expression of known cytokinin primary response genes based on microarray analysis. Target genes are as described (Taniguchi et al., 2007) and include type A ARRs, the cytokinin oxidase CKX4, the cytokinin hydroxylase CYP735A2, a putative disease resistance response protein (DRRP), the expansin EXP1, and a transferase family protein. (D) Real-time RT-PCR analysis of the cytokinin-regulated genes ARR5, ARR7, ARR15, CKX4, and AHK4 in wild-type and arr1-3 arr10-5 arr12-1 (arr) mutant shoots. The key is the same as that used in (C).