Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Abstract

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.

Figure 1.

Salt- and Drought-Tolerant Phenotypes of the CK-Deficient CKX Overexpresser Plants. (A) Plants were grown on GM plates for 10 d and were transferred on 0.5× MS/0 mM NaCl medium for 6 d. WT, wild type. (B) Plants were grown on GM plates for 10 d and were transferred on 0.5× MS/200 mM NaCl medium for 6 d. (C) Survival rates and se values (error bars) were calculated from the results of three independent experiments (n = 30 plants/genotype). (D) Two-week-old wild-type and CKX overexpresser plants were transferred from GM plates to soil and grown for 1 additional week. (E) Three-week-old plants were exposed to drought stress for 14 d and plants were photographed 3 d subsequent to rewatering and after removal of inflorescences. (F) Survival rates and se values (error bars) were calculated from the results of three independent experiments (n = 30 plants/genotype). Asterisks indicate significantly higher survival rates than the wild type as determined by Student’s t test (* P < 0.01; ** P < 0.005; *** P < 0.001).

Figure 2.

Salt- and Drought-Tolerant Phenotypes of the CK-Deficient ipt1 3 5 7 Mutant. (A) Plants were grown on GM plates for 10 d and were transferred to 0.5× MS/0 mm NaCl medium for 6 d. WT, wild type. (B) Plants were grown on GM plates for 10 d and were transferred to 0.5× MS/200 mm NaCl medium for 6 d. (C) Survival rates and se values (error bars) were calculated from the results of three independent experiments (n = 30 plants/genotype). Asterisks indicate significantly higher survival rates than wild type as determined by Student’s t test (*** P < 0.001). (D) Two-week-old wild-type and ipt1 3 5 7 plants were transferred from GM plates to soil and grown for 1 additional week. (E) Three-week-old plants were exposed to drought stress for 14 d and plants were photographed 3 d subsequent to rewatering and after removal of inflorescences. (F) Soil relative moisture contents were monitored during the drought tolerance test of the ipt1 3 5 7 mutant.

Figure 3.

RWC, Electrolyte Leakage, Stomatal Aperture, and Stomatal Density of the CK-Deficient Plants. (A) The wild type (WT), ipt1 3 5 7, 35S:CKX1, and 35S:CKX3 plants were grown and exposed to drought stress as described in Figures 2D and 2E. At the indicated time points, plants were harvested for measurement of RWC. Error bars = se values (n = 5). (B) Average stomatal aperture of rosette leaves from 4-week-old wild-type and CK-deficient plants in the presence or absence of ABA. Error bars = se values (n > 17). (C) Average stomatal density of rosette leaves from 4-week-old wild-type and CK-deficient plants. Error bars = se values (n > 3). (D) The wild-type and CK-deficient plants were grown and exposed to drought stress as described in Figures 2D and 2E. At the indicated time points, plants were harvested for measurement of relative electrolyte leakage. Error bars = se values (n = 5). Asterisks indicate significant differences as determined by a Student’s t test (* P < 0.05; ** P < 0.01; *** P < 0.001).[See online article for color version of this figure.]

Figure 4.

Altered ABA Biosynthesis in CK-Deficient Plants. (A) Average levels of ABA in 10-d-old plants grown on GM plates. Error bars = se values of three biological replications. Asterisks indicate significant differences as determined by a Student’s t test (** P < 0.01). FW, fresh weight; WT, wild type. (B) Expression of ABA biosynthetic genes ABA1, ABA2, AAO3, and NCED3 in 10-d-old wild-type and CK-deficient plants grown on GM plates. Mean relative expression levels were normalized to a value of 1 in wild-type plants. Error bars = se values of three biological replicates.

Figure 5.

ABA Response of CK-Deficient Plants. (A) Response of the CK-deficient plants to exogenous ABA as determined by a growth inhibition assay. Relative fresh weights of plants were determined after 14 d of incubation at 22°C. Errors bars = se values (n = 7, where each measurement represents the weight of six plants). WT, wild type. (B) Response of the CK-deficient plants to exogenous ABA as determined by a germination assay. Germination rates were determined after 6 d of incubation at 22°C by counting the number of open cotyledons. se values (error bars) were calculated from the results of four independent experiments. Asterisks indicate significant differences as determined by a Student’s t test (* P < 0.05; ** P < 0.01; *** P < 0.001). (C) Endogenous ABA levels in the CK-deficient plants under dehydration and salt stresses. Error bars = se values of three biological replications. Printed numbers represent the fold change over the untreated sample from the same genotype. FW, fresh weight. (D) Expression of ABA signaling marker genes in CK-deficient plants under 2- and 5-h dehydration and salt stress. Data represent the means and se values of three independent biological replicates.

Figure 6.

Expression of Arabidopsis IPT and CKX Genes in 2-Week-Old Wild-Type Plants under Various Stress and Hormone Treatments. Relative expression levels for IPT and CKX genes were normalized to a value of 1 in the respective mock control plants. Data represent the means and se values of three independent biological replicates. The stress-inducible RD26 gene was used to confirm that the treatments were effective in stimulating stress-responsive gene expression.

Figure 7.

CK Contents in Stressed Wild-Type Plants and Expression of CKX Genes in CK-Deficient Plants. (A) Endogenous CK levels in the wild-type Arabidopsis plants under dehydration (D2, 2-h dehydration; D5, 5-h dehydration) and salt stress (S2, 2-h salt stress; S5, 5-h salt stress). Data were calculated from three independent biological replicates. se values for each CK metabolite are available in Supplemental Data Set 2 online. Asterisks indicate significant differences as determined by a Student’s t test (* P < 0.05; ** P < 0.01; *** P < 0.001). FW, fresh weight. (B) Downregulated expression of the CKX genes in CK-deficient plants. Ten-day-old plants were grown on GM plates and were harvested for expression analysis. Relative expression levels of the CKX genes were normalized to a value of 1 in the wild-type (WT) plant. Data represent the means and se values of three independent biological replicates.

Figure 8.

Model for the Role of Bioactive CKs under Stresses. Upon stress, IPT gene expression is reduced, leading to a decrease in CK accumulation. The stress-induced ABA levels also downregulate the expression of IPT genes, which results in a further decrease in CK contents. Because of a reduction in CK content, the inhibitory effect of CK signaling on the expression of stress responsive genes is alleviated (dotted bar), leading to enhanced plant survival.