Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response

Abstract

Cytokinin is an essential phytohormone controlling various biological processes, including environmental stress responses. In Arabidopsis, although the cytokinin (CK)-related phosphorelay--consisting of three histidine kinases, five histidine phosphotransfer proteins (AHPs), and a number of response regulators--has been known to be important for stress responses, the AHPs required for CK signaling during drought stress remain elusive. Here, we report that three Arabidopsis AHPs, namely AHP2, AHP3, and AHP5, control responses to drought stress in negative and redundant manner. Loss of function of these three AHP genes resulted in a strong drought-tolerant phenotype that was associated with the stimulation of protective mechanisms. Specifically, cell membrane integrity was improved as well as an increased sensitivity to abscisic acid (ABA) was observed rather than an alteration in ABA-mediated stomatal closure and density. Consistent with their negative regulatory functions, all three AHP genes' expression was down-regulated by dehydration, which most likely resulted from a stress-induced reduction of endogenous CK levels. Furthermore, global transcriptional analysis of ahp2,3,5 leaves revealed down-regulation of many well-known stress- and/or ABA-responsive genes, suggesting that these three AHPs may control drought response in both ABA-dependent and ABA-independent manners. The discovery of mechanisms of activation and the targets of the downstream components of CK signaling involved in stress responses is an important and challenging goal for the study of plant stress regulatory network responses and plant growth. The knowledge gained from this study also has broad potential for biotechnological applications to increase abiotic stress tolerance in plants.

Fig. 1.

Loss-of-function of AHP2, AHP3, and AHP5 results in enhanced drought tolerance. (A) Two-week-old WT and ahp2,3,5 plants were transferred from germination medium (GM) plates to soil and grown for an additional week. (B) Three-week-old plants were exposed to drought stress for 13 d and photographed 3 d subsequent to rewatering and after the removal of inflorescences. (C) For control purposes, 2-wk-old WT and ahp2,3,5 plants were transferred from GM plates to trays and grown under well-watered conditions in parallel with the drought test as shown in (B). (D) Soil relative moisture contents and relative humidity were monitored during the drought tolerance test (A and B). (E) Survival rates and SEs (error bars) were calculated from the results of three independent experiments (n = 30 plants/genotype). Asterisks indicate significantly higher survival rates than WT as determined by a Student’s t test analysis (***P < 0.001). (F) Two-week-old WT and ahp2,3,5 plants were transferred to soil in an alternate order and grown for an additional week. (G) Three-week-old plants were exposed to drought stress; plants were photographed 20 d after the withholding of water. (H) For control purposes, WT and ahp2,3,5 plants were grown in an alternate order in parallel under well-watered conditions.

Fig. 2.

CK-dependent repression of AHP2, AHP3, and AHP5 genes under stress conditions. (A) Expression of AHP2, AHP3, and AHP5 genes in 2-wk-old WT plants after exposure to dehydration, salt stress (250 mM), and ABA (100 µM) treatments. Relative expression levels were normalized to a value of 1 in the respective mock control plants. Data represent the means and SEs of three independent biological replicates. The stress-inducible RD26 gene was used as ABA, NaCl, and dehydration stress markers to confirm the efficacy of our stress treatments. (B) Down-regulated expression of AHP2, AHP3, and AHP5 genes in 10-d-old WT and CK-deficient plants. Relative expression levels were normalized to a value of 1 in WT plants. Data represent the means and SE of four independent biological replicates.

Fig. 3.

Comparison of RWC, electrolyte leakage, stomatal aperture, and density of WT and ahp2,3,5 plants. (A) The WT and ahp2,3,5 plants were grown and exposed to drought stress as described in Fig. 1 A and B. At the indicated time points, plants were harvested for measurement of RWC. Error bars represent SEs (n = 5). (B) Soil relative moisture contents and relative humidity were recorded just before sample collection for measurements of RWC and electrolyte leakage. (C) Average stomatal aperture of rosette leaves from 4-wk-old WT and ahp2,3,5 plants in the presence or absence of ABA. Error bars represent SEs (n > 28). (D) Average stomatal density of rosette leaves from 4-wk-old WT and ahp2,3,5 plants. Error bars represent SE (n = 25). (E) Electrolyte leakage of the WT and ahp2,3,5 plants exposed to drought stress as described in (A). Error bars represent SEs (n = 5). (F) Comparison of electrolyte leakage levels between WT and ahp2,3,5 plants collected at a similar RWC during drought stress. Error bars represent SE (n = 5). Asterisks indicate significant differences as determined by a Student’s t- test analysis (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 4.

Response of the ahp double and triple mutants to treatment with exogenous ABA. Seeds were sown on germination medium-1% sucrose containing the indicated ABA concentrations; germination rates were quantified after 4 d of incubation by counting the number of open cotyledons. Error bars indicate SEs that were calculated from the results of four independent experiments. Asterisks indicate significant differences as determined by a Student’s t test analysis (*P < 0.05, **P < 0.01, ***P < 0.001).