The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner

Abstract

A number of Arabidopsis (Arabidopsis thaliana) lesion-mimic mutants exhibit alterations in both abiotic stress responses and pathogen resistance. One of these mutants, constitutive expresser of PR genes22 (cpr22), which has a mutation in two cyclic nucleotide-gated ion channels, is a typical lesion-mimic mutant exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense-related genes, and enhanced resistance to various pathogens; the majority of its phenotypes are SA dependent. These defense responses in cpr22 are suppressed under high-humidity conditions and enhanced by low humidity. After shifting plants from high to low humidity, the cpr22 mutant, but not the wild type, showed a rapid increase in SA levels followed by an increase in abscisic acid (ABA) levels. Concomitantly, genes for ABA metabolism were up-regulated in the mutant. The expression of a subset of ABA-inducible genes, such as RD29A and KIN1/2, was down-regulated, but that of other genes, like ABI1 and HAB1, was up-regulated in cpr22 after the humidity shift. cpr22 showed reduced responsiveness to ABA not only in abiotic stress responses but also in germination and stomatal closure. Double mutant analysis with nahG plants that degrade SA indicated that these alterations in ABA signaling were attributable to elevated SA levels. Furthermore, cpr22 displayed suppressed drought responses by long-term drought stress. Taken together, these results suggest an effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses in cpr22.

Figure 1.

Effect of humidity on cpr22 phenotypes. A, Morphology, chlorotic phenotypes, and spontaneous cell death formation of 4-week-old cpr22 and Ws-wt plants grown under 65% RH (top row), under 95% RH (middle row), or under 95% RH and shifted to 65% RH for 2 d (bottom row) at 22°C. B and C, Growth of H. arabidopsidis Emwa1 on Ws-wt and cpr22 plants grown under 65% or 95% RH conditions. Cotyledons of 7-d-old seedlings were inoculated with H. arabidopsidis Emwa1 (10⁶ spores mL⁻¹). At 7 d post infection, infected cotyledons were stained by trypan blue to visualize pathogen growth (B) or the number of sporangiophores on two cotyledons per plant was determined (C). Experiments were done three times with similar results. Bars = 1 cm (plants) and 125 μm (trypan blue staining).

Figure 2.

Quantitative real-time PCR analysis of NCED3, AAO3, PP2C, ABI1, RD29A, and KIN1/2 expression in cpr22 at 0, 12, and 24 h after shift from 95% RH to 65% RH conditions. Transcript levels were normalized to the expression of 18S RNA. Shown is the ratio of cpr22 to Ws-wt. Each bar represents the mean level of three replicates ± se. The experiment was repeated three times with similar results.

Figure 3.

ABA and SA increase after humidity shift in cpr22. ABA (A) and SA (B) levels in Ws-wt (white bars) and cpr22 (gray bars) at 0, 12, 24, and 36 h after the shift from 95% RH to 65% RH conditions were measured. The data represent mean levels of three extracts. Each extract was made from three to five plants. Experiments were repeated four times with similar results. DW, Dry weight.

Figure 4.

cpr22 displays enhanced dehydration. Plants were weighed at various times after detachment from their roots. A, Ws-wt (black circles) and cpr22 heterozygous (white squares) plants grown under ambient humidity conditions. B, Ws-wt (black circles) and cpr22 homozygous (white squares) plants grown under 95% RH conditions. C, Ws-wt plants 16 h after soil drenching with 1 mm SA (white circles), 4-hydroxybenzoic acid (4-HBA; white squares), or control solution (0.1% methanol in water, the same solution in which SA was dissolved; black diamonds). D, F2 progeny of cross-pollination between nahG transgenic (Nö background) and cpr22 (Ws background) plants. −/−/−/− plants carry neither cpr22 nor nahG (black diamonds), −/−/N/± plants carry nahG but not cpr22 (white squares), c/−/N/± plants are heterozygous for cpr22 and carry nahG (white triangles), and c/−/−/− plants are heterozygous for cpr22 but do not carry nahG (black circles). All plants were genotyped prior to experiments by PCR-based molecular markers (see “Materials and Methods”). Each bar represents the mean level of five plants ± se. All experiments were repeated three times with similar results. FW, Fresh weight.

Figure 5.

cpr22 plants display reduced sensitivity to ABA. A, Impairment in ABA-induced stomatal closure of Ws-wt (white bars) and cpr22 (gray bars) plants in the presence or absence of 10 μm ABA. Stomatal aperture is shown in μm (left panel) and in percentage of untreated plants (right panel); n = 3 experiments, 30 stomata per condition per experiment. Student's t test shows significant difference between the wild type and cpr22. B, Effects of ABA on germination of Ws-wt (white bars) and cpr22 homozygous (gray bars) seeds. The experiment was conducted with approximately 100 seeds per plate. Each bar represents the mean level of three plates ± se. Student's t test shows significant difference between Ws-wt and cpr22. The experiment was repeated more than three times with similar results. C, Four-week-old plants grown on soil were sprayed with 0, 10, and 100 μm ABA. Samples were taken at 0, 6, and 24 h after treatment, and gene expression was analyzed by semiquantitative RT-PCR. D, Three-week-old plants grown on MS agar plates were soaked with 10 or 100 μm ABA solution (for details, see “Materials and Methods”). Samples were taken at 0, 2, and 10 h after treatment, and gene expression was analyzed by semiquantitative RT-PCR. Actin (ACT2) served as a loading control in both experiments. All experiments were repeated three times with similar results.

Figure 6.

The nahG transgene rescues ABA-related phenotypes in cpr22. A, Quantitative real-time PCR analysis of NCED3, ABI1, and PP2C expression in F2 progeny of cross-pollination between nahG transgenic and cpr22 plants at 0, 12, and 24 h after shift from 95% RH to 65% RH. Transcript levels were normalized to the expression of 18S rRNA (multiplied by 1,000 for clarity). Each bar represents the mean level of three replicates ± se. Experiments were repeated three times with similar results. B, Elevated SA levels cause ABA accumulation in cpr22. F2 progeny of cross-pollination of nahG transgenic (Nö background) and cpr22 (Ws background) plants were used: −/−/−/− plants carry neither cpr22 nor nahG, −/−/N/± plants carry nahG but not cpr22, c/−/−/− plants are heterozygous for cpr22 but do not carry nahG, and c/−/N/± plants are heterozygous for cpr22 and carry nahG. All plants were grown under 95% RH conditions and then shifted to 65% RH conditions. ABA levels at 0 and 24 h after the shift from 95% RH to 65% RH conditions were measured. All plants were genotyped prior to experiments (see “Materials and Methods”). The data represent mean levels of three extracts. Each extract was made from three to five plants. Student's t test shows significant difference between cpr22 and cpr22/nahG plants. DW, Dry weight.

Figure 7.

cpr22 plants display an attenuated drought stress response. Moderate drought stress was applied by terminating irrigation using 4-week-old plants. The first sample was taken when the soil surface became dry (0 h). A, ABA levels in Ws-wt (white bars) and cpr22 heterozygous (gray bars) plants. The data represent mean levels of three extracts. Each extract was made from three to five plants. B, Quantitative real-time PCR analysis of NCED3, PP2C, RD29A, and KIN1/2 expression in Ws-wt (white bars) and cpr22 (gray bars) plants. Transcript levels were normalized to the expression of 18S rRNA (multiplied by 1,000 for clarity) measured in the same samples. Each bar represents the mean level of three replicates ± se. Experiments were repeated three times with similar results. DW, Dry weight.