Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis

Abstract

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.

Figure 1.

max2 is strongly hypersensitive to drought stress and exhibits reduced sensitivity to ABA-induced stomatal closure. A, max2 is strongly hypersensitive to drought stress. Ten-day-old seedlings (left column) were subjected to drought conditions by withholding water for 7 d and then rewatered. Photographs were taken 2 d after rewatering (right column). B, max2 alleles lost water faster than the wild type (Wt). Leaves of the same developmental stages were excised and weighed at various time points after detachment. Values are means ± sd of three individual plants per genotype. Experiments were repeated at least three times with similar results. C, max2 is hyposensitive to ABA in a stomatal closure assay. Aperture ratios (width-length) were measured 2 h after the addition of 1 or 10 µm ABA. Error bars represent se for three independent experiments with 45 stomata per data point. D, max2 is hyposensitive to drought-induced stomatal closure. Stomatal aperture was observed at various time points after detachment. Values are means ± sd of three individual plants per genotype. Experiments were repeated at least three times with similar results. Significant differences between the wild type and max2-1 are shown by asterisks (**P < 0.01, calculated by Student’s t test). [See online article for color version of this figure.]

Figure 2.

The cuticle thickness of max2 is thinner than that of the wild type. A, TEM images of cuticle membranes of leaf and stem. Leaves and inflorescence stems of 4-week-old wild-type Col-0 and max2-1 mutant plants grown in soil were subjected to TEM. B, Leaf and stem cuticle thickness analysis by TEM. Each value is the mean ± se of five independent measurements. Significant differences between the wild type and max2-1 are shown by asterisks (**P < 0.01, calculated by Student’s t test). C, Chlorophyll leaching assay of the wild type and the max2-1 mutant. Extracted chlorophyll contents at individual time points were expressed as percentages of total chlorophyll extracted at 24 h after initial immersion. Each value is the mean ± se of five independent measurements.

Figure 3.

The expression of stress-responsive genes was impaired in the max2 mutant. The expression of stress-responsive genes was assayed by qRT-PCR in seedlings of the wild type and max2-1. Two-week-old seedlings were subjected to drought stress for 0, 1, and 3 h. Total RNA was extracted at the indicated times. Transcript levels were quantified by qRT-PCR using PP2A as a control. Data shown are means ± sd of three independent experiments.

Figure 4.

The expression of genes involved in ABA biosynthesis, catabolism, transport, and early signaling is impaired in the max2 mutant compared with the wild type. The expression of genes involved in ABA biosynthesis, catabolism, transport, and early signaling was assayed by qRT-PCR in seedlings of Col-0 and max2-1. Two-week-old seedlings were subjected to drought stress for 0, 1, and 3 h. Total RNA was extracted at the indicated times. Transcript levels were quantified by qRT-PCR using PP2A as a control. Data shown are means ± sd of three independent experiments.

Figure 5.

The max2 mutant was hypersensitive to ABA during early seedling development. A, Time course for cotyledon greening of the Col-0, max2-1, max2-2, and pps genotypes grown on medium containing 0.5 µm ABA. Data shown are means ± sd of three replicates. At least 100 seeds per genotype were scored in each replicate. B, Cotyledon greening percentage of the indicated genotypes grown on different concentrations of ABA. Greening percentage was recorded for 3-d-old seedlings. Data shown are means ± sd of three replicates. At least 100 seeds per genotype were scored in each replicate. C, Bar graph showing the root lengths of wild-type and max2-1 seedlings grown on MS medium containing different concentrations of ABA. Germinated seedlings with approximately 1-cm root length were transferred to ABA-containing plates. Root lengths were measured after 7 d of growth. D, Photographs of wild-type and max2-1 seedlings grown on an MS plate (top panel), an MS plate containing 20 µm ABA (middle panel), and an MS plate containing 50 µm ABA (bottom panel). [See online article for color version of this figure.]

Figure 6.

Phenotypic characterization of max2/abi double mutants. A, max2 is epistatic to abi3 and abi5 for the hypocotyl elongation phenotype under white light. The bar graph shows the hypocotyl lengths of the wild type and max2, abi3, abi5, max2/abi3, and max2/abi5 mutants grown on MS medium under continuous white light. B, abi3 is epistatic to max2 for the ABA-regulated seedling growth response. Photographs show wild-type, max2-1, abi3-8, and max2-1/abi3-8 seedlings grown on an MS plate (top panel) and an MS plate containing 0.8 µm ABA (bottom panel). C, abi5 is epistatic to max2 for the ABA-regulated seedling growth response. Photographs show wild-type, max2-1, abi5-7, and max2-1/abi5-7 seedlings grown on an MS plate (top panel) and an MS plate containing 0.8 µm ABA (bottom panel). [See online article for color version of this figure.]

Figure 7.

ABA-regulated genes have enhanced expression in max2 seeds. The expression of ABI3, ABI5, and AtEM1 in dry seeds (DS) and during seed imbibition was determined by qRT-PCR. Imbibed seeds were sown on medium with or without ABA (5 µm) under constant light at 22°C for germination. Total RNA was extracted from the seeds at the indicated times. Transcript levels were quantified by qRT-PCR using PP2A as a control. Data shown are means ± sd of three independent biological repeats.

Figure 8.

ABA regulates the expression of MAX2. A, MAX2 expression pattern in dry seeds (DS) and during seed imbibition determined by qRT-PCR. Imbibed seeds were sown on medium with or without ABA (5 µm) in constant light at 22°C for germination. AtEM1 was used as a control. Total RNA was extracted at the indicated times. Transcript levels were quantified by qRT-PCR using PP2A as a control. Data shown are means ± sd of three independent biological replicates. B, ABA-mediated repression of MAX2 expression revealed by qRT-PCR. Two-week-old wild-type seedlings were treated with 50 µm ABA for 0, 3, and 6 h. Transcript levels were quantified by qRT-PCR using PP2A as a control. Data shown are means ± sd of three independent biological replicates. C, MAX2 expression pattern in dry seeds of the wild type and abi3, abi4, and abi5 mutants determined by qRT-PCR. AtEM1 was used as a control. Total RNA was extracted from the dry seeds. Transcript levels were quantified by qRT-PCR using PP2A as a control. Data shown are means ± sd of three independent biological replicates.

Figure 9.

max2 was hypersensitive to osmotic stress during seed germination. A, C, and E, Time course for germinative cotyledon greening of Col-0, max2-1, max2-2, and pps seedlings grown on medium containing 150 mm NaCl (A), 300 mm mannitol (C), or 4% Glc (E). Data shown are means ± sd of three replicates. At least 100 seeds per genotype were scored in each replicate. B, D, and F, Germinative cotyledon-greening percentage of the indicated genotypes grown on different concentrations of NaCl (B), mannitol (D), or Glc (F). Cotyledon greening was recorded for 3-d-old seedlings. Data shown are means ± sd of three replicates. At least 100 seeds per genotype were scored in each replicate.

Figure 10.

Strigolactone is not necessary for the ABA and osmotic stress response pathways. A, Photographs of Col-0, max1, max2-1, max3, and max4 genotypes grown on medium containing 0.5 µm ABA, 150 mm NaCl, or 4% Glc. Photographs were taken for 3-d-old seedlings. B, D, and F, Time course for germinative cotyledon greening of Col-0, max1, max2-1, max3, and max4 seedlings grown on medium containing 0.5 µm ABA (B), 150 mm NaCl (D), or 4% Glc (F). Data shown are means ± sd of three replicates. At least 100 seeds per genotype were scored in each replicate. C, E, and G, Percentage of germinative cotyledon greening for the indicated genotypes grown on different concentrations of ABA (C), NaCl (E), or Glc (G) recorded for 3-d-old seedlings. Data shown are means ± sd of three replicates. At least 100 seeds per genotype were scored in each replicate. [See online article for color version of this figure.]

Figure 11.

Strigolactone is not necessary for the drought response. A, Strigolactone biosynthetic mutants are not defective in drought stress. Two-week-old seedlings on soil were subjected to withholding water for 10 d to induce drought stress. The top panel shows seedlings before drought treatment, and the bottom panel shows seedlings after 10 d of drought treatment. B, Strigolactone biosynthetic mutants lose water similar to wild-type controls. Leaves of the same developmental stages were excised and weighed at various time points after detachment. Values are means ± sd of three individual plants per genotype. Experiments were repeated at least three times with similar results. [See online article for color version of this figure.]