Figure 1.
Hot leaf phenotype of has aba3-1 and has mutants visualised by thermal imaging.
False colour infrared image of the temperature of drought stressed plants. (A–C) leaf temperature of has aba3-1 mutants compared to wild-type (WT) and the aba3-1 mutant. (D–F) leaf temperature of has mutants compared to wild-type. Plants were 14 days old and watering had been withheld for 2 days. Scale indicates temperature (°C).
Figure 2.
has aba3-1 suppressor mutants show reduced water loss on progressive drought stress.
Watering was withheld from three-week old plants for seven days and soil allowed to dry, not watered, or continued, water control. Water content (WC) was calculated as the % (w/w) of rosette weight corresponding to water, as determined from plant weight before and after freeze-drying. WT, wild-type. Error bars represent SE values (n = 4). Similar results were obtained in 2 independent experiments.
Table 1.
Segregation analysis of three HAS loci in backcrosses to original aba3-1 mutant indicates that the three mutations are recessive.
Table 2.
Chromosome location of three HAS loci.
Figure 3.
The has loci do not modify ABA accumulation.
A, The ABA-deficient phenotype is not suppressed in has aba3-1 mutants. Results presented are representative of those obtained in 2 independent experiments. B, has mutants do not have higher ABA levels than wild-type. ABA contents were determined in rosette leaves after 4 h of dehydration (dehydrated) and compared with non-dehydrated (control) plants. No significant difference was found in Student t-tests comparing has aba3-1 to aba3-1 or has to wild-type. WT, wild-type; DW, dry weight. Error bars represent SE values (n = 3).
Figure 4.
Reduced rosette size and root length of has2 and has3 mutants.
Wild-type has1, has2 and has3 A, rosettes grown in long-day photoperiod for 14–18 days. B, rosettes grown in short-day photoperiod for 55 to 73 days. C, primary root length of 13 d old seedlings grown in long-day photoperiod. WT, wild-type. Error bars represent SE values (b, n = 4; c, n = 24). Similar results were obtained in 2 independent experiments. Student t-test p<1%, ** or p<0.1%, ***.
Figure 5.
The has1 mutation improves resistance to rapid dehydration and causes the overexpression of RD29B, RD29A and RD22.
A, Rapid dehydration of plants carrying the has1 mutation alone or with the aba3-1 mutation. Water loss is expressed as a percentage of the initial fresh weight (FW). Error bars represent SE values (n = 4). Results presented are representative of those obtained in 4 independent experiments. Quantitative RT-PCR analysis of drought inducible gene expression in wild-type and has mutants, B, RD29B, C, ERD1, D, RD29A and E, RD22 transcript abundance in leaves after 4 h at a water-deficit equivalent to the loss of 25% (w/w) of the fresh weight (stressed) compared to controls (non-stressed). Steady-state mRNA levels are represented as a percentage of the constitutive EF1α-4a gene (EF) abundance. Error bars represent SE values (n = 3). WT, wild-type. Similar results were obtained for samples derived from 2 independent plants.
Figure 6.
has mutants show ABA-hypersensitivity compared to wild-type.
A, Induction of stomatal closure by ABA in wild-type and has mutants. Stomata aperture ratios (width/length) were measured after a 2 h pre-treatment in the light in stomata opening solution followed by a 3 h incubation with or without ABA at the concentrations indicated. Data are means ± SE of 3 independent experiments with 40 apertures measured per experiment and condition. Significance in Student t-tests comparing samples with and without ABA for the same genotype; **, p<1% or ***, p<0.1% level. B, ABA inhibition of root growth. Primary root length of 13 d old seedlings grown in long day photoperiod ABA was included in the growth media at the concentrations indicated. Error bars represent SE values (n = 24). Results presented are representative of those obtained in 2 independent experiments. C, ABA inhibition of seed germination after 7 d at 25°C. ABA was included in the growth media at the concentrations indicated. Germination was determined from the number of seedlings with green cotyledons compared to the total number of seeds sown. Error bars represent SE values (n = 3). Results presented are representative of those obtained in 2 independent experiments. Significance in Student t-test when comparing mutant and wild-type at a given ABA concentration, p<0.1%, ***. WT, wild-type.
Figure 7.
Level of Dickeya dadantii bacterial infection of has mutants.
Degree of infection of wild-type, has1, has2 and has3 plants (40 to 48 individuals) and its evolution over 5 days after inoculation. The scale of infection is denoted by: 0: no symptom; 1: maceration limited to the inoculation point; 2: maceration extends from the infection point; 3: maceration covers half the lamina; 4: maceration spread over the whole lamina; 5: maceration spread over the whole leaf (lamina and petiole). Similar results were obtained in 2 independent experiments. *, significant differences, p<0.05%, between wild-type and mutants in Fisher tests comparing infection scores of ≥3.
Figure 8.
Schematic representation of the HAS loci mode of action as putative negative regulators in ABA signalling responses to Dickeya dadantii infection and water deficit.
Responses to water deficit and Dickeya dadantii infection are highlighted in blue or orange, respectively. Line width reflects extent of locus effect on a given response.