Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis

Abstract

AtMYB44 belongs to the R2R3 MYB subgroup 22 transcription factor family in Arabidopsis (Arabidopsis thaliana). Treatment with abscisic acid (ABA) induced AtMYB44 transcript accumulation within 30 min. The gene was also activated under various abiotic stresses, such as dehydration, low temperature, and salinity. In transgenic Arabidopsis carrying an AtMYB44 promoter-driven beta-glucuronidase (GUS) construct, strong GUS activity was observed in the vasculature and leaf epidermal guard cells. Transgenic Arabidopsis overexpressing AtMYB44 is more sensitive to ABA and has a more rapid ABA-induced stomatal closure response than wild-type and atmyb44 knockout plants. Transgenic plants exhibited a reduced rate of water loss, as measured by the fresh-weight loss of detached shoots, and remarkably enhanced tolerance to drought and salt stress compared to wild-type plants. Microarray analysis and northern blots revealed that salt-induced activation of the genes that encode a group of serine/threonine protein phosphatases 2C (PP2Cs), such as ABI1, ABI2, AtPP2CA, HAB1, and HAB2, was diminished in transgenic plants overexpressing AtMYB44. By contrast, the atmyb44 knockout mutant line exhibited enhanced salt-induced expression of PP2C-encoding genes and reduced drought/salt stress tolerance compared to wild-type plants. Therefore, enhanced abiotic stress tolerance of transgenic Arabidopsis overexpressing AtMYB44 was conferred by reduced expression of genes encoding PP2Cs, which have been described as negative regulators of ABA signaling.

Figure 1.

Northern blots of AtMYB44 expression. A, Induction of AtMYB44 by ABA. Sterilized water (nontreatment; NT), 100 μm methyl jasmonate (MJ), 100 μm ABA, or 50 μm ethephon (ET) was applied to the surface of solid Murashige and Skoog agar medium in which 2-week-old Arabidopsis seedlings were growing. Total RNA was extracted from plants harvested at the indicated times after each treatment. B, Induction of AtMYB44 and RD29A by abiotic stresses. Two-week-old seedlings were dried on Whatman 3MM paper (Dry), treated with 250 mm NaCl (NaCl), or incubated at 4°C (Cold).

Figure 2.

Localization of AtMYB44 expression. A, Histochemical GUS assay. An approximately 3.0-kb fragment of the AtMYB44 promoter was fused to the GUS gene and transformed into Arabidopsis. Histochemical assays for GUS activity in transgenic plants were performed as described by Jefferson et al. (1987). GUS staining patterns were confirmed by observing at least eight different transgenic lines. 1, Rosette leaf; 2, flower; 3, inflorescence; 4, floral nectar; 5, stamen; 6, carpel; 7, petal; 8, sepal. B, GUS activity in transgenic Arabidopsis seedlings grown on Murashige and Skoog medium. 1, One-week-old whole seedling; 2, root tip (1 week old); 3, paradermal section of the abaxial epidermis (200×) from 2-week-old plant. Scale bar = 20 μm. C, Subcellular localization of AtMYB44 protein. AtMYB44 cDNA was fused to GFP and the construct was expressed in transgenic Arabidopsis under the control of the CaMV 35S promoter. GFP fluorescence patterns were confirmed by observing at least five different transgenic lines under a confocal laser-scanning microscope. 1, GFP fluorescence; 2, differential interference contrast (DIC; optical microscopic image); 3, merged image (GFP + DIC); 4, GFP from 35S:GFP control plant. Scale bars = 20 μm for the images from the 35S:AtMYB44-GFP plant (1, 2, and 3) and 10 μm for that from the 35S:GFP plant (4), respectively.

Figure 3.

Blot analyses of transgenic Arabidopsis. AtMYB44 cDNA was fused to the CaMV 35S promoter and transformed into Arabidopsis (35S:AtMYB44). T-10, T-14, T-17, T-18, and T-21 denote the transgenic line. The atmyb44 knockout line (SALK_039074) was obtained from the SALK collection. A, Southern blot indicating copy numbers of the inserted T-DNA. Genomic DNA was digested with XbaI (X) and EcoRI (E), and the blot was hybridized with a NEOMYCIN PHOSPHOTRANSFERASEII (NPTII) probe. B, Northern blot demonstrating the constitutive expression of AtMYB44 in transgenic plants. C, Western blot showing the AtMYB44 protein levels in transgenic plants. Asterisk, AtMYB44 protein band (approximately 33 kD); arrowheads, bands of two unknown cross-reacted proteins (approximately 40 and 29 kD, respectively).

Figure 4.

Growth of transgenic Arabidopsis overexpressing AtMYB44. A, One-week-old seedlings grown on Murashige and Skoog medium. Scale bar = 1 cm. B, Growth of rosette leaves after growing on soil. Scale bars = 1 cm for all the images. C, Appearance of transgenic plants 5 weeks after sowing. D, Flowering time of 35S:AtMYB44 plants. The time (DAS) at which the main inflorescence shoot had elongated to 1 was recorded. In addition, the number of rosette leaves when plants were flowering was counted. In all cases, 20 plants were counted to calculate the average ± sd. [See online article for color version of this figure.]

Figure 5.

Responses of 35S:AtMYB44 and atmyb44 knockout plants to ABA. A, Germination rate. Seeds were germinated and grown on Murashige and Skoog agar plates with or without ABA for 7 d. B, Size of stomatal apertures. Stomata were fully opened prior to ABA treatment. Rosette leaves of 5-week-old plants were detached and floated abaxial-side down on opening solution for 2 h prior to ABA treatment. Leaves were then treated with ABA for 2 h by adding it to the solution. Stomatal apertures in epidermal peels were observed under a microscope and measured. The sizes of at least 50 stomatal apertures were measured for each treatment.

Figure 6.

Abiotic stress tolerance tests of 35S:AtMYB44 plants. A, Transpiration rates. For water-loss measurements, the aerial part of 5-week-old plants was separated from the roots, placed on weighing dishes, and allowed to dry slowly on the laboratory bench (25°C, 60% relative humidity). Weights of the samples were recorded at regular intervals. B, Drought tolerance test. Watering of 4-week-old plants was stopped for 12 d and then resumed for 3 d. C, Salt tolerance test. Four-week-old plants were watered for 12 d at 4-d intervals with increasing concentrations of NaCl: 100 mm, 200 mm, and 300 mm. In B and C, survival rates (%) were calculated from the numbers of surviving plants per total plants tested in 10 independent experiments and are indicated under each of the plant lines. [See online article for color version of this figure.]

Figure 7.

Northern blots of salt-inducible genes in 35S:AtMYB44 plants. Five-week-old plants were treated with 250 mm NaCl and harvested at the indicated times. cDNA probes used were EST clones obtained from TAIR.