LWR1 and LWR2 are required for osmoregulation and osmotic adjustment in Arabidopsis

Abstract

With the goal of identifying molecular components of the low-water-potential response, we have carried out a two-part selection and screening strategy to identify new Arabidopsis mutants. Using a system of polyethylene glycol-infused agar plates to impose a constant low-water-potential stress, putative mutants impaired in low-water-potential induction of the tomato (Lycopersicon esculentum) le25 promoter were selected. These lines were then screened for altered accumulation of free Pro. The seedlings of 22 mutant lines had either higher or lower Pro content than wild type when exposed to low water potential. Two mutants, designated low-water-potential response1 (lwr1) and lwr2, were characterized in detail. In addition to higher Pro accumulation, lwr1 seedlings had higher total solute content, greater osmotic adjustment at low water potential, altered abscisic acid content, and increased sensitivity to applied abscisic acid with respect to Pro content. lwr1 also had altered growth and morphology. lwr2, in contrast, had lower Pro content and less osmotic adjustment leading to greater water loss at low water potential. Both lwr1 and lwr2 also had altered leaf solute content and water relations in unstressed soil-grown plants. In both mutants, the effects on solute content were too large to be explained by the changes in Pro content alone, indicating that LWR1 and LWR2 affect multiple aspects of cellular osmoregulation.

Figure 1.

Wild-type response to low-ψ_w treatment. Three-day-old wild-type seedlings (le25:ADH Ben) were transferred to either −0.25 MPa (control) or −0.75 MPa PEG-infused agar plates, and low-ψ_w responses were measured over a 96-h period. A, Seedling RWC. Data are means ± se (n = 6). B, Seedling ψ_s. Dotted line shows the agar ψ_w of the −0.25 MPa treatment. Dashed line shows the agar ψ_w of the −0.75 MPa treatment. Data are means ± se (n = 3–4). C, Seedling Pro content. Data are means ± se (n = 4–6). Error bars are not shown when smaller than symbols.

Figure 2.

ADH activity and allyl alcohol responses of le25:ADH Ben seedlings. A, Induction of ADH activity by low ψ_w. ADH activity was assayed 48 h after seedlings were transferred to a range of agar ψ_w. Data are means ± se (n = 5–7). B, Appearance of le25:adh and Ben adh null seedlings 7 d after a 2-h, 1.2 mm allyl alcohol treatment. Prior to the allyl alcohol treatment, 3-d-old seedlings were exposed to either control (−0.25 MPa) or low-water-potential stress (−1.2 MPa) for 2 d. After the allyl alcohol treatment, seedlings were transferred to control plates. Scale bars in pictures indicate 1 mm. The nylon mesh used to transfer seedlings between plates can be seen in the background.

Figure 3.

Allyl alcohol resistance and Pro content of 22 mutant lines. A, Percent survival of each mutant line to allyl alcohol treatment. Allyl alcohol selection was performed as in Figure 2 except that 3.0-mm allyl alcohol was used. Dashed line across the bottom of the figure indicates the wild type (unmutagenized le25:ADH Ben) response (21% survival). B, Pro content of each mutant line. Three-day-old seedlings were transferred to −1.2 MPa PEG-infused plates for 3 d. Pro content was determined and expressed relative to the wild-type control. The dashed line indicates the wild-type level (100%, which in these experiments was 6.42 ± 0.35 μmol g FW⁻¹). The same mutant lines are depicted in the same order in both panels. Therefore, the allyl alcohol resistance and Pro content for the same line can be compared by looking at the bars in the same horizontal position in the two sections. The lwr1 and lwr2 mutants are individually labeled. Data are means ± se (n = 3–6) in both A and B.

Figure 4.

Pro contents of wild type (Ben), lwr1, and lwr2 in response to a range of agar ψ_w. Pro content was assayed 96 h after transfer of 3-d-old seedlings to the indicated ψ_w. Data are means ± se (n = 5–8). Significant differences between mutant and wild type (P ≤ 0.05) are indicated by asterisks in the figure.

Figure 5.

Analysis of osmotic adjustment of wild type (Ben), lwr1, and lwr2. A, RWC of mutants and wild type after 96 h of exposure to a range of agar ψ_w. Data are means ± se (n = 4–10). Significant differences between mutant and wild type are indicated by an asterisk in the figure (P < 0.05). B, ψ_s of wild-type and mutant seedlings measured 96 h after transfer to a range of agar ψ_w. The dashed diagonal line indicates where seedling ψ_s equals ψ_w of the agar plate. Data are means ± se (n = 4–10). Error bars are smaller than symbols for all data points. Significant differences between mutant and wild type are indicated by an asterisk in the figure (P < 0.001). C, ψ_s at 100% RWC (ψ_s100) of mutant and wild-type seedlings calculated from data in A and B. Lines are regression lines fitted to the data for each genotype (r² > 0.97 in all cases). The regression lines of both lwr1 and lwr2 were significantly different from that of wild type (F test; P = 0.0003 for lwr1 and wild type and P = 0.003 for lwr2 and wild type). Inset, Osmotic adjustment (slope of each regression line converted to millimolar solute concentration) per MPa decrease in ψ_w.

Figure 6.

Concentration of K⁺ in unmutagenized le25:ADH (Ben), lwr1, and lwr2 seedlings at −0.25 and −0.75 MPa. Data are means ± se (n = 4–8). lwr1 was significantly different from wild type (P = 0.05) at −0.75 MPa (indicated by an asterisk).

Figure 7.

ABA content and response of wild type (Ben), lwr1, and lwr2 seedlings to applied S(+)-ABA. A, ABA content of 3-d-old seedlings after 96 h at the indicated ψ_w. Data are means ± se (n = 4–9). Regression lines were fitted to the combined data of wild type and lwr2 (r² = 0.99) and lwr1 (r² = 0.97). The two regression lines were significantly different (F test, P = 5 × 10⁻¹²). B, ABA content expressed as a function of RWC. Regression lines were fitted for each genotype (Ben, r² = 0.99; lwr1, r² = 0.44 [this line is not shown in the figure]; lwr2, r² = 0.97). The wild-type regression line was significantly different from that of lwr2 (F test, P = 0.0003) C, Pro content 96 h after transfer to plates containing the indicated concentrations of S(+)-ABA. Data are means ± se (n = 7). Significant differences between mutant and wild type are indicated by an asterisk in the figure (P < 0.002).

Figure 8.

Leaf water relations of wild type (Ben), lwr1, and lwr2 under well-watered conditions (soil ψ_w = −0.24 MPa). A, Leaf ψ_w, ψ_s, and calculated ψ_p. Data are means ± se (n = 4−5). Significant differences (P < 0.05) of mutants versus wild type are marked with an asterisk. B, Water loss of detached leaves. Data are means ± se (n = 14). Error bars are not shown when smaller than symbols.

Figure 9.

Morphological differences of lwr1 compared to wild type (Ben). A, Five-day-old seedlings of Ben, lwr1, and lwr2 at −0.25 MPa. Scale bar indicates 1 mm. B, Fully expanded rosette leaves of Ben and lwr1. Scale bar indicates 1 cm. C, Rosette stage plants of Ben and lwr1. Scale bar indicates 1 cm. D, Flowering plants of Ben and lwr1. Scale bar indicates 2 cm.