Uncoupling the effects of abscisic acid on plant growth and water relations. Analysis of sto1/nced3, an abscisic acid-deficient but salt stress-tolerant mutant in Arabidopsis

Abstract

We have identified a T-DNA insertion mutation of Arabidopsis (ecotype C24), named sto1 (salt tolerant), that results in enhanced germination on both ionic (NaCl) and nonionic (sorbitol) hyperosmotic media. sto1 plants were more tolerant in vitro than wild type to Na(+) and K(+) both for germination and subsequent growth but were hypersensitive to Li(+). Postgermination growth of the sto1 plants on sorbitol was not improved. Analysis of the amino acid sequence revealed that STO1 encodes a 9-cis-epoxicarotenoid dioxygenase (similar to 9-cis-epoxicarotenoid dioxygenase GB:AAF26356 [Phaseolus vulgaris] and to NCED3 GB:AB020817 [Arabidopsis]), a key enzyme in the abscisic acid (ABA) biosynthetic pathway. STO1 transcript abundance was substantially reduced in mutant plants. Mutant sto1 plants were unable to accumulate ABA following a hyperosmotic stress, although their basal ABA level was only moderately altered. Either complementation of the sto1 with the native gene from the wild-type genome or supplementation of ABA to the growth medium restored the wild-type phenotype. Improved growth of sto1 mutant plants on NaCl, but not sorbitol, medium was associated with a reduction in both NaCl-induced expression of the ICK1 gene and ethylene accumulation. Osmotic adjustment of sto1 plants was substantially reduced compared to wild-type plants under conditions where sto1 plants grew faster. The sto1 mutation has revealed that reduced ABA can lead to more rapid growth during hyperionic stress by a signal pathway that apparently is at least partially independent of signals that mediate nonionic osmotic responses.

Figure 1.

The sto1 mutation enhances germination on media supplemented with NaCl or KCl. A, Photographs are representative of wild-type and sto1 mutant plants. Seeds were placed on MS medium or MS medium supplemented with 160 mm NaCl or KCl and allowed to germinate and grow for 14 d. B, Percentage of germinated seeds after 14 d (significant difference at 99% confidence level). Germination was assessed on 60 seeds of wild-type or sto1 mutant plants distributed in three replica plates per each treatment (20 seeds per each genotype).

Figure 2.

Growth response of wild-type and sto1 seedlings and plants at increasing NaCl concentrations. A, Seeds were placed in petri plates on MS medium or MS medium supplemented with increasing concentrations of NaCl and allowed to germinate and grow. Plant fresh weight was measured after 21 d. Values are means of 60 plants ± se. B, Seeds were germinated and grown in soil at saturated atmospheric humidity (see “Materials and Methods” for details) and irrigated every day with saline water with different NaCl concentrations. After 28 d, shoot fresh weight was measured. Values are means of 20 plants ± se.

Figure 3.

sto1 mutant plants are tolerant to KCl and NaCl and hypersensitive to LiCl stresses. A, Seeds were germinated in petri plates on standard MS medium and 7-d-old seedlings were subsequently transferred to MS medium supplemented with 160 mm NaCl, KCl, or 20 mm LiCl and allowed to grow for an additional 20 d. Photographs are representative of wild-type and sto1 mutant plants after 20 d from transferring onto saline medium. B, Plant fresh weights were measured after 20 d of growth on indicated medium. Values are means of 60 plants ± se.

Figure 4.

The sto1/nced3 mutation inhibits growth on media supplemented with sorbitol. A, Percentage of germinated wild-type and sto1/nced3 seeds on MS medium supplemented with 300 mm sorbitol over a 21-d time period. Germination was assessed on 60 seeds of wild-type and sto1/nced3 plants distributed on three replica plates (20 seeds per each genotype). B, Root length of 14-d-old seedlings growing on sorbitol-containing medium. Values are means of 60 plants ± se. C, Fresh weight of 14-d-old seedlings growing on sorbitol-containing medium. Values are means of 60 plants ± se. D, Seeds were germinated in petri plates on standard MS medium and 3-d-old seedlings were subsequently transferred on MS medium (not shown) or MS medium supplemented with 300 mm sorbitol and allowed to grow for an additional 20 d and then photographed.

Figure 5.

sto1 mutant plants are sensitive to soil desiccation. A, Representative photograph of wild-type and sto1 mutant plants exposed to desiccation. Plants were grown in soil under a standard irrigation regime until four to five fully expanded leaves were formed, at which stage irrigation was stopped. After 15 d, in coincidence with the appearance of clear symptoms of leaf desiccation, plants were rewatered and left to recover for 48 h, at which time pictures were taken. B, Shoot fresh weights of desiccation-stressed wild-type and sto1 mutant plants after rewatering. Values are means of 20 plants ± se.

Figure 6.

PCR analysis and genome location of T-DNA insertion in sto1/nced3 mutants. A, Secondary TAIL-PCR product (lane 2) and shift of the tertiary PCR product (lane 3) in sto1. B, Correct genomic integration of the T-DNA insertion was verified by diagnostic PCR; lanes 1 (marker); 2 (DNA template, wild type; primers, T-DNA LB [3′] and STO1-specific primer [5′]); 3 (DNA template, sto1; primers, T-DNA LB [3′] and STO1-specific primer [5′]); 4 (DNA template, wild type; primers, sto1-specific primer [3′] and STO1-specific primer [5′]); 5 (DNA template, sto1; primers, STO1-specific primer [3′] and STO1-specific primer [5′]). Oligonucleotide sequences are reported in Table V. C, Physical map of the sto1 locus and insertion site of the T-DNA. Solid line represents fragment of the bacterial artificial chromosome clone MOA2.4. Black box indicates the coding region of the gene; arrow indicates the predicted transcription direction.

Figure 7.

NCED3 transcript abundance is increased by NaCl treatment and is reduced in sto1/nced3 mutant plants. Ten micrograms of total RNA were isolated from 21-d-old sto1/nced3 mutant and wild-type plants that were germinated and grown on 145 mm NaCl, separated on a denaturing formaldehyde-agarose gel, and blotted onto nylon membrane. The membrane-bound RNA was hybridized with DIG-labeled DNA probe (Roche, Indianapolis). The probe was produced by PCR reaction using the primers listed in Table V.

Figure 8.

Complementation with the STO1/NCED3 gene reverts the soil desiccation-sensitive phenotype of the sto1/nced3 mutant. A, STO1/NCED3 transcript abundance detected by RT-PCR in wild type, sto1/nced3, lines 3 to 14 (pBI vector control), and lines 4 to 6 (expressing STO1/NCED3). One microliter of cDNA was used as a template for the first PCR amplification (20 cycles). B, Representative photograph of lines 3 to 14 (pBI vector control) and lines 4 to 6 (pBI::STO1/NCED3). Top, sto1/nced3 plants complemented with STO1/NCED3 (lines 4–6) grown under standard irrigation regime (left) and exposed to desiccation (right) showing the reverted desiccation-sensitive phenotype; Bottom, sto1/nced3 plants complemented with vector only (lines 3–14) grown under standard irrigation regime (left) and exposed to desiccation (right). C, Responses of C24, sto1/nced3, gl1, and Atnced3 (T5004) mutant seedlings to NaCl and sorbitol treatments. Seeds were germinated and grown in petri plates on standard MS medium or supplemented with 160 mm NaCl or 300 mm sorbitol. Plant fresh weights (mg) after 21 d were as follows: MS, 36.2 ± 1.7 (C24); 37.5 ± 1.6 (sto1); 35.5 ± 1.5 (gl1); 36.5 ± 1.3 (Atnced3). NaCl, 4.3 ± 0.34 (C24); 8.5 ± 0.3 (sto1); 3.9 ± 0.32 (gl1); 7.7 ± 0.24 (A-nced3). Sorbitol, 9.35 ± 0.15 (C24); 3.92 ± 0.68 (sto1); 7.2 ± 0.08 (gl1); 5.2 ± 0.31 (Atnced3). (Values are means ± se, n = 20.)

Figure 9.

ABA treatment abolishes the enhanced germination of sto1/nced3 seeds on NaCl medium. Seeds of wild type and sto1/nced3 mutant plants were surface sterilized and placed on MS medium supplemented with 145 mm NaCl (A) or with 145 mm NaCl + 20 μm ABA (B). Germination on standard MS medium was also included (not shown). For each genotype (wild type and sto1/nced3), the number of germinated seeds (out of 60) was assessed over 21 d and expressed as a percentage.

Figure 10.

ABA reverts the LiCl sensitivity of sto1/nced3 seedlings to wild type. Wild-type and sto1 seeds were surface sterilized and germinated on standard MS medium or MS medium supplemented with 20 mm LiCl or 20 mm LiCl + 20 μm ABA. A representative photograph of 15-d-old seedlings (from three replica plates per treatment) is displayed.

Figure 11.

Expression of the ICK1 gene in sto1/nced3 and wild-type plants. Total RNA was extracted from whole seedlings that were grown for 7 d on MS medium or MS medium supplemented with 145 mm NaCl. Twenty micrograms of total RNA were loaded in each lane. The probe was produced by PCR reaction using the primers listed in Table V. Bottom, ethidium bromide-stained total RNA gel image as a loading control.

Figure 12.

Ethylene-treated wild-type plants mimic the sto1/nced3 phenotype. A, Representative photographs of 15-d-old wild-type and sto1/nced3 seedlings germinated and grown in the presence or absence of 20 ppm of C₂H₄ on petri plates containing standard MS medium, or MS medium supplemented with 145 mm NaCl or 20 mm LiCl. B, Number of new leaves per plant formed after 15-d treatment. Values are means of 100 plants ± se.

Figure 13.

Transpiration of wild-type and sto1/nced3 mutant plants. A, Transpiration of wild-type and sto1/nced3 mutant plants was measured as daily water loss over a 7-d time interval. Each value represents the average daily water loss of three plants ± se. B, Daily fluctuations of rate of water loss in wild-type and sto1/nced3 mutant plants. The diurnal patterns displayed are representative of three independent experiments.