The low-oxygen-induced NAC domain transcription factor ANAC102 affects viability of Arabidopsis seeds following low-oxygen treatment

Abstract

Low-oxygen stress imposed by field waterlogging is a serious impediment to plant germination and growth. Plants respond to waterlogging with a complex set of physiological responses regulated at the transcriptional, cellular, and tissue levels. The Arabidopsis (Arabidopsis thaliana) NAC domain-containing gene ANAC102 was shown to be induced under 0.1% oxygen within 30 min in both roots and shoots as well as in 0.1% oxygen-treated germinating seeds. Overexpression of ANAC102 altered the expression of a number of genes, including many previously identified as being low-oxygen responsive. Decreasing ANAC102 expression had no effect on global gene transcription in plants but did alter expression patterns in low-oxygen-stressed seeds. Increasing or decreasing the expression of ANAC102 did not affect adult plant survival of low-oxygen stress. Decreased ANAC102 expression significantly decreased germination efficiency following a 0.1% oxygen treatment, but increased expression had no effect on germination. This protective role during germination appeared to be specific to low-oxygen stress, implicating ANAC102 as an important regulator of seed germination under flooding.

Figure 1.

Expression of ANAC102 in response to low oxygen. A, Time course of ANAC102 expression subjected to 0.1% oxygen. Plants of ecotype Col-0 in liquid MS medium were placed in chambers containing 0.1% oxygen for 0.5, 2, 4, 8, or 24 h. Expression changes were monitored in both root (black triangles) and shoot (white triangles) tissues. Data are expression at different times relative to the 0-h time point (±se; n = 9). B, Expression of ANAC102 and ADH1 in imbibed seeds under ambient and 0.1% oxygen (6 d of exposure) relative to 3-week-old root tissue in ambient oxygen (±se; n = 9).

Figure 2.

Tissue-specific expression, induction of ANAC102, and vital staining of low-oxygen-treated seeds. An ANAC102 promoter∷GUS fusion line was constructed and plants were stained with 5-bromo-4-chloro-3-indolyl-β-glucuronic acid to localize ANAC102 expression. A, Untreated whole plant. B, Untreated roots. C, Closeup of untreated roots. D, Untreated leaf and developing flowers. E, Seedling at 2 d after germination. F, Vital staining of an Arabidopsis embryo that failed to germinate following a 6-d treatment of 0.1% oxygen. The embryo was isolated from the seed coat prior to staining. Green tissue is alive, and red-stained tissue is dead. C, Embryonic cotyledons; R, embryonic radicle. G, Untreated 3-week-old plant stained for 8 h. H, Three-week-old plant treated with 0.1% oxygen for 4 h, stained for 8 h. I, Expression of native ANAC102 gene and ANAC102∷GUS fusion relative to ANAC102 expression in untreated plants. Black bars represent expression in untreated plants, and hatched bars represent expression in plants treated with 0.1% oxygen for 4 h. Error bars represent 1 se (n = 9).

Figure 3.

QRT-PCR analysis of selected genes up-regulated in ANAC102-modified expression lines. Changes in expression were monitored for a set of genes in 3-week-old Arabidopsis plants from each of Col-0, C24, KO-1 (white squares), and OX-1 (black squares) lines. Plants were grown exposed to 0.1% oxygen in anaerobic chambers and in the dark for the various times specified. Data presented are expression log₂-transformed ratios for each of KO-1 and OX-1 relative to their wild-type lines, Col-0 and C24, respectively, at each time point. An expanded version of this figure, including expression data for Col-0 and C24, is available in Supplemental Figure S2.

Figure 4.

Survival after exposure to 0.1% oxygen. Plants with T-DNA insertions within the ANAC102 gene (KO-1, KO-2, and Col-0 background) and plants overexpressing the ANAC102 gene (OX-1, OX-2, and C24 background) and their respective parental ecotypes were subjected to low-oxygen treatments. A, Plants were treated with 0.1% oxygen in the dark for 3 d. Survival percentages are averages over five experiments (±se; n = 15). Data points with different lowercase letters denote lines with survival percentages significantly different from each other as determined by ANOVA and Tukey's honestly significant difference test (P < 0.05). B, Plants were treated for 1 d with 5% oxygen, followed by 3 d with 0.1% oxygen. Survival percentages are averages over three experiments (±se; n = 9).

Figure 5.

Effects on germination of various abiotic stresses. Seeds were scored for germination at intervals up to 1 week. Data represent average numbers of germinated seeds from three replicates containing 20 to 50 seeds each (±se; n = 3). Black symbols represent data for untreated seeds, and white symbols represent treated seeds. Data points with different lowercase letters denote lines with germination percentages significantly different from each other (only differences between lines within a treatment are shown) as determined by ANOVA and Tukey's honestly significant difference test (P < 0.05). A, Seeds treated with 0.1% oxygen for 6 d. Germination was scored after removal to ambient oxygen levels. B, Seeds sown on medium containing 200 mm NaCl. C, Seeds sown on medium containing 5% (w/v) mannitol. D, Seeds sown on medium containing 15 mm ABA.

Figure 6.

Relative expression of selected genes in seeds exposed to 0.1% oxygen for 4 or 6 d. Seeds of each line were imbibed, cold stratified, and then placed in a 0.1% oxygen atmosphere for 4 or 6 d. Expression ratios for selected genes are given relative to expression levels in untreated wild-type (Col-0 for Col-0 and KO-1; C24 for C24 and OX-1) imbibed seeds. Bars represent expression ratios for Col-0 (black columns), KO-1 (dotted columns), C24 (shaded columns), and OX-1 (diagonal lined columns; ±se; n = 9). Data points with different letters denote lines with relative expression ratios significantly different from each other as determined by two-way ANOVA (line × time) and Tukey's honestly significant difference test (P < 0.05). Uppercase letters differentiate between Col-0 and KO-1 at the different times, while lowercase letters differentiate between C24 and OX-1 at the different times. No comparisons were made between lines in the Col-0 background and lines in the C24 background.