Mutations affecting induction of glycolytic and fermentative genes during germination and environmental stresses in Arabidopsis

Abstract

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is known to be induced by environmental stresses and regulated developmentally. We used a negative-selection approach to isolate mutants that were defective in regulating the expression of the ADH gene during seed germination; we then characterized three recessive mutants, aar1-1, aar1-2, and aar2-1, which belong to two complementation groups. In addition to their defects during seed germination, mutations in the AAR1 and AAR2 genes also affected anoxic and hypoxic induction of ADH and other glycolytic genes in mature plants. The aar1 and aar2 mutants were also defective in responding to cold and osmotic stress. The two allelic mutants aar1-1and aar1-2 exhibited different phenotypes under cold and osmotic stresses. Based on our results we propose that these mutants are defective in a late step of the signaling pathways that lead to increased expression of the ADH gene and glycolytic genes.

Figure 1

GUS and ADH activities of AG2 and of the aar mutants during seed germination. Seedlings of AG2 and aar1–1, arr1–2, and aar2–1 at different developmental stages were harvested and assayed for GUS and ADH activities as described in Methods. A, GUS activity is expressed as pmol 4-methylumbelliferone min⁻¹ mg⁻¹ protein. B, One unit of ADH enzyme is defined as an increase in A₃₄₀ of 0.01 per min. The data presented are the averages of three independent treatments. Plants grown at different times were used for replicate treatments. Bar graphs at each time point (from left to right) represent activities for AG2, aar1–1, aar1–2, and aar2–1. Bars indicate sd.

Figure 2

Anoxic and hypoxic induction of GUS activity in mature plants. Twenty-day-old AG2 and aar1–1, aar1–2, and aar2–1 plants were subjected to anoxic or hypoxic treatment and assayed for GUS activity as described in Methods. The data presented are the averages of three independent treatments. Bars indicate sd.

Figure 3

RT-PCR analysis of anoxic and hypoxic induction of ADH. Total RNA (1 μg) from anoxic- or hypoxic-treated plants was used in a 100-μL reaction for first-strand cDNA synthesis. One-tenth of the synthesized cDNA from each treatment was then used in subsequent PCR reactions. A, RT-PCR products for ADH were analyzed by agarose-gel electrophoresis. The sizes of PCR products for each gene are 525 bp for ADH and 350 bp for βATP. The notations on top of each lane represent treatment conditions of normoxia (N), anoxia (A), and hypoxia (H). B, Digitized images of the ADH bands in A were quantified and normalized to the βATP band in each lane. The normalized mRNA levels from normoxic-treated plants (solid bars) of AG2 and each aar mutant were used as the reference levels to calculate magnitudes of induction. Checked and striped bars correspond to anoxic- and hypoxic-treated samples, respectively. The data presented are the averages of three independent treatments. Bars indicate sd.

Figure 4

RT-PCR analysis of anoxic and hypoxic induction of PDC1 and PDC2. Analysis of RT-PCR products (A) and quantification of levels of induction (B) of PDC1 and PDC2 were performed as described in Figure 3. The symbols used are as described in the Figure 3 legend. The sizes of PCR products are 682 bp for PDC1 and 740 bp for PDC2.

Figure 6

Effects of cold and mannitol on ADH and PDC1 expression. A, RNA samples from AG2 and aar mutants grown under normal conditions (lanes N), cold treatment (lanes C), and mannitol treatment (lanes M) were subjected to RT-PCR analysis. B, Quantification of cold and mannitol induction of ADH and PDC1 was performed as described for Figure 3B. Solid bars correspond to samples from control experiments, whereas checked and striped bars correspond to samples from cold and mannitol treatments, respectively. The data presented are the averages of three independent treatments. Bars indicate sd.

Figure 5

Effect of aar mutations on anoxic and hypoxic induction of GAPC. A, Levels of GAPC mRNA from normoxic-treated (lanes N), anoxic-treated (lanes A), and hypoxic-treated (lanes H) AG2 plants and aar mutants were analyzed by relative RT-PCR. The sizes of the PCR products are 670 bp for GAPC and 350 bp for βATP. B, Quantification of levels of induction of GAPC by anoxia and hypoxia in AG2 and aar mutants were calculated as described for Figure 3B.