Thioredoxin-regulated beta-amylase (BAM1) triggers diurnal starch degradation in guard cells, and in mesophyll cells under osmotic stress

Abstract

BAM1 is a plastid-targeted β-amylase of Arabidopsis thaliana specifically activated by reducing conditions. Among eight different chloroplast thioredoxin isoforms, thioredoxin f1 was the most efficient redox mediator, followed by thioredoxins m1, m2, y1, y2, and m4. Plastid-localized NADPH-thioredoxin reductase (NTRC) was also able partially to restore the activity of oxidized BAM1. Promoter activity of BAM1 was studied by reporter gene expression (GUS and YFP) in Arabidopsis transgenic plants. In young (non-flowering) plants, BAM1 was expressed both in leaves and roots, but expression in leaves was mainly restricted to guard cells. Compared with wild-type plants, bam1 knockout mutants were characterized by having more starch in illuminated guard cells and reduced stomata opening, suggesting that thioredoxin-regulated BAM1 plays a role in diurnal starch degradation which sustains stomata opening. Besides guard cells, BAM1 appears in mesophyll cells of young plants as a result of a strongly induced gene expression under osmotic stress, which is paralleled by an increase in total β-amylase activity together with its redox-sensitive fraction. Osmotic stress impairs the rate of diurnal starch accumulation in leaves of wild-type plants, but has no effect on starch accumulation in bam1 mutants. It is proposed that thioredoxin-regulated BAM1 activates a starch degradation pathway in illuminated mesophyll cells upon osmotic stress, similar to the diurnal pathway of starch degradation in guard cells that is also dependent on thioredoxin-regulated BAM1.

Fig. 1.

Specificity of BAM1 for chloroplast thioredoxin isoforms. Activation kinetics of oxidized BAM1 were analysed by measuring PNPG5-depedent activity after incubation of the pre-oxidized enzyme in the presence of 0.5 mM reduced DTT alone or plus 1 μM thioredoxins. Activities obtained under same conditions but in the absence of BAM1 were subtracted. Symbols: full circles, thioredoxin f1; open circles, thioredoxin m1; open squares, thioredoxin m2; open triangles, thioredoxin m3; full triangles, thioredoxin m4; full squares, thioredoxin x; stars, thioredoxin y1 and half full squares, thioredoxin y2.

Fig. 2.

Activation of BAM1 by NTRC. Activation of oxidized BAM1 (0.5 μM) was analysed by measuring PNPG5-dependent activity after 3 h of incubation at room temperature in the presence of NTRC (5 μM) and 0.5 mM NADPH. The release of p-nitrophenol from PNPG5 in the absence of BAM1 was null. Data represent mean values ±SD of three independent experiments made on different BAM1 preparations.

Fig. 3.

Localization of YFP fluorescence under the control of the BAM1 promoter, in a representative 7-d-old seedling. (A–C) Shoot; (D–F) cotyledon (magnification of the portion delimited by the blue rectangle); (G–I) first leaf (magnification of the portion delimited by the red rectangle); (L–N) primary root tip; (O–Q) developing secondary root.

Fig. 4.

Localization of BAM1promoter::GUS activity during different stages of Arabidopsis development. The figure shows a typical expression pattern obtained from two independent experiments (scale bar=100 μm). (A, A') 10-d-old plants; (B, B’) 17-d-old plants; (C, C’) 24-d-old plants; (D, D') 31-d-old plants; (E, E') 38-d-old plants; (G, G') 45-d-old plants.

Fig. 5.

Beta-amylase activity in wild-type and bam1 plants, hydroponically grown under normal conditions. Total PNPG5-activity was measured on soluble proteins extracted from leaves after 1 h of incubation in the presence of 20 mM reduced DTT. Redox-sensitive activity was obtained by subtracting from total PNPG5-activity the same activity measured after 1 h of incubation with oxidized DTT. Values are the means ±SE (n=30 independent protein extractions). Statistical analysis by ANOVA underlined significant differences (P <0.001) between wild-type and bam1 plants. A Duncan's Test was conducted to compare means and statistically significantly different values (P <0.05) are indicated by different letters in the graphs.

Fig. 6.

Starch content in guard cells and stomatal aperture analysis on wild-type and bam1 mutant plants. Leaves were collected from plants hydroponically grown for 26 d, after 6 h of light exposure. Both measurements were performed throughout digital image processing. After iodine staining, starch content (upper graph) was quantified as the total pixel area of the starch accumulations visible in chloroplasts of single guard cells. Values are means ±SE (n=72 guard cells). In the pictures on the right the typical starch accumulation observed in wild-type and bam1 guard cells. In the lower graph, stomatal aperture measurement was determined as the ratio between width and length of the stomatal pore. Values are means ±SE (n=70 stomata). Statistical analysis by ANOVA and a Duncan's Test (P <0.01) were conducted. Statistically significant differences are indicated by three stars in the graphs. (This figure is available in colour at JXB online.)

Fig. 7.

Induction of BAM1promoter::GUS activity under control conditions and in the presence of 450 mM mannitol. Treatment was applied 3 h after beginning of the light period. GUS activity was measured after 5 min, 1 h, 2 h, 4 h, 6 h, and 8 h of treatment (scale bar=100 μm).

Fig. 8.

Localization of YFP fluorescence in leaves of plants transformed with BAM1promoter::YFP. (A) Representative control leaf; (B) representative leaf after 10 h of 450 mM mannitol treatment. Each image represents a 3D reconstruction from a Z stack confocal acquisition.

Fig. 9.

Beta-amylase activity in wild-type and bam1 mutant plants, hydroponically grown under normal conditions (control) or in the presence of 450 mM mannitol for 4 h (mannitol). (A) Soluble proteins extracted from leaves were incubated for 1 h in the presence of 20 mM reduced DTT before measuring PNPG5-activity; (B) redox-sensitive fraction obtained by subtracting from total PNPG5-activity (A) the same activity measured after 1 h of incubation with oxidized DTT. Values are the means ±SE (n=30 independent protein extractions). Statistical analysis by ANOVA underlined significant differences (P <0.001) between wild-type and bam1 plants. A Duncan's Test was conducted to compare means and statistically significantly different values (P <0.05) are indicated by different letters in the graphs.

Fig. 10.

Starch content in wild-type and bam1 plants under osmotic stress conditions. Twenty-six-day-old plants were osmotically stressed 3 h after switching on the light, by adding 450 mM mannitol to the hydroponic solution. Starch was quantified as described by Smith and Zeeman (2006). Five independent experiments were performed. Data are means values ±SE (n=15).