Involvement of five catalytically active Arabidopsis β-amylases in leaf starch metabolism and plant growth

Abstract

Starch degradation in chloroplasts requires β-amylase (BAM) activity, but in Arabidopsis, there are nine BAM proteins, five of which are thought to be catalytic. Although single-gene knockouts revealed the necessity of BAM3 for starch degradation, contributions of other BAMs are poorly understood. Moreover, it is not possible to detect the contribution of individual BAMs in plants containing multiple active BAMs. Therefore, we constructed a set of five quadruple mutants each expressing only one catalytically active BAM, and a quintuple mutant missing all of these BAMs (B-Null). Using these mutants, we assessed the influence of each individual BAM on plant growth and on leaf starch degradation. Both BAM1 and BAM3 alone support wild-type (WT) levels of growth. BAM3 alone is sufficient to degrade leaf starch completely whereas BAM1 alone can only partially degrade leaf starch. In contrast, BAM2, BAM5, and BAM6 have no detectable effect on starch degradation or plant growth, being comparable with the B-Null plants. B-Null plant extracts contained no measurable amylase activity, whereas BAM3 and BAM1 contributed about 70% and 14% of the WT activity, respectively. BAM2 activity was low but detectable and BAM6 contributed no measurable activity. Interestingly, activity of BAM1 and BAM3 in the mutants varied little developmentally or diurnally, and did not increase appreciably in response to osmotic or cold stress. With these genetic lines, we now have new opportunities to investigate members of this diverse gene family.

Keywords: cold stress; osmotic stress; starch; starch degradation; β‐amylase.

Figure 1

Starch content in leaves of 4‐, 6‐, and 8‐week‐old wild‐type (WT) and mutant Arabidopsis plants. Plants were grown under a 12/12 hr‐light/dark photoperiod, harvested at the end of the night period and stained with iodine. (a) WT and single BAM mutants. (b) WT and quadruple mutants lacking all but one BAM gene, and B‐Null lacking all five BAM genes. For each treatment, 3–5 leaves from at least two different plants were analyzed and representative leaves are shown

Figure 2

Effect of individual BAM genes on plant mass at 5 weeks of age when grown under a 12‐hr‐light/12‐hr‐dark photoperiod. Data are means ± SD of the six largest plants from two pots per genotype. WT, B1‐Q, and B3‐Q means are not significantly different from each other, as are the remaining four genotypes, but the two groups are significantly different (p < .01)

Figure 3

Appearance of WT and B‐Null plants at 5 weeks of age. The B‐Null plant contains mutations in BAM1, ‐2, ‐3, ‐5, and ‐6 genes

Figure 4

Total amylase activity in crude extracts from leaves of WT and mutant lines of Arabidopsis grown under a 12‐hr‐light/12‐hr‐dark photoperiod. All extracts were assayed at 25℃ in 50 mM MES buffer, pH 6, with 80 mg/ml soluble starch and 100 mM KCl. (a) Plants harvested at 6 weeks of age. (b) Plants harvested at 7 weeks of age. Values are means ± SD (n = 3). Means that were significantly different from the B‐Null activity are labeled with *p < .05, **p < .01

Figure 5

Effect of developmental age on amylase activity in crude extracts from leaves of WT, B1‐Q and B3‐Q plants grown under a 12‐hr‐light/12‐hr‐dark photoperiod. All extracts were assayed at 25℃ in 50 mM MES buffer, pH 6, using 40 mg/ml soluble starch. Values are means ± SD (n = 3). Means that were significantly different between weeks are labeled with *p < .05, **p < .01

Figure 6

Total amylase activity in crude extracts from leaves of 5‐week‐old B3‐Q and B1‐Q plants over a diurnal period. Extracts were assayed at 25℃ in 50 mM MES buffer, pH 6, with 40 mg/ml soluble starch. Each point represents the activity in one extract prepared from leaves of 3 plants

Figure 7

Effect of 300 mM mannitol applied as a soil drench on day zero on amylase activity and reducing sugar levels in B1‐Q plants. (a) Amylase activity in leaf extracts assayed at 25℃ in 50 mM MES buffer, pH 6, with 40 mg/ml soluble starch. (b) Reducing sugar levels in the same extracts used in a. Values are means ± SD (n = 3). Means that were significantly different from the controls of that time point are labeled with *p < .05, **p < .01

Figure 8

Effect of 4°C cold treatment for up to 4 days on amylase activity and reducing sugar levels in B3‐Q plants. (a) Amylase activity in leaf extracts assayed at 25℃ in 50 mM MES buffer, pH 6, using 40 mg/ml soluble starch. (b) Reducing sugar levels in the same extracts used in a. Values are means ± SD (n = 3). Means that were significantly different from the controls of that time point are labeled with **p < .01