The ram1 mutant of Arabidopsis exhibits severely decreased beta-amylase activity

Abstract

Despite extensive biochemical analyses, the biological function(s) of plant beta-amylases remains unclear. The fact that beta-amylases degrade starch in vitro suggests that they may play a role in starch metabolism in vivo. beta-Amylases have also been suggested to prevent the accumulation of highly polymerized polysaccharides that might otherwise impede flux through phloem sieve pores. The identification and characterization of a mutant of Arabidopsis var. Columbia with greatly reduced levels of beta-amylase activity is reported here. The reduced beta-amylase 1 (ram1) mutation lies in the gene encoding the major form of beta-amylase in Arabidopsis. Although the Arabidopsis genome contains nine known or putative beta-amylase genes, the fact that the ram1 mutation results in almost complete loss of beta-amylase activity in rosette leaves and inflorescences (stems) indicates that the gene affected by the ram1 mutation is responsible for most of the beta-amylase activity present in these tissues. The leaves of ram1 plants accumulate wild-type levels of starch, soluble sugars, anthocyanin, and chlorophyll. Plants carrying the ram1 mutation also exhibit wild-type rates of phloem exudation and of overall growth. These results suggest that little to no beta-amylase activity is required to maintain normal starch levels, rates of phloem exudation, and overall plant growth.

Figure 1

The ram1 mutant exhibits severely reduced levels of β-amylase activity. Wild-type (WT), pgm1, and ram1 pgm1 gl1 plants were grown in soil under continuous light for 3 to 5 weeks. Cuttings (leaves or stem sections) were then removed and placed in water (0 mm Suc) or 88 mm Suc. After approximately 3 d, total proteins were prepared from the leaf or stem sections and assayed for β-amylase activity (1 unit [U] = the amount of β-amylase required to generate 1 nmol of reducing sugar per min from amylopectin). Note that, although pgm1 mutants grown under a 12-h photoperiod accumulate high levels of soluble sugars leading to induction of β-amylase activity, pgm1 mutants grown under continuous light exhibit wild-type levels of soluble sugars and of β-amylase activity (Caspar et al., 1985, 1989). The data presented represent the combined results of multiple independent experiments. Values indicate the means ± sd (n = 2–6).

Figure 2

The ram1 mutant has wild-type levels of β-amylase mRNA. Wild-type and ram1 pgm1 seedlings were grown on solid minimal Arabidopsis media for approximately 2 weeks and then for an additional 2 weeks in soil under continuous light conditions. Plants were then removed from the soil, rinsed with water, and placed with their root systems in either water (Suc −) or an 88 mm Suc solution (Suc +). After 3 d under 66 to 80 μmol photons m⁻² s⁻¹ continuous light, rosette leaves were harvested and used to prepare total RNA. A northern blot prepared using this RNA was probed with labeled DNA corresponding to an Arabidopsis β-amylase cDNA. The blot was then stripped and reprobed with labeled DNA corresponding to the 28S rDNA.

Figure 3

Severe decreases in β-amylase activity do not affect leaf starch or soluble sugar levels. Wild-type (WT) and ram1 plants were grown under continuous light for 44 d (= 24-h photoperiod plants); the final 5 d was at a light intensity of 140 to 150 μmol photons m⁻² s⁻¹. Additional plants were grown for 33 d under continuous light and then for an additional 11 d under an 8-h photoperiod (= 8-h photoperiod plants); the final 5 d was at a light intensity of 220 to 230 μmol photons m⁻² s⁻¹. Leaves were harvested from the plants growing in the 8-h photoperiod at the end of the light period, 5.2 h after the end of the light period and 9.3 h after the end of the light period. Two rosette leaves, each from a different plant, were used for each sugar/starch assay. The sugar assays measured the combined amounts of Glc, Suc, and Fru present in a particular sample. The ram1 plants used in these experiments lack the pgm1 mutation and are from a line that has been back-crossed four times to wild-type plants. Values indicate the means ± sd (n = 8). This experiment was repeated, with similar results. fwt, Fresh weight.

Figure 4

The ram1 mutant exhibits wild-type (WT) rates of phloem exudation. Wild-type and ram1 (lacking the pgm1 mutation) plants were grown under continuous light for 46 d; the final 5 d was at a light intensity of 200 to 210 μmol photons m⁻² s⁻¹ (= 24-h photoperiod plants). Additional plants were grown for 35 d under continuous light and then for an additional 11 d under an 8-h photoperiod; the final 5 d was at a light intensity of 220 to 230 μmol photons m⁻² s⁻¹ (= 8-h photoperiod plants). Phloem exudates were collected over different time intervals from groups of three leaves. The phloem exudates were analyzed to determine the amount of soluble sugar (Suc, Glc, and Fru) exuded from each group of leaves within a particular time period. The amount of soluble sugar in each sample was then divided by the combined fresh weights (fwt) of the three leaves from which that sample was collected. Values indicate the means ± sd (n = 8). This experiment was repeated, with similar results.

Figure 5

Anthocyanin and chlorophyll accumulation are unaffected by the ram1 mutation. A, For anthocyanin determinations, plants were grown under continuous light for 2 weeks on minimal Arabidopsis medium supplemented with 30 mm Suc and then for an additional week on medium containing either 30 or 180 mm Suc. Anthocyanin levels were divided by sample fresh weight (fwt) and scaled relative to values obtained for wild-type (WT) seedlings on 180 mm Suc. B, For chlorophyll determinations, plants were grown under continuous light for 2 weeks on minimal Arabidopsis medium supplemented with either 15 or 150 mm Suc. Total shoot systems of three seedlings were used for each anthocyanin and chlorophyll assay. Values indicate the means ± sd (n = 3). These experiments were repeated, with similar results.

Figure 6

The ram1 mutant exhibits wild-type (WT) growth rates. Wild-type and ram1 plants were grown in soil at 19°C to 20°C under either continuous light (24-h photoperiod) or an 8-h photoperiod. At regular intervals, shoot systems (corresponding to all above ground parts) of groups of five plants were harvested and weighed. The ram1 plants used in these experiments lack the pgm1 mutation and are from a line that has been back-crossed four times to wild-type plants. The values presented represent the weights of individual shoot systems. Values indicate the means ± sd (n = 5–6). This experiment was repeated, with similar results.