Altered regulation of β-amylase activity in mutants of Arabidopsis with lesions in starch metabolism

Abstract

Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants wgs due to an up to 40-fold increase in the activity of an extrachloroplast j8-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of (3-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that fi-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.One of the few experimental treatments of higher plants that may result in an increase in the rate of photosynthetic CO2 fixation is to remove some of the leaves from plants at the stage during which seeds are developing (1, 2). In many species, this causes an increase in the rate of photosynthetic CO2 fixation in the remaining leaves. The mechanisms that regulate the altered rate of photosynthesis are not known, but they have been suggested to be either a hormonal signal produced by the developing seeds or a metabolic effect of the altered carbohydrate pools that occur in the leaf cells because of an imbalance between synthesis and export of carbohydrate. To investigate this phenomenon, we have previously identified three classes of mutants of Arabidopsis that either lack leaf starch (3, 4), contain reduced levels of starch (5), or have elevated levels of starch. These mutants have substantial alterations in leaf carbohydrate metabolism that may mimic some of the effects of experimentally induced changes in the ratio of carbohydrate-exporting tissue to carbohydrateimporting tissue. However, by contrast with invasive methods, the specificity and stability of the genetic differences between the mutants and the wild type facilitates a detailed analysis of the secondary metabolic responses that are directly associated with carbohydrate metabolism.A previous analysis of a mutant that was unable to synthesize starch because of a lack of chloroplast phosphoglucomutase activity (3) indicated that it had increased leaf sugar content and altered photosynthesis and dark respiration rates, which were secondary effects of an inability to synthesize starch. In a further analysis of the secondary effects of this and related mutations on other aspects of leaf metabolism, we have identified a major effect on the leaf 3-amylase (EC 3.2.1.2) activity in these mutants. Elucidation of the mechanisms responsible for this effect may provide fresh insights into the regulation of l...

Monogenic Recessive Mutations Causing Both Late Floral Initiation and Excess Starch Accumulation in Arabidopsis.

“…The degree of changes in these latter activities varied under different growth and light conditions (data not shown). Because similar changes were observed in starchless mutants (Lin et al, 1988b;Caspar et al, 1989), this increased activity is a secondary rather than a direct effect of the primary mutation. However, the different extent of the decrease in a-amylase activity during plant development agrees well with the increasing amount of starch accumulated in the leaves.…”

“…We also examined enzyme activities of PGI, PGM, ADGase, branching enzyme, disproportioning enzyme (D-enzyme), and starch phosphorylase by native gel electrophoresis, demonstrating that the cam? and gi mutants possess all of the isozymes Because a variety of both starch-deficient and starch-excess mutants in Arabidopsis has a considerably elevated leve1 of extrachloroplastic amylase activity (Caspar et al, 1989), we examined the caml mutant for amylase activity. Using an in vitro assay based on starch digestion and staining of the remaining starch with iodine, we detected a slightly higher general amylase activity for the caml mutant (data not shown).…”

The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

“…␤-Amylase activity is induced by sugars (Caspar et al, 1989;Nakamura et al, 1991;Mita et al, 1995). Mutations that affect sugar-induced ␤-amylase expression are expected to be useful in elucidating the molecular mechanisms by which sugar-regulated gene expression occurs in plants.…”

“…The major form of ␤-amylase in Arabidopsis has been shown to be localized to phloem sieve elements (Wang et al, 1995) and to be inducible by sugars (Caspar et al, 1989;Mita et al, 1995). Based on these findings, the major form of Arabidopsis ␤-amylase has been postulated to facilitate phloem transport by preventing accumulation of polysaccharides in sieve elements.…”

Sucrose-Induced Accumulation of β-Amylase Occurs Concomitant with the Accumulation of Starch and Sporamin in Leaf-Petiole Cuttings of Sweet Potato

“…However, the presence of such a-glucans needs to be established (3,27), and the amount of j3-amylase that is synthesized seems to be more than sufficient for such a role. As an alternative explanation, Giese and Hejgaard (5) suggested that f3-amylase may have a nitrogen-storage function in barley seeds, since the profile of accumulation of ,B-amylase in developing seed resembles to that of the storage protein, hordein, and the synthesis of 3-amylase responds to increased supply of nitrogen.…”