Subcellular Localization and Characterization of Amylases in Arabidopsis Leaf

Abstract

Amylolytic enzymes of Arabidopsis leaf tissue were partially purified and characterized. Endoamylase, starch phosphorylase, D-enzyme (transglycosylase), and possibly exoamylase were found in the chloroplasts. Endoamylase, fraction A2, found only in the chloroplast, was resolved from the exoamylases by chromatography on a Mono Q column and migrated with an RF of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, Al, has an RF of 0.23 on the polyacrylamide gel. Viscometric analysis showed that Al has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. Al, however, can be distinguished from A3 by having much higher amylolytic activity in succinate buffer than acetate buffer, and having much less reactivity with amylose. Al probably is also localized in the chloroplast, and contributes to the 30 to 40% higher amylolytic activity of the chloroplast preparation in succinate than acetate buffer at pH 6.0. The high activity of n-enzyme compared to the amylolytic activity in the chloroplast suggests that transglycosylation probably has an important role during starch degradation in Arabidopsis leaf. Extrachloroplastic amylase, A3, has an RF of 0.55 on 7% electrophoretic gel and constitutes 80% of the total leaf amylolytic activity. The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic.

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

“…This suggests a lowered a-amylase activity and an increased P-amylase activity. Native gel assays confirmed these data; the only known chloroplast amylases, A1 (p) and A2 (a) (Lin et al, 1988b), appeared unchanged. But higher activities could be detected for the extrachloroplastic p-amylase A3 and also (although less pronounced) for the A5 isozyme.…”

“…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.…”

“…The ADGase and UDP-glucose pyrophosphorylase gel assays were performed following the procedure of Weaver et al (1972). Amylases were detected as described by Lin et al (1988b).…”

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

“…Although #-amylase activity has been detected in chloroplasts from spinach leaves (23), several studies with other plants have 'Supported in part by Grants-in-Aid for Scientific Research on Priority Areas ("Totipotency of Plant Cells" and "Metabolite Accumulation in Higher Plants") from the Ministry of Education, Science and Culture, Japan. indicated that most, if not all, ofthe cellular 3-amylase activity is extrachloroplastic (13,14,18,25,26) and may be localized in the vacuole (15, 3 1). Precursors to the fl-amylase of barley (1 1) and soybean (19) seeds and a precursor to the subunits of 3-amylase in sweet potato tuberous roots (30) do not contain N-terminal transit peptide sequences that play a role in the transport of proteins into plastids.…”

Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves (Diurnal Oscillation of Amylolytic Activity and Starch Content in Chloroplasts)

“…The chloroplastic and extrachloroplastic amylases differ partly in their substrate specificity (Okita and Preiss, 1980). The extrachloroplastic amylase in spinach leaves has been identified as an a-amylase (Okita and Preiss, 1980), whereas in Vicia faba (Chapman et al, 1972), pea (Ziegler, 1988), barley (Jacobsen et al, 1986), wheat (Ziegler and Beck, 1986), and Arabidopsis thaliana (Lin et al, 1988) most if not a11 of the P-amylase activity was associated with the extrachloroplastic fraction and may be localized in the vacuole (Ziegler and Beck, 1986). Spinach leaves, like pea leaves, contained one phosphorylase form, which appeared also to be located outside of the chloroplast (Steup and Latzko, 1979).…”