Purification and Characterization of Pea Epicotyl β-Amylase

118

110

The levels of P-amylase activity and of the mRNA for P-amylase in rosette leaves of Arabidopsis thaliana (1.) Heynh. increased significantly, with the concomitant accumulation of starch, when whole plants or excised mature leaves were supplied with sucrose. A supply of glucose or fructose, but not of mannitol or sorbitol, to plants also induced the expression of the gene for j?-amylase, and the induction occurred not only in rosette leaves but also in roots, stems, and bracts. These results suggest that the gene for P-amylase of Arabidopsis is subject to regulation by a carbohydrate metabolic signal, and expression of the gene in various tissues may be regulated by the carbon partitioning and sink-source interactions in the whole plant. P-Amylase (EC 3.2.1.2) is abundant in some starch-storing organs of plants, such as seeds and tuberous roots. In addition to these reproductive organs, 0-amylase is also present in various vegetative tissues (Beck and Ziegler, 1989). However, the physiological roles of p-amylase in plants in vivo are not known at present in spite of extensive enzymological studies of the breakdown of starch in vitro. Native starch granules are not attacked by plant P-amylases without the prior digestion by other enzymes or the solubilization by boiling (Beck and Ziegler, 1989). Precursors to P-amylases from plants ( Kreis et al., 1987;Monroe et al., 1991;Yoshida and Nakamura, 1991) do not contain the N-terminal transit peptide sequences that are responsible for targeting proteins to plastids. Indeed, results of most of the previous studies of vegetative tissues from various plant species indicate that P-amylase is present outside the '

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Sugar-Inducible Expression of a Gene for [beta]-Amylase in Arabidopsis thaliana

Mita¹,

Suzuki-Fujii²,

Nakamura³

1995

118

110

“…The precursor to the subunit of j3-amylase in sweet potato does not contain a presequence at its N terminus (30), suggesting that j3-amylase is localized outside of the plastids as are the fiamylases in vegetative tissues of other plants (13-15, 26, 30). Furthermore, ,B-amylase is unable to attack native starch granules without their prior digestion by other enzymes or solubilization by boiling (15,27). Therefore, it is unlikely that 3-amylase plays an essential role in the metabolism of transitorily accumulated and reserve starch within the plastid.…”

Section: Effects Of Various Sugars On Inductionmentioning

confidence: 99%

“…Extrachloroplastic malto-dextrins have been suggested to be the substrates ofB-amylase in vivo (15). 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.…”

Section: Effects Of Various Sugars On Inductionmentioning

confidence: 99%

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

Nakamura¹,

Ohto²,

Yoshida³

et al. 1991

106

ABSTRACT,8-Amylase of sweet potato (Ipomoea batatas L.), which constitutes about 5% of the total soluble protein of the tuberous root, is absent or is present in only small amounts in organs other than the tuberous roots of the normal, field-grown plants. However, when leaf-petiole cuttings from such plants were supplied with a solution that contained sucrose, the accumulation of 8-amylase was induced in both leaf and petiole portions of the explants. The sucrose-induced accumulation of fl-amylase in leaf-petiole cuttings occurred concomitant with the accumulation of starch and of sporamin, the most abundant storage protein of the tuberous root. The accumulation of fl-amylase, of sporamin and of starch in the petioles showed similar dependence on the concentration of sucrose, and a 6% solution of sucrose gave the highest levels of induction when assayed after 7 days of treatment. The induction of mRNAs for f-amylase and sporamin in the petiole could be detected after 6 hours of treatment with sucrose, and the accumulation of j6-amylase and sporamin polypeptides, as well as that of starch, continued for a further 3 weeks. In addition to sucrose, glucose or fructose, but not mannitol or sorbitol, also induced the accumulation of fl-amylase and sporamin, suggesting that metabolic effects of sucrose are important in the mechanism of this induction. Treatment of leaf-petiole cuttings with water under continuous light, but not in darkness, also caused the accumulation of small amounts of these components in the petioles, probably as a result of the endogenous supply of sucrose by photosynthesis. These results suggest that the expression of the gene for f-amylase is under metabolic control which is coupled with the expression of sink function of cells in the sweet -otato.

“…,B-Amylase, the major amylolytic and the major exoamylolytic enzyme in pea leaves (1,16,20) appears to be primarily vacuolar (28). Of the extrachloroplastic glucans surveyed, none have been found to act as substrates for either a-amylase (2) or j3-amylase (20), and no substantiated explanations have been tendered to account for the presence of these enzymes outside the chloroplast.…”

mentioning

confidence: 99%

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Saeed

Duke²

1990

Self Cite

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or ,B-limit dextrin containing gel and KI/12 staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar fI-amylase (EC 3.2.1.2) and the primarily apoplastic aamylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily Bl-amylase) activity and considerably higher ratios of ,-to aamylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and fl-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of a-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher a-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in aamylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the a-amylase that was greatly increased in activity was the primarily apoplastic aamylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic a-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar 8-amylase activity.The physiological roles of amylases in vegetative tissues of higher plants are poorly understood. One reason for this current state of confusion is that at the cellular level most of the amylolytic activity in plant vegetative tissues is extrachloroplastic, away from the site of starch synthesis and degradation. More than 90% of the total amylolytic activity in pea leaves has been reported to be extrachloroplastic (16).