Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs

Abstract

A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes. Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low anity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in¯owers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds. This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds. Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation. The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates. Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined. A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species. In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates. These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.

Starch Biosynthesis. 3: The Glycogen Precursor Mechanism Using Phosphorylase in the Production of the Precursor Glycogen

“…Thus as pointed out by Duwenig et al [37] the observation that the presence of polygalacturonic acid, even though it activates bamylase for the degradation of starch, does not have any inductive effect on the activity of plastidic phosphorylase for starch degradation. Thus Duwenig et al [37] state that "… this result might be interpreted as an argument against a role of plastidic phosphorylase in starch degradation, as the gene is instead coexpressed with other genes that encode starch biosynthetic enzymes".…”

“…More recent results by Duwenig et al [37], on spinish leaves showed that the activity of the apparently single plastidic phosphorylase in spinach is increased when net starch synthesis occurs. As pointed out by Duwenig et al [37] these results, which show that the activity of phosphorylase is higher at a time when starch synthesis occurs, agree with the studies of 1) Van Berkel et al [38] who in 1991 showed that the activity of plastidic phosphorylase is higher in the developing seeds of pea cotyledones (when starch is being produced) as compared to these germinating seeds (when starch is being degraded); 2) Kossman et al [39] who in 1991 observed that plastidic phosphorylase transcript level is higher in growing potato tubers (when starch is being synthesized), as compared to that in potato sprouting tubers; and 3) Steup [40] who in 1988 showed that an increase in the activity of phosphorylase is associated, not only with starch degradation, but also with starch synthesis.…”

Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase,

“…cDNAs encoding the plastidic forms of SP enzymes have been isolated and characterized from a variety of higher plants (Steup, 1988). Studies with potato (Brisson et al, 1989;St-Pierre and Brisson, 1995), spinach (Duwenig et al, 1997a), and pea (van Berkel J et al, 1991) have shown that the expression of plastidic SP genes correlates with starch biosynthesis. The fact that the 112-kD SP enzyme was enriched in the amyloplast stroma where other starch synthetic enzymes are localized supports the hypothesis that SP may play some role in starch biosynthesis.…”

“…The insertion sequence is thought to reflect the affinity of plastidic SP enzymes for ␣-glucan substrates (Mori et al, 1993). The three peptides of the 112-kD protein also aligned closely with sequences from the plastidic SP enzymes from potato leaf (Sonnewald et al, 1995), sweet potato (Lin et al, 1991), and spinach leaf (Duwenig et al, 1997a). The N-terminal amino acid sequence of the 112-kD protein was unique and did not align with the N terminus of the plastidic SP enzymes or with any sequences in the GenBank database.…”

“…Affinity electrophoresis (Shimomura and Fukui, 1980;Takeo, 1984;Duwenig et al, 1997a) was performed with 7.5% (w/v) polyacrylamide tube gels in the absence or presence of the indicated glucan substrates at 4°C. Electrophoresis was performed for 1 h at 3 mA per tube, and then for 3 h at 5 mA per tube.…”