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 affinity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in flowers 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-related enzymes during potato tuber dormancy and sprouting

“…On the other hand, STP itself may also be involved in starch synthesis [6], and in this case, both enzymes would form starch in the direct neighbour hood of the vascular tissue. Duwenig et al [24] reported parallels in the regulation of expression of phosphorylase and AGPase genes in spinach and pota toes and use this finding to support their suggestion that STP is involved in starch synthesis. However, such evidence can equally well indicate that STP partici pates in starch cycling.…”

“…However, such evidence can equally well indicate that STP partici pates in starch cycling. If it is so, such cycling is likely to involve the plastid isoform of STP, since Duwenig et al [24] presented evidence that the cytosolic isoform was not involved in starch breakdown during storage. One may then hypothesize that amylases degrade starch in storage parenchyma cells, the resulting sugars being transported towards the vascular tissue, and starch is resynthesized around the vascular tissue.…”

Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization

“…The other type, Pho2 (or,previously,Pho H), is restricted to the cytosol and possesses an extremely high anity towards branched polyglucans such as glycogen (Steup 1988). Plant Pho1-type phosphorylases are unique in two aspects: First, all ®ve proteins of this type sequenced so far contain a large insertion of approximately 80 amino acid residues that is located between the C-terminal and the N-terminal domain (Nakano and Fukui 1986;Lin et al 1990;Sonnewald et al 1995;Buchner et al 1996;Duwenig et al 1997). The insertion has not been found in any phosphorylase from bacteria or animals (Newgard et al 1989) or in the plant Pho2-type enzymes (Mori et al 1991;Buchner et al 1996).…”

“…In potato leaf discs, expression both of the Pho1a-type phosphorylase and of ADPglucose pyrophosphorylase is induced by exogenous sugars such as sucrose or glucose (Duwenig et al 1997).…”

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

“…However, observations have shown that the accumulation of starch is connected with the increase of SP activity in potato tubers (Mingo-Castel et al 1976;Albrecht et al 2001), maize endosperm (Liu and Shannon 1981), rice (Baun et al 1970), wheat (Schupp and Ziegler 2004), and sweet potato roots (Chang et al 2000). Starch content is also in proportion to the expression of SP in potato (Brisson et al 1989;St-Pierre and Brisson 1995;Duwenig et al 1997;Albrecht et al 2001), spinach (Duwenig et al 1997), and pea (Van Berkel et al 1991). We have reported that the proteolytic modification of L-SP on L78 has a regulatory role for its catalytic behavior (Chen et al 2002).…”