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

Abstract

Higher plants contain two types of phosphorylase (EC 2.4.1.1). One type is plastidic (Phol) and the other resides in the cytosol (Pho2). For Solanum tuberosum L., two highly homologous Pho1-type sequences (designated as Pho1a and Pho1b, respectively) have been described that occur both in a homodimeric, (Pho1a)2, and a heterodimeric, Pho1a-Pho1b, state [U. Sonnewald et al. (1995) Plant Mol Biol 27:567 576; T. Albrecht et al. (1998) Eur J Biochem 251:981-991]. We present a spatial and temporal analysis of the expression patterns of the Pho1-type phosphorylases in S. tuberosum. Expression was analyzed at transcript, protein and activity levels. The specificity of both the probes and the antibodies used was carefully determined to ensure selectivity of detection. For both the Pho1a and Pho1b probes the degree of cross-hybridization was estimated. Peptide scanning identified the epitopes of the anti-Pho 1a and anti-Pho 1b antibodies. Expression of the two Pho1-type genes was analyzed in various organs of the potato plant. In all organs studied the Pho1a transcript levels exceeded those of Pho1b. Furthermore, leaves of a given developmental stage were sampled during the light period and were analyzed for transcript and protein levels and for various carbohydrate pools as well. The data show that in leaves the Pho1a gene expression closely corresponds to starch accumulation, suggesting that the enzyme fulfils a metabolic function within the process of starch biosynthesis. In tubers, Pho1a is constitutively expressed in the parenchyma cells whereas expression of the Pho1b, gene is restricted to cells in close vicinity of the vascular tissue.

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

“…On the other hand, SP had never been reported as having any regulation mechanism during the past decades, and its physiological role remains unclear (Nelson and Pan 1995), although pea SP has been reported as inhibited by the physiological concentrations of ADPglucose (Kruger and Ap Rees 1983). 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).…”

“…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).…”

Plastidial α-Glucan Phosphorylase Is Not Required for Starch Degradation in Arabidopsis Leaves But Has a Role in the Tolerance of Abiotic Stress

“…Antisense repression of a gene encoding a plastidial isoform of phosphorylase in potato (Solanum tuberosum) decreased the detectable phosphorylase activity in leaves but had no major impact on the accumulation of starch (Sonnewald et al, 1995). However, the nature of the antisense technique and the fact that potato contains a second plastidial isoform, which is expressed in leaves (Albrecht et al, 2001), mean that an important function for phosphorylase in starch metabolism could not be ruled out.…”

Identification of Polysaccharide Binding Proteins by Affinity Electrophoresis in Inhomogeneous Polyacrylamide Gels and Subsequent SDS–PAGE/Matrix-Assisted Laser Desorption Ionization–Time of Flight Analysis

“…In potato tubers only one Pho1-type phosphorylase isoform is detectable (3), which is retarded noticeably if the separation gel contains a high concentration of glycogen. In contrast, potato leaves express two pho1-type genes, designated Pho1a and Pho1b (11). In leaf extracts both a Pho1-type homodimer [(Pho1a) 2 ] and a heterodimer [Pho1a-Pho1b] are detectable (3).…”