Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers.

Starch phosphorylase has been cloned from a lambda gt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding-induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.     

Starch-accumulating Sweet Potato Callus Tissue Devoid of β-Amylase but with Two Starch Phosphorylase Isozymes

By controlling the concentrations of kinetin, auxin, and sucrose in the Murashige–Skoog medium, starch contents in callus culture induced from sweet potato tissues could be manipulated. Activity staining and Western analysis on PAGE plates and activity assays made on starch phosphorylase in the presence and absence of mercuric ions showed that β-amylase is absent in callus cultures regardless of whether their starch content is high or low. This would imply that β-amylase induction in sweet potato calli is not linked to the metabolic control through which the expression of storage function is associated, as proposed by Nakamura et al. [Plant Physiol., 96, 902 (1991)] for sweet potato leaf-petiole cuttings. Analyses of starch phosphorylase in crude extracts suggested the presence of a new starch phosphorylase in tuberous root and callus tissue. This phosphorylase is immunologically different from the tuberous root and leaf enzymes that we studied previously.     

A second L-type isozyme of potato glucan phosphorylase: cloning,antisense inhibition and expression analysis

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81–84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1–81) and the insertion sequence (amino acids 463–570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively.Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.     

Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods

Phosphorylase was purified from young and senescent potato tubers. Antibodies raised against the enzyme from young tubers crossreacted with phosphorylase from old tissue, although the latter exhibited different physico-chemical properties. In polyacrylamide gel electrophoresis it migrated with higher mobility, its subunit molecular weight was determined in the range of 40,000 in contrast to 100,000 of the phosphorylase in young tubers. The enzyme of senescent tubers displayed an isoelectric point of 5.4 different from the one of young tubers with 5.0, and the diffusion coefficients of the two enzymes varied. The appearance of the phosphorylase form typical for senescent tissue is connected with changes in the intracellular localization as revealed by immunofluorescence. Before massive starch accumulation is initiated, non-vacuolated subepidermal cells contain antigenically active material in their cytoplasm. During starch accumulation in fully differentiated storage parenchyma, only amyloplasts fluoresce, indicating the presence of adsorbed phosphorylase protein. Cytoplasmic phosphorylase can be detected in the continuance of senescence and, finally, after 16 months of tuber storage, the particle-bound enzyme had mostly disappeared. Simultaneously, we observed membrane destruction and decomposition on the ultrastructural level. The phosphorylase from senescent potatoes is a converted molecule and seems to be formed by proteolytic cleavage. The location of phosphorylase in the amyloplasts during starch synthesis indicates that it also plays a role in starch synthesis and not only in its degradation.Abbreviations PBS phosphate buffered saline - FITC fluorescein-isothiocyanate - IgG immunoglobuline G Dedicated to Professor Dr. A. Frey-Wyssling on the occasion of his 80th birthday     

Engineered plant phosphorylase showing extraordinarily high affinity for various alpha-glucan molecules.

alpha-Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78-residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78-residue insertion and its flanking regions are replaced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574-5581), but when two and four unconserved residues in the N-terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Auxin and Kinetin on the Development and Differentiation of Potato Tissue Cultured in vitro

The present investigation deals with the influence of auxin and kinetin on cultures of potato tuber tissue, and the effect of the size of the explants on the organ formation. Exogenous application of auxin is indispensable for initiating callus formation of the tissue, but kinetin is not necessarily required. Kinetin serves to maintain the callus development, indicating that the stimulation of callus growth due to exogenous auxin would presumably be mediated by the addition of kinetin to the medium. A relatively low concentration of auxin in the medium is required for root formation on the culture. In this case, IAA is markedly superior to any other auxin in root forming activity. NAA is somewhat less effective than IAA, and 2,4-D shows no stimulation on root formation. The growth and organ formation of potato tissue are remarkably affected By the variation in sizes of the explants. Spontaneous formation of a sprout was observed on the large explant in the medium without auxin. Small explants form only callus or roots in the presence of auxin and kinetin irrespective of combination or concentration. It seems likely that a sufficient amount of stimulus for initiation of bud formation may be contained in the large sized explant itself.     

Induction of alcohol dehydrogenase in explants of potato tuber (Solanum tuberosum L.)

Plant Cell Reports - During callus formation a huge increase in alcoholdehydrogenase activity was observed in potato tuber tissue discs. Callus formation was no prerequisite for this increase;...     

Isoperoxidases as markers of the wound-induced differentiation pattern in potato tuber

Isoperoxidases induced by wounding in potato tuber tissue were separated by starch gel electrophoresis and found to be distributed in a highly specific spatial pattern. This pattern of molecular differentiation correlates well with the pattern of cell differentiation associated with wound healing. Although wound induced isoperoxidases were found to vary between three varieties of potato, they were distributed in the same spatial pattern. The combination of isozymes extracted from various parts of a potato plant was specific for each of nine organs and tissues, and all combinations were different from the isozymes from wounded tuber tissue. Isoperoxidases can thus be considered as highly specific molecular markers of cell differentiation.     

Assimilate Conversion in Potato Tubers in Relation to Starch Deposition and Cell Growth

The activity of enzymes involved in the conversion of sucrose to starch together with the distribution of ¹⁴C-labelled photosynthate and ⁴C-sucrose was studied in potato tubers showing a range of growth rates and growth patterns. Within a particular tuber the uptake of ¹⁴C from labelled photosynthate and the conversion to ethanol-insoluble ¹⁴C was greatest in the apical tissue where both the rate of production of new storage cells and starch synthesis were likely to be greatest. Uptake and conversion of ¹⁴C was lowest in the older tissue of the tuber base. Pre-treatment of tubers with gibberellic acid reduced the total input of ¹⁴C from labelled photosynthate, reversed the gradient in ¹⁴C uptake between apical and basal tuber tissue, increased the amount of ¹⁴C per g fresh weight in the basal tissue and decreased the conversion of labelled sugars to starch. For tubers with different growth rates both the total uptake of ¹⁴C from labelled photosynthate and the ratio ethanol-insoluble ¹⁴C/ethanol-soluble ¹⁴C appeared to be correlated with growth rate. In contrast when tubers were fed directly with ¹⁴C-sucrose via the tuber surface, total uptake was independent of growth rate but the correlation between growth rate and the ratio ethanol-insoluble ¹⁴C/ethanol-soluble ¹⁴C persisted. Within a particular tuber there was a decreasing gradient in sucrose synthetase activity between youngest tissue of the tuber apex and the older tissue at the tuber base but there was no clear correlation between mean enzyme activity and tuber growth rate. ADPG-pyrophosphorylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase showed some correlation with tuber growth rate. Starch synthase, starch phosphorylase and UDPG-pyro-phosphorylase activities per g fresh weight of tuber tissue appeared to be relatively constant. The results suggest that the transport of sugar from the phloem sieve tubes to the tuber storage parenchyma cells, in particular the phloem unloading step, and the conversion of sugar into starch are subject to separate regulation in the potato tuber.     

Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.     

Light-mediated changes in the plastidic phosphorylase patterns in shoots of Pisum sativum

α-1,4-Glucan phosphorylase (EC 2.4.1.1) forms from light or dark grown shoots of Pisum sativum L. cv. 'Kleine Rheinländerin' have been studied using various electrophoretic techniques. The phosphorylase patterns of green and etiolated shoots differed. Etiolated shoots contained two enzyme forms, one residing inside and the other outside the etioplast; this was shown by electrophoresis of extracts of isolated etioplasts. Purity and intactness of the organelle preparation were ascertained by electron microscopy. Light-grown shoots contained, in addition to these two enzyme forms, a third phosphorylase which appears to be chloroplast-specific. The two plastidic phosphorylase forms differed slightly in their apparent molecular masses (as determined by non-denaturing polyacrylamide gel electrophoresis) and in their affinities towards branched polyglucans (as revealed by affinity electrophoresis). The apparent affinity of the extrachloroplastic phosphorylase form to these polyglucans was orders of magnitude higher than that of the two plastidic enzyme forms. The development of the chloroplast-specific phosphorylase pattern is under photocontrol. Investigations performed with red or far-red illuminated wild-type plants and with a pale mutant which has a highly reduced pigment and thylakoid content suggest that this photocontrol is mediated by phytochrome.     

α-1,4-glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.)

Peptide patterns and immunological properties of the cytoplasmic and chloroplastic -1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves have been studied and were compared with those of phosphorylases from other sources. The two spinach leaf phosphorylases were immunologically different; a limited cross-reactivity was observed only at high antigen or antibody concentrations. Peptide mapping of the two enzymes resulted in complex patterns composed of more than 20 fragments; but no peptide was electrophoretically identical in both proteins. Approximately 13 to 15 of the fragments exhibited antigeneity but no cross-reactivity of any peptide was observed. Therefore, the two compartment-specific phosphorylase forms from spinach leaves represent isoenzymes possessing different primary structures. Peptide patterns of potato tuber and rabbit muscle phosphorylase were different from those of the two spinach leaf enzymes. Although the potato tuber phosphorylase resides in the plastidic compartment and is kinetically closely related to the chloroplastic spinach enzyme, it reacted more strongly with the anti-cytoplasmic-phosphorylase immunoglobulin G. Similar results were obtained with rabbit muscle phosphorylase. These observations support the assumption that the chloroplast-specific phosphorylase isoenzyme has a higher structural diversity than does the cytoplasmic counterpart.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PEG polyethylene glycol (approx. MW 8000) I=Schächtele and Steup 1986     

Affinity of glucose analogs for alpha-glucan phosphorylases from rabbit muscle and potato tubers

The action of phosphorylase b from rabbit muscle and potato phosphorylase was inhibited to various extents by several glucose analogs. Like glucose itself, all of the glucosidic oxygen-substituted analogs tested in kinetic experiments showed a nonlinear competitive inhibition for muscle phosphorylase b and a linear competitive one for potato phosphorylase. 5-Thio-D-glucose, one of the ring oxygen-substituted analogs, also inhibited the action of muscle phosphorylase b in the same manner, while the inhibition pattern of 5-amino-D-glucose (nojirimycin) was of a linear noncompetitive type. Since the conformation of 5-amino-D-glucose in aqueous solution is half-chair (Reese et al. (1971) Carbohyd. Res. 18, 381-388), the unusual kinetic behavior of the compound toward muscle phosphorylase b was supposed to be due to its half-chair conformation. In the glucosidic oxygen-substituted analogs, the affinity for both muscle phosphorylase b and potato phosphorylase decreased with decreasing order of magnitude of electronegativity of the glucosidic atom. The strong positive correlation between the affinity and the electronegativity suggests that D-glucose-1-P, the substrate, may bind to phosphorylase with the formation of a hydrogen bond between its glucosidic oxygen and a hydrogen donor of the enzyme.     

Continous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum dem/ssum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculatedin vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis denovo of PSTV was substantiated by the incorporation of [³H]uridine and of [³²P]orthophosphate into viroid RNA.     

Potato Lectin: A Cell-Wall Glycoprotein

The activity and the amount of potato lectin were measured inpotato tuber slices (Solanum tuberosum cv. Huinkul) aeratedfor 48 h. Lectin was found in a soluble form, liberated to themedium and associated with insoluble structures. Polyacrylamidegel electrophoresis in denaturating conditions and immunologicaltechniques indicated that the lectins associated to cell wall,soluble or liberated to the medium, were identical. The cell-wallfraction was found to contain 65% of total lectin in the tuber.The possible role of potato lectin in tubers was discussed. (Received June 5, 1985; Accepted September 3, 1985)     

Polyphenol oxidase in potato. A multigene family that exhibits differential expression patterns.

Polyphenol oxidase (PPO) activity in potato (Solanum tuberosum) plants was high in stolons, tubers, roots, and flowers but low in leaves and stems. PPO activity per tuber continued to increase throughout tuber development but was highest on a fresh weight basis in developing tubers. PPO activity was greatest at the tuber exterior, including the skin and cortex tissue 1 to 2 mm beneath the skin. Flowers had high PPO activity throughout development, particularly in the anthers and ovary. Five distinct cDNA clones encoding PPO were isolated from developing tuber RNA. POT32 was the major form expressed in tubers and was found in all parts of the tuber and at all stages of tuber development. It was also expressed in roots but not in photosynthetic tissues. POT33 was expressed in tubers but mainly in the tissue near the skin. POT72 was detected in roots and at low levels in developing tubers. NOR333 was identical with the P2 PPO clone previously isolated from potato leaves (M.D. Hunt, N.T. Eannetta, Y. Haifeng, S.M. Newman, J.C. Steffens [1993] Plant Mol Biol 21: 59-68) and was detected in young leaves and in tissue near the tuber skin but was highly expressed in flowers. The results indicate that PPO is present as a small multigene family in potato and that each gene has a specific temporal and spatial pattern of expression.     

Subcellular immuno-localization, amino acid composition and partial amino acid sequences of α-l,4-glucan phosphorylase of Gracilaria spp (Rhodophyta)

Antibodies have been raised against an α-l,4-glucan phosphorylase (EC 2. 4. 1. 1) purified from the red alga Gracilaria chilensis. Localization of α-l,4-glucan phosphorylase in thin sections of G. chilensis and G. tenuistipitata was performed using immuno-gold labelling and transmission electron microscopy. The enzyme was localized in the cytosol and around the cytosolic starch granules of the algal cells. The labelling was not associated with the chloroplast or the cell wall. Amino acid composition of the red algal phosphorylase was quite similar to that of potato tuber and rabbit muscle phosphorylases. Partial amino acid sequences showed 48, 54 and 65% homology with the rabbit, potato and Escherichia coli enzymes, respectively.     

The presence of glycogen synthase in phosphorylase kinase preparations isolated from rabbit skeletal muscles

Phosphorylase kinase isolated from rabbit skeletal muscle contains a protein whose molecular mass as determined by polyacrylamide gel electrophoresis is 571 000 Da. The protein was found to possess a higher affinity for glycogen as compared to phosphorylase kinase and phosphorylase. The protein separated from kinase by chromatography on a DEAE-cellulose column produced during SDS electrophoresis one protein band corresponding to Mr of 95 200 Da. The above properties of the protein and the glycogen synthetase activity revealed in the presence of glucose-6-phosphate suggest that phosphorylase kinase preparations contain a hexameric form of glycogen synthetase.     

Effect of cold on glucose metabolism by callus and tubers of Solanum tuberosum

This work was done in order to discover the immediate effects of low temperature on glucose metabolism by tissue of Solanum tuberosum. [U-¹⁴C]-Glucose was supplied to tubers, and to callus derived from tubers, for 3 hr at 2 and 25°. The detailed distribution of label showed that lowering the temperature of both callus and tuber tissue to 2° caused a striking increase in the percentage of the metabolized label that was recovered in glucose-6-phosphate, fructose-6-phosphate. and glucose-1-phosphate. It is suggested that these results, together with the cold-lability of glycolytic enzymes, indicate that lowering the temperature of potato tissue reduces glycolysis in relation to the activities of other reactions involving hexose phosphates.