Changes in the levels of free IAA and cytokinins in potato tubers during dormancy and sprouting

Changes in the levels of free indol-3-ylacetic acid (IAA) and free cytokinins were determined in the course of dormancy and sprouting period in potato tubers(Solanum tuberosum L., cv. Nevskii) stored at 4 °C. The same analyses were performed in potato tubers after Ethrel application, which prolongs dormancy. No significant changes were found in free IAA level during dormancy followed by a rapid decrease during sprouting. After Ethrel application a significant lower IAA level was found 3 weeks after application, but further on no changes in free IAA level between treated and non-treated tubers were detected. Cytokinin level was relatively low and constant till sprouting and increases then by about 55 %, mainly due to an increase in the level of zeatin riboside and isopentenyladenosine. Ethrel application decreased cytokinin level during dormancy slightly, but postpones the increase coupled with sprouting by about 1 month. Thus, IAA does not seem to have a significant effect on tuber dormancy, while cytokinins are probably necessary for sprouting initiation.     

Maturation and subcellular compartmentation of potato starch phosphorylase.

The subcellular localization and maturation of starch phosphorylase (EC 2.4.1.1) was studied in developing potato tubers. The enzyme is localized inside the stroma of amyloplasts in young tubers, whereas in mature tubers it is found within the cytoplasm in the immediate vicinity of the plastids. A phosphorylase cDNA clone was isolated and used in RNA gel blot experiments to demonstrate that phosphorylase mRNAs are of the same size and abundance in both young and mature tubers. In vitro translation of mRNAs followed by immunoprecipitation with a phosphorylase antiserum indicates that the enzyme is synthesized as a higher molecular weight precursor in both young and mature tubers. The presence of a transit peptide at the N terminus of the protein was confirmed by the sequencing of the phosphorylase cDNA clone. The transit peptide has several structural features common to transit peptides of chloroplast proteins but contains a surprisingly large number of histidine residues. The mature form of the enzyme is present in both young and mature tubers, suggesting that a similar processing of the transit peptide may take place in two different subcellular locations.     

The catecholamine potentiates starch mobilization in transgenic potato tubers.

In human and animal cells, the catecholamines are involved in glycogen mobilization. Since the compounds are found in a potato, their function in starch mobilization was hypothesized. In order to verify this hypothesis, the transgenic potato plants Solanum tuberosum L. cv. Desiree overexpressing tyrosine decarboxylase (TD EC 4.1.1.25) cDNA from parsley has been generated. The cDNA expression was judged by the northern blot analysis and the enzyme activity measurements. Four independent transgenic lines with the highest TD mRNA expression were selected and analyzed. The expected substantial decrease in tyrosine content was followed by significant increase in tyramine and dramatic enhancement of norepinephrine synthesis was detected. The level of L-3,4-dihydroxyphenylalanin (L-Dopa) was only slightly increased and dopamine significantly decreased in most cases in these plants. The increase in norepinephrine was accompanied by changes in carbohydrate metabolism. The significant increase in glucose and sucrose and the decrease in starch content were characteristic features of TD overexpressed transgenic potato tubers. The features mentioned above indicate that catecholamines potentiate starch mobilization in potato plants in common with animal cells. The decrease in tyrosine content in transgenic plants is also compensated by significant increase in chlorogenic acid synthesis thus potentially increasing the antioxidant capacity of transgenic tubers. The glycoalkaloids content is changed in the transformants. This may originate from glucose accumulation and glycolysis activation. The obtained transgenic potato provides material for further detailed studies of the physiological function of catecholamines in plants.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.     

Concurrent synthesis and degradation of alcohol dehydrogenase in elicitor-treated and wounded potato tubers

The accumulation of alcohol dehydrogenase (ADH) in arachidonic acid-elicited potato (Solanum tuberosum L.) tuber discs was studied. In accordance with our previous report of the accumulation of Adh mRNA beginning 2 hours after elicitor treatment (DP Matton, CP Constabel, N Brisson [1990] Plant Mol Biol 14: 775-783), immunoprecipitation of ADH from in vivo labeled discs indicated that ADH synthesis occurred as early as 12 hours after treatment. However, levels of ADH activity and protein, as shown by enzyme assay and immunoblot, did not rise in parallel but decreased during the first 24 hours of treatment. After 24 hours, ADH activity and protein began to increase, reaching a several-fold increase at 96 hours after elicitation. Water-treated control discs showed a similar though delayed and less pronounced pattern. These results imply a turnover of ADH following elicitor treatment of potato tuber discs. As shown by nondenaturing gel electrophoresis, the synthesis and degradation involved the same ADH isozyme.     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

Growth ring formation in the starch granules of potato tubers

Starch granules from higher plants contain alternating zones of semicrystalline and amorphous material known as growth rings. The regulation of growth ring formation is not understood. We provide several independent lines of evidence that growth ring formation in the starch granules of potato (Solanum tuberosum) tubers is not under diurnal control. Ring formation is not abolished by growth in constant conditions, and ring periodicity and appearance are relatively unaffected by a change from a 24-h to a 40-h photoperiod, and by alterations in substrate supply to the tuber that are known to affect the diurnal pattern of tuber starch synthesis. Some, but not all, of the features of ring formation are consistent with the involvement of a circadian rhythm. Such a rhythm might operate by changing the relative activities of starch-synthesizing enzymes: Growth ring formation is disrupted in tubers with reduced activity of a major isoform of starch synthase. We suggest that physical as well as biological mechanisms may contribute to the control of ring formation, and that a complex interplay of several factors may by involved.     

Time course and spatial distribution of phenylalanine ammonia-lyase and peroxidase activity in wounded potato tuber tissue

The time course and spatial distribution of wound-induced activities of phenylalanine ammonia-lyase and peroxidase were determined to establish correlations between molecular and cellular aspects of the wound-induced pattern of cell differentiation in potato (Solanum tuberosum L.) tissue. A high correlation between peroxidase activity and suberization was observed. Peroxidase activity increased for several days after wounding. Peroxidase content of suberizing cells was more than 10 times higher than that of the immediately adjacent dividing cells. Suberizing and dividing cells contained different isoperoxidases. Neither time course nor spatial distribution of the wound-induced activity of phenylalanine ammonia-lyase was directly correlated with the wound-induced pattern of cell differentiation.     

The three-dimensional distribution of minerals in potato tubers

Background and Aims

The three-dimensional distributions of mineral elements in potato tubers provide insight into their mechanisms of transport and deposition. Many of these minerals are essential to a healthy human diet, and characterizing their distribution within the potato tuber will guide the effective utilization of this staple foodstuff.

Methods

The variation in mineral composition within the tuber was determined in three dimensions, after determining the orientation of the harvested tuber in the soil. The freeze-dried tuber samples were analysed for minerals using inductively coupled plasma-mass spectrometry (ICP-MS). Minerals measured included those of nutritional significance to the plant and to human consumers, such as iron, zinc, copper, calcium, magnesium, manganese, phosphorus, potassium and sulphur.

Key Results

The concentrations of most minerals were higher in the skin than in the flesh of tubers. The potato skin contained about 17 % of total tuber zinc, 34 % of calcium and 55 % of iron. On a fresh weight basis, most minerals were higher in tuber flesh at the stem end than the bud end of the tuber. Potassium, however, displayed a gradient in the opposite direction. The concentrations of phosphorus, copper and calcium decreased from the periphery towards the centre of the tuber.

Conclusions

The distribution of minerals varies greatly within the potato tuber. Low concentrations of some minerals relative to those in leaves may be due to their low mobility in phloem, whereas high concentrations in the skin may reflect direct uptake from the soil across the periderm. In tuber flesh, different minerals show distinct patterns of distribution in the tuber, several being consistent with phloem unloading in the tuber and limited onward movement. These findings have implications both for understanding directed transport of minerals in plants to stem-derived storage organs and for the dietary implications of different food preparation methods for potato tubers.     

Identification of the major starch synthase in the soluble fraction of potato tubers.

The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA. The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described. Immunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for approximately 80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules. Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers. However, they caused a profound change in the morphology of starch granules, indicative of important underlying changes in the structure of starch polymers within the granule.     

Enzymatic activities of starch synthesis in potato tubers of different sizes

The objective of this study was to determine the relationship between tuber weight and enzymatic activities involved in tuber starch synthesis. As tuber weight increased, the activities of sucrose synthetase, UDPG pyrophosphorylase, and granular starch synthetase escalated, whereas the activities of soluble starch synthetase and ADPG pyrophosphorylase stayed constant and that of phosphorylase declined. This suggests that when samples are taken to determine specific enzymatic activities, the sampling procedure should ensure that results do not vary because of differences in the tuber weight or size distribution.     

Biochemical and molecular characterization of a novel starch synthase from potato tubers

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity. The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.     

Growth of14C-labelled starch granules in potato tubers as revealed by autoradiographs

Summary Experiments have been presented which indicate that the layers of potato starch granules are built up by a gradual process of apposition. This process is dependent upon the supply of carbohydrates to the amyloplasts. The stripping film technique has made it possible for the first time to make starch granule growth more directly visible.     

Regulation of sucrose and starch metabolism in potato tubers in response to short-term water deficit

To investigate the effect of water stress on carbon metabolism in growing potato tubers (Solanum tuberosum L.), freshly cut and washed discs were incubated in a range of mannitol concentrations corresponding to external water potential between 0 and −1.2 MPa. (i) Incorporation of [¹⁴C]glucose into starch was inhibited in water-stressed discs, and labeling of sucrose was increased. High glucose overrode the changes at low water stress (up to −0.5 MPa) but not at high water stress. (ii) Although [¹⁴C]sucrose uptake increased in water-stressed discs, less of the absorbed [¹⁴C]sucrose was metabolised. (iii) Analysis of the sucrose content of the discs confirmed that increasing water deficit leads to a switch, from net sucrose degradation to net sucrose synthesis. (iv) In parallel incubations containing identical concentrations of sugars but differing in which sugar was labeled, degradation of [¹⁴C]sucrose and labeling of sucrose from [¹⁴C]glucose and fructose was found at each mannitol concentration. This shows that there is a cycle of sucrose degradation and resynthesis in these tuber discs. Increasing the extent of water stress changed the relation between sucrose breakdown and sucrose synthesis, in favour of synthesis. (v) Analysis of metabolites showed a biphasic response to increasing water deficit. Moderate water stress (0–200 mM mannitol) led to a decrease of the phosphorylated intermediates, especially 3-phosphoglycerate (3PGA). The decrease of metabolites at moderate water stress was not seen when high concentrations of glucose were supplied to the discs. More extreme water stress (300–500 mM mannitol) was accompanied by an accumulation of metabolites at low and high glucose. (vi) Moderate water stress led to an activation of sucrose phosphate synthase (SPS) in discs, and in intact tubers. The stimulation involved a change in the kinetic properties of SPS, and was blocked␣by protein phosphatase inhibitors. (vii) The amount of ADP-glucose (ADPGlc) decreased when discs were incubated on 100 or 200 mM mannitol. There was a strong correlation between the in vivo levels of ADPGlc and 3PGA when discs were subjected to moderate water stress, and when the sugar supply was varied. (viii) The level of ADPGlc increased and starch synthesis was further inhibited when discs were incubated in 300–500 mM mannitol. (ix) It is proposed that moderate water stress leads to an activation of SPS and stimulates sucrose synthesis. The resulting decline of 3PGA leads to a partial inhibition of ADP-glucose pyrophosphorylase and starch synthesis. More-extreme water stress leads to a further alteration of partitioning, because it inhibits the activities of one or more of the enzymes involved in the terminal reactions of starch synthesis. Received: 26 August 1996 / Accepted: 5 November 1996