Metabolite profiling of potato (Solanum tuberosum L.) tubers during wound-induced suberization

Suberin is a specific cell wall-associated biopolymer characterized by the deposition of both a poly(phenolic) domain (SPPD) associated with the cell wall, and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. In planta, suberin functions to prevent plants from desiccation and pathogen attack. Although the chemical identity of the monomeric components of the SPPD and SPAD are well known, their concerted biosynthesis and assembly into the suberin macromolecule is poorly understood. To expand our knowledge of suberin biosynthesis, a GC/MS-based metabolite profiling study was conducted, using wound healing potato (Solanum tuberosum L.) tubers as a model system. A time series of both non-polar and polar metabolite profiles were created, yielding a broad-based, dynamic picture of wound-induced metabolism, including suberization. Principal component analysis revealed a separation of metabolite profiles according to different suberization stages, with clear temporal differences emerging in the non-polar and polar profiles. In the non-polar profiles, suberin-associated aliphatics contributed the most to cluster formation, while a broader range of metabolites (including organic acids, sugars, amino acids and phenylpropanoids) influenced cluster formation amongst polar profiles. Pair-wise correlation analysis revealed strong correlations between known suberin-associated compounds, as well as between suberin-associated compounds and several un-identified metabolites in the profiles. These data may help to identify additional, as yet unknown metabolites associated with suberization process.     

Accumulation of vitamin E in potato (Solanum tuberosum) tubers

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). α-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in α-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less α-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.     

Subcellular pyrophosphate metabolism in developing tubers of potato (Solanum tuberosum)

PPi has previously been implicated specifically in the co-ordination of the sucrose–starch transition and in the broader context of its role as co-factor in heterotrophic plant metabolism. In order to assess the compartmentation of pyrophosphate (PPi) metabolism in the potato tuber we analysed the effect of expressing a bacterial pyrophosphatase in the amyloplast of wild type tubers or in the cytosol or amyloplast of invertase-expressing tubers. The second and third approaches were adopted since we have previously characterized the invertase expressing lines to both exhibit highly altered sucrose metabolism and to contain elevated levels of PPi (Farré et al. (2000a) Plant Physiol 123:681) and therefore this background rendered questions concerning the level of communication between the plastidic and cytosolic pyrophosphate pools relatively facile. In this study we observed that the increase in PPi in the invertase expressing lines was mainly confined to the cytosol. Accordingly, the expression of a bacterial pyrophosphatase in the plastid of either wild type or invertase-expressing tubers did not lead to a decrease in total PPi content. However, the expression of the heterologous pyrophosphatase in␣the cytosol of cytosolic invertase-expressing tubers led to strong metabolic changes. These results are discussed both with respect to our previous hypotheses and to current models of the compartmentation of potato tuber metabolism.     

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Alkyl ferulates in wound healing potato tubers

Seven ferulic acid esters of 1-alkanols ranging in carbon length from C16 to C28 were synthesized and an HPLC protocol for their separation developed. Extracts prepared from wound healing potato (Solanum tuberosum) tubers and analysed by HPLC indicated that alkyl ferulate esters begin to accumulate 3-7 days after wound treatment. Of the nine esters identified by EIMS, (including two esters of odd chain length alkanols) hexadecyl and octadecyl ferulates were predominant. Alkyl ferulate esters were restricted to the wound periderm.     

Metabolic regulation of pathways of carbohydrate oxidation in potato (Solanum tuberosum) tubers

In the present article we evaluate the consequence of tuber-specific expression of yeast invertase, on the pathways of carbohydrate oxidation, in potato ( Solanum tuberosum L. cv. Desiree). We analysed the relative rates of glycolysis and the oxidative pentose phosphate pathway that these lines exhibited as well as the relative contributions of the cytochrome and alternative pathways of mitochondrial respiration. Enzymatic and protein abundance analysis revealed concerted upregulation of the glycolytic pathway and of specific enzymes of the tricarboxylic acid cycle and the alternative oxidase but invariant levels of enzymes of the oxidative pentose phosphate pathway and proteins of the cytochrome pathway. When taken together these experiments suggest that the overexpression of a cytosolic invertase (EC 3.2.1.26) results in a general upregulation of carbohydrate oxidation with increased flux through both the glycolytic and oxidative pentose phosphate pathways as well as the cytochrome and alternative pathways of oxidative phosphorylation. Moreover these data suggest that the upregulation of respiration is a consequence of enhanced efficient mitochondrial metabolism.     

Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato (Solanum tuberosum)

An anionic peroxidase (EC 1.11.1.7), thought to be involved in suberization, was purified 110-fold from wound-healing slices of Solanum tuberosum by a combination of ammonium sulfate fractionation, Sephadex G-100 gel filtration, isoelectric focusing, and phenyl-Sepharose CL-4B chromatography in 24% yield. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and horizontal thin-layer polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 47,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This peroxidase was found to be a glycoprotein containing about 17% carbohydrate, approximately one-quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.15. An anionic peroxidase was also isolated from abscisic acid-treated callus tissue culture of S. tuberosum by the above purification procedure. The two enzymes were shown to be immunologically similar, if not identical, based on their cross-reactivity with rabbit antibody prepared against the peroxidase from wound-healing slices, whereas the major cationic peroxidase from wound-healing slices did not cross-react with this antibody. The anionic enzyme from both sources showed very similar specific activities when assayed with a range of substrates, whereas the specific activities found for the cationic isozyme isolated from wound-healing slices were quite different.     

Pigment formation in potato tubers (Solanum tuberosum) exposed to light followed by darkness

Potato tubers ( Solanum tubersoum cvs Bintje and King Edward). never exposed to light, lack chlorophyllous pigments. Continuous irradiation results in chlorophyll (Chl) formation and induces the ability for protochlorophyll (Pchl) formation when the tubers are brought back to darkness. Pigment synthesis takes place in both blue and red light, but blue light is more effective than red in starting the greening process. The pigment formation is strongest in the layers just below the periderm with a steep gradient inwards. Small amounts of Chl formed after irradiation. slowly fade away during extended darkness. However, the Chl formed after long time of irradiation is remarkably stable. Irradiated potatoes, placed in darkness, form Pchl with a fluorescence emission peak at 633 nm. A maximal level is reached after ca 7 days. Resolution of the Pchl spectrum suggests the presence of small amounts of a pigment with an emission maximum at around 642 nm. No sign of the Pchl with emission maximum at 657 nm, which dominates in etiolated leaves, is found. A faint Chl fluorescence indicates that some Pchl, probably the 642 nm form, is phototransformed into Chl in weak light. The Chl formation in the potato tuber is discussed in relation to that of roots and leaves.     

Aldehyde oxidase isoenzymes (E.C. 1.2.3.1) in potato tubers (Solanum tuberosum)

Two isoenzymes of aldehyde oxidase (E.C. 1.2.3.1 [EC] ) can be separatedfrom potato tubers (Solanum tuberosum) by polyacrylamide gelelectrophoresis. The pH optima of these two isoenzymes werepH 7.5. Both enzymes can oxidize different aldehydes, e.g. crotonaldehyde,Propionaldehyde, acetaldehyde, formaldehyde, glyoxal and benzyl-aldehyde.The isoenzymes could not use xanthine as a substrate. Formaldehydewas oxidized only in the presence of phosphate ions. A substratedependent inhibition of the enzyme activity is possible throughchloral hydrate. PMS, FMN, riboflavine, cytochrome c and O₂ serve as electronacceptors. (Received December 12, 1973; )     

Differential induction of polar and non‐polar metabolism during wound‐induced suberization in potato (Solanum tuberosum L.) tubers

Wound‐induced suberin deposition involves the temporal and spatial coordination of phenolic and fatty acid metabolism. Phenolic metabolism leads to both soluble metabolites that accumulate as defense compounds as well as hydroxycinnamoyl derivatives that form the basis of the poly(phenolic) domain found in suberized tissue. Fatty acid metabolism involves the biosynthesis of very‐long‐chain fatty acids, 1‐alkanols, ω‐hydroxy fatty acids and α,ω‐dioic acids that form a poly(aliphatic) domain, commonly referred to as suberin. Using the abscisic acid (ABA) biosynthesis inhibitor fluridone (FD), we reduced wound‐induced de novo biosynthesis of ABA in potato tubers, and measured the impact on the expression of genes involved in phenolic metabolism (StPAL1, StC4H, StCCR, StTHT), aliphatic metabolism (StCYP86A33, StCYP86B12, StFAR3, StKCS6), metabolism linking phenolics and aliphatics (StFHT) or acyl chains and glycerol (StGPAT5, StGPAT6), and in the delivery of aliphatic monomers to the site of suberization (StABCG1). In FD‐treated tissue, both aliphatic gene expression and accumulation of aliphatic suberin monomers were delayed. Exogenous ABA restored normal aliphatic suberin deposition in FD‐treated tissue, and enhanced aliphatic gene expression and poly(aliphatic) domain deposition when applied alone. By contrast, phenolic metabolism genes were not affected by FD treatment, while FD + ABA and ABA treatments slightly enhanced the accumulation of polar metabolites. These data support a role for ABA in the differential induction of phenolic and aliphatic metabolism during wound‐induced suberization in potato.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Purification of potato lectin (Solanum tuberosum agglutinin) from tubers or fruits using chromatofocusing

The lectin from potato tubers (Solanum tuberosum agglutinin) has been purified to homogeneity by a procedure involving chromatofocusing followed by gel filtration. By subjecting tuber and fruit extracts from an individual plant to this purification scheme, it was demonstrated that the lectins from those two tissues, though similar, are not identical.     

Pectic epitopes are differentially distributed in the cell walls of potato (Solanum tuberosum) tubers

Six monoclonal antibodies (mAbs) were used to map the distribution of pectic epitopes in the cell walls of potato ( Solanum tuberosum L. cvs Kardal and Karnico) tuber tissue in both light and electron microscopes. Unesterified (mAb JIM 5 epitope) and methyl-esterified (mAb JIM 7 epitope) pectins were abundant and equally distributed in all parenchymal and vascular cell walls. Homogalacturonans (HGAs) involved in Ca²⁺-cross-linking (mAb 2F4 epitope) were localised to the middle lamella and abundant at cell corners. The tuber cortex was densely labelled, but parenchymal cell walls in the perimedullary region contained few epitopes of calcium pectate except at corners and pit fields. In contrast, pectic side-chains were not detectable in the middle lamella of all parenchymal cell walls, except in the cortex where mAb LM6 (arabinan epitope) labelled the entire wall. The galactan epitope (mAb LM5) was localised to a zone very close to the plasmalemma in cortical cell walls and was also less abundant at pit fields and in vascular cell walls. MAb CCRC-M2 (rhamnogalacturonan I epitope) did not cross-react. Our results show that the cell walls of potato tubers are not homogeneous structures and that the pectic composition of the walls is spatially regulated.     

The inhibition of potato tubers wound reparation

The multiple washing of the wound surface of potato tubers by water adversely affected the protective properties of wound periderm. Immune inhibitor β-1,3-β-1,6 glucan had a property of local effect and inhibited the process of wound healing. The pentasaccharide of xyloglucan caused necrosis of potato tuber tissue and prevented the wound reparation process.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

The ascorbate system and lipid peroxidation in stored potato (Solanum tuberosum L.) tubers

The changes in the components of the ascorbateglutathione systemduring the storage of potato (Solanum tuberosum L. cv. Spunta)tubers for 40 weeks at both 3C and 9C were studied in relationto lipid peroxidation. The level of malondialdehyde was foundto be higher at 3C than at 9C throughout storage. Thus, lipidperoxidation, which is the main cause of membrane deterioration,was accelerated at the lower temperature. Catalase activityincreased throughout storage independently of temperature. Theascorbate content of tubers decreased during storage both at3C and at 9C, as in other ageing processes. However, ascorbateperoxidase activity reached a maximum after about 8 weeks ofstorage, then declined at 9C, but held a higher level at 3C.The dehydroascorbic content also reached a maximum after about8 weeks and was significantly higher in tubers stored at 3C.These findings indicate a greater utilization of ascorbate byascorbate peroxidase at the lower temperature. Ascorbate freeradical reductase, dehydroascorbate reductase and glutathionereductase, the enzymes involved in the regeneration of ascorbate,were not affected by temperature and remained quite unchangedthroughout storage. It can be concluded that the ascorbate systemis involved in the scavenging of the free-radicals responsiblefor lipid peroxidation in stored potato tubers, at least atlow temperatures and in the first period of storage. Key words: Ascorbate, lipid peroxidation, potato tubers, Solanum tuberosum L