Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Vacuolar Invertase Gene Silencing in Potato (Solanum tuberosum L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Impact of invertase overexpression on cell size, starch granule formation and cell wall properties during tuber development in potatoes with modified carbon allocation patterns

Transgenic potato tubers that overexpressed either a cytosolic or an apoplastic invertase in the wild type or AGPase antisense background were used to analyse the effect of invertase activity on cell expansion, starch granule formation and turgor pressure during tuber development. Although the transgenic plants did not develop a visible phenotype in aerial regions the size and number of tubers were significantly modified in the various lines. Transmission electron and light microscopy were performed to monitor starch grain size and number, cell size and cell wall thickness. Water potential, osmotic pressure, and indirectly, turgor pressure were determined during the final stages of tuber development. Glucose levels were high in transgenic tubers that overexpressed a yeast-derived invertase. The number of starch grains per cell was almost identical in all transgenic lines. However, the amount of starch was modified in the transgenics as compared to the wild type. As expected, the size of starch grains was reduced in all lines that expressed an AGPase antisense mRNA. These results indicate that invertase activity and glucose levels do not affect initiation of starch grain formation during the early stages of tuber development, but growth of starch corns in the later stages of tuber maturation.     

DNA variation at the invertase locus invGE/GF is associated with tuber quality traits in populations of potato breeding clones

Starch and sugar content of potato tubers are quantitative traits, which are models for the candidate gene approach for identifying the molecular basis of quantitative trait loci (QTL) in noninbred plants. Starch and sugar content are also important for the quality of processed products such as potato chips and French fries. A high content of the reducing sugars glucose and fructose results in inferior chip quality. Tuber starch content affects nutritional quality. Functional and genetic models suggest that genes encoding invertases control, among other things, tuber sugar content. The invGE/GF locus on potato chromosome IX consists of duplicated invertase genes invGE and invGF and colocalizes with cold-sweetening QTL Sug9. DNA variation at invGE/GF was analyzed in 188 tetraploid potato cultivars, which have been assessed for chip quality and tuber starch content. Two closely correlated invertase alleles, invGE-f and invGF-d, were associated with better chip quality in three breeding populations. Allele invGF-b was associated with lower tuber starch content. The potato invertase gene invGE is orthologous to the tomato invertase gene Lin5, which is causal for the fruit-sugar-yield QTL Brix9-2-5, suggesting that natural variation of sugar yield in tomato fruits and sugar content of potato tubers is controlled by functional variants of orthologous invertase genes.     

StGA2ox1 is induced prior to stolon swelling and controls GA levels during potato tuber development

The formation and growth of a potato ( Solanum tuberosum ) tuber is a complex process regulated by different environmental signals and plant hormones. In particular, the action of gibberellins (GAs) has been implicated in different aspects of potato tuber formation. Here we report on the isolation and functional analysis of a potato GA 2-oxidase gene ( StGA2ox1 ) and its role in tuber formation. StGA2ox1 is upregulated during the early stages of potato tuber development prior to visible swelling and is predominantly expressed in the subapical region of the stolon and growing tuber. 35S-over-expression transformants exhibit a dwarf phenotype, reduced stolon growth and earlier in vitro tuberization. Transgenic plants with reduced expression levels of StGA2ox1 showed normal plant growth, an altered stolon swelling phenotype and delayed in vitro tuberization. Tubers of the StGA2ox1 suppression clones contain increased levels of GA₂₀, indicating altered GA metabolism. We propose a role for StGA2ox1 in early tuber initiation by modifying GA levels in the subapical stolon region at the onset of tuberization, thereby facilitating normal tuber development and growth.     

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.     

Sucrose metabolism in plastids

The question whether sucrose (Suc) is present inside plastids has been long debated. Low Suc levels were reported to be present inside isolated chloroplasts, but these were argued to be artifacts of the isolation procedures used. We have introduced Suc-metabolizing enzymes in plastids and our experiments suggest substantial Suc entry into plastids. The enzyme levansucrase from Bacillus subtilis efficiently synthesizes fructan from Suc. Targeting of this enzyme to the plastids of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants leads to high-level fructan accumulation in chloroplasts and amyloplasts, respectively. Moreover, introduction of this enzyme in amyloplasts leads to an altered starch structure. Expression of the yeast invertase in potato tuber amyloplasts results in an 80% reduction of total Suc content, showing efficient hydrolysis of Suc by the plastidic invertase. These observations suggest that Suc can enter plastids efficiently and they raise questions as to its function and metabolism in this organelle.     

Effect of temperature on invertase, invertase inhibitor, and sugars in potato tubers

The accumulation of reducing sugars in potato tubers exposed to low temperatures occurs with concomitant formation of the enzyme invertase. During the initial period of cold treatment when reducing sugars increase rapidly, invertase formation proceeds until the level of enzyme exceeds that of an endogenous macromolecular invertase inhibitor, resulting in a basal invertase activity. As the rate of sugar accumulation decreases and the sugar level becomes nearly constant, total invertase decreases, the basal activity disappears, and a low excess of inhibitor develops. On transfer of cold-stored tubers to warmer temperatures, sugars and invertase decrease sharply and a large excess of inhibitor develops. These changes in sugars, invertase and inhibitor occur reversibly when the tubers are subjected to alternating temperatures.     

Enhancing vacuolar sucrose cleavage within the developing potato tuber has only minor effects on metabolism

Modification of tuber carbohydrate metabolism by the tuber-specific expression of a yeast invertase targeted to the cytosol or apoplast has previously been demonstrated to have diverse effects on tuber growth and metabolism. In the current study, we generated plants exhibiting tuber-specific expression of the same enzyme targeted to the vacuole. Enzymatic analysis of the carbohydrate levels of the tuber revealed dramatic decreases in sucrose content coupled with large increases in the levels of glucose and hexose phosphates, but unaltered starch content in the transformants. Analysis of the key enzyme of glycolysis suggests that this pathway is down-regulated in the transformants. Despite these changes in metabolite pools and enzyme activity, few consistent changes could be observed in the estimated metabolic fluxes following incubation of isolated tuber discs in labelled glucose. The analysis of the relative levels of a wide range of metabolites using a gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling method revealed large changes in the levels of fructose and decreases in a range of other sugars, but very few changes in the contents of organic and amino acids. This metabolic profile is remarkably consistent with that obtained following expression of the invertase in the apoplastic compartment, providing circumstantial evidence for the endocytotic trafficking of sugars within potato tuber parenchyma. Finally, the results of this study are compared with those from other plant species and the relative roles of the vacuolar isoform of the enzyme are contrasted.     

Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria

3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3- were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3- plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents.     

Natural diversity of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) invertases

Background  

Invertases are ubiquitous enzymes that irreversibly cleave sucrose into fructose and glucose. Plant invertases play important roles in carbohydrate metabolism, plant development, and biotic and abiotic stress responses. In potato (Solanum tuberosum), invertases are involved in 'cold-induced sweetening' of tubers, an adaptive response to cold stress, which negatively affects the quality of potato chips and French fries. Linkage and association studies have identified quantitative trait loci (QTL) for tuber sugar content and chip quality that colocalize with three independent potato invertase loci, which together encode five invertase genes. The role of natural allelic variation of these genes in controlling the variation of tuber sugar content in different genotypes is unknown.     

QTL mapping of potato chip color and tuber traits within an autotetraploid family

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid crop species, and this creates challenges for traditional line development and molecular breeding. Recent availability of a single-nucleotide polymorphism (SNP) array with 8303 features and software packages for linkage and association mapping in autotetraploid species present new opportunities for the identification of genomic regions that contribute to high-value traits in cultivated potato. A biparental tetraploid potato population was evaluated across three field seasons and storage trials in order to identify quantitative trait loci (QTL) for multiple tuber traits including fried chip color after 5.5–7.2 °C storage. Tetra-allelic dosage information was used to construct a genetic linkage map that covered 1041 cM and contained 2095 SNP markers with a median marker interval of 0.4 cM. A total of 41 QTL were identified for flower color, tuber yield, tuber number per plant, tuber weight, tuber size, and chip color after various storage regimes. Moderate effect QTL for chip color at 3 months were identified that co-localized with candidate genes vacuolar invertase (VInv), invertase inhibitor (INH2), and apoplastic invertase (Inv _ap -b). A separate QTL for chip color after 6 months of storage was identified in the short arm of chromosome 2, and this locus may contribute to variation in senescent sweetening resistance. QTL for tuber weight, length, and width co-localized with a known QTL for plant maturity on chromosome 5. Genome-wide association mapping using a polyploid model detected the tuber size QTL and identified a number of candidate SNPs, but was unable to detect markers significantly associated with chip color.     

The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers

In the present paper we investigated the effect of the sucrose (Suc) analog palatinose on potato (Solanum tuberosum) tuber metabolism. In freshly cut discs of growing potato tubers, addition of 5 mM palatinose altered the metabolism of exogenously supplied [U-14C]Suc. There was slight inhibition of the rate of 14C-Suc uptake, a 1.5-fold increase in the rate at which 14C-Suc was subsequently metabolized, and a shift in the allocation of the metabolized label in favor of starch synthesis. The sum result of these changes was a 2-fold increase in the absolute rate of starch synthesis. The increased rate of starch synthesis was accompanied by a 3-fold increase in inorganic pyrophosphate, a 2-fold increase in UDP, decreased UTP/UDP, ATP/ADP, and ATP/AMP ratios, and decreased adenylate energy charge, whereas glycolytic and Krebs cycle intermediates were unchanged. In addition, feeding palatinose to potato discs also stimulated the metabolism of exogenous 14C-glucose in favor of starch synthesis. In vitro studies revealed that palatinose is not metabolized by Suc synthases or invertases within potato tuber extracts. Enzyme kinetics revealed different effects of palatinose on Suc synthase and invertase activities, implicating palatinose as an allosteric effector leading to an inhibition of Suc synthase and (surprisingly) to an activation of invertase in vitro. However, measurement of tissue palatinose levels revealed that these were too low to have significant effects on Suc degrading activities in vivo. These results suggest that supplying palatinose to potato tubers represents a novel way to increase starch synthesis.     

Expression of a bacterial xylose isomerase in potato tubers results in an altered hexose composition and a consequent induction of metabolism

Here we investigate the role of hexoses in the metabolism of the developing potato (Solanum tuberosum) tuber by the expression of a bacterial xylose isomerase which catalyzes the interconversion of glucose and fructose. Previously, we found that glycolysis was induced in transgenic tubers expressing a yeast invertase in the cytosol and postulated that this was due either to the decreased levels of sucrose or to effects downstream of the sucrose cleavage. In the present study xylose isomerase was expressed under the control of the tuber-specific patatin promoter. Selected transformants exhibited minor changes in the levels of tuber glucose and fructose but not in sucrose. Analysis of the enzyme activities of the glycolytic pathway revealed minor yet significant increases in the maximal catalytic activities of aldolase and glyceraldehyde 3-phosphate dehydrogenase but no increase in the activities of other enzymes of glycolysis. These lines were also characterized by an elevated tuber number, glycolytic and sucrose synthetic fluxes and in some metabolite levels downstream of glycolysis. When considered together these data suggest that the perturbation of hexose levels can result in increased glycolytic and sucrose (re)synthetic fluxes in the potato tuber even in the absence of changes in the level of sucrose. The consequences of altering hexose levels in the tuber are, however, not as severe as those observed following perturbation of the level of tuber sucrose.     

Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

Theobroxide induces tubers in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) and flower buds in morning glory (<Emphasis Type="Italic">Pharbitis nil</Emphasis>) under non-inductive high temperatures

Induction of some plant organs including tubers and flower buds begins with sensing environmental cues, such as photoperiod and temperature in the leaves. Theobroxide has been shown to induce potato tuberization and flower-bud formation in morning glory under non-inductive photoperiodic conditions, stimulating the activity of lipoxygenase (LOX) and the synthesis of jasmonic acid (JA). In the present study, the ability of theobroxide to overcome the inhibitory effect of unfavorable high temperature on the induction of tubers in potato and flower buds in morning glory was examined. Both tuber induction and flower-bud formation under non-inductive high temperatures were promoted by the application of theobroxide at a high concentration. However, although theobroxide treatment resulted in an increase in fresh weight during potato tuber growth at 30°C, morning glory plants treated with theobroxide at 35°C failed to bloom, implying that theobroxide may assist only in flower-bud formation.