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Foliar application of selenite and selenate to potato (Solanum tuberosum): effect of a ligand agent on selenium content of tubers.

The effect of a foliar spray of selenium on potatoes was investigated for 2 years. Amounts of 0, 50, and 150 g of Se ha(-)(1) were applied both as sodium selenate and as sodium selenite in water, either pure or with the addition of 0.15% of soluble leonardite as a source of humic acids (pH 7). Tuber selenium concentration increased with the application levels, both with sodium selenate and with sodium selenite, when only aqueous solutions were used. When humic acids were added, the tuber selenium level rose more markedly after the application of sodium selenate as compared to the case of the aqueous solutions; however, in the case of sodium selenite, the level showed a large increase only after the application of 50 g of Se ha(-)(1). Kinetics showed that humic acids raised the selenate availability, but no differences were found in the distribution of selenium in the tuber fractions. Foliar application of selenium with humic acids was proven to be a good way to increase the selenium content of potatoes, but the assimilation process of selenium was simpler with selenate than with selenite.

Humic Substances↗

Esterified phenolics of the cell walls of chufa (Cyperus esculentusL. ) tubers and their role in texture.

Chufas (Cyperus esculentus) are edible tubers that, like Chinese waterchestnut (CWC), are very crisp when raw and do not soften when cooked. The present study compares the mechanical properties of chufas with those of potato and CWC in relation to the carbohydrate and phenolic compositions of the cell walls. The cutting toughness of raw chufa was higher than that of raw CWC and potato; its value decreased on boiling, as also observed with CWC, but remained over twice that of raw potato. Chufa cell walls were rich in xylose, arabinose, glucose, uronic acid, and galactose, with minor quantities of mannose. The cell walls of the parenchyma exhibited a uniform pH-dependent autofluorescence indicating the presence of cinnamic acid derivatives. Analysis of these revealed that peeled tuber cell walls are rich in ferulic acid, whereas p-coumaric acid dominates the monomeric phenol fraction of the skin. Cell wall material from both skin and peeled tubers contains a significant amount of different diferulic acids ( approximately 20% of the wall ferulic acid), consisting mainly of the 8-O-4'-, 8-5'-, and 5-5'-dimers. These are potentially available to form thermally stable cross-links between polysaccharides within the wall and between cells. This may confer thermal stability of texture.

Carbohydrates↗

Repression of the 14-3-3 gene affects the amino acid and mineral composition of potato tubers.

Recently, transgenic potato plants were created showing underexpression of the 20R isoform of the 14-3-3 protein. The transgenic plants grown in tissue culture showed a significant increase in nitrate reductase activity and a decrease in nitrate level. The transgenic line with the lowest 14-3-3 quantity was field-trialed (1997-2000) and analyzed. The reduction in the 14-3-3 protein level consistently resulted in a starch content increase and in an increase in the ratio of soluble sugars to starch in the tubers, although the latter was only barely visible. The determination of amino acid composition in the tubers showed a significant increase in methionine, proline, and arginine content and a slight but consistent increase in hydrophobic amino acid and lysine content in the cells of the transgenic potato plants. We also observed an increase in the crude protein content, from 19 to 22.1% of the control value in consecutive years. It is proposed that all of these changes might have resulted from the downregulation of nitrate reductase and sucrose phosphate synthase activities by 14-3-3, although other potential mechanisms cannot be excluded (e.g., an increase in enzyme protein level). 14-3-3-repressed transgenic plants showed a significant increase in calcium content in their tubers. It is thus proposed that a function of the isolated 14-3-3 isoform is in the control of amino acid synthesis and calcium metabolism. However, the mechanism of this control is as yet unknown.

14-3-3 Proteins↗

Investigation of the tuber constituents of maca (Lepidium meyenii Walp.).

Lepidium meyenii, known in South America as maca, has received attention worldwide as a powerful energizer that improves physical and mental conditions and increases fertility. Because of these reports, we investigated the secondary metabolites of the tuber of maca. The methanol extract of the tuber of maca contained, in addition to free sugars and amino acids, the following: uridine, malic acid and its benzoyl derivative, and the glucosinolates, glucotropaeolin and m-methoxyglucotropaeolin. Because glucosinolates and their derived products have received increasing attention due to their biological activities, the occurrence of glucosinolate degradation products in the hexane extract was also investigated, and benzylisothiocyanate and its m-methoxy derivative were isolated. The two glucosinolates were semiquantified by HPLC, and benzylisothiocyanate was semiquantified by GC/MS. The methanol extract of maca tuber also contained (1R,3S)-1-methyltetrahydro-beta-carboline-3-carboxylic acid, a molecule which is reported to exert many activities on the central nervous system.

Amino Acids↗

ADP ribosylation factor regulates metabolism and antioxidant capacity of transgenic potato tubers.

In our recent studies we have evidenced that repression of ADP-ribosylation factor (ARF) in potato plants results in 14-3-3 gene activation. The significant alteration in plant phenotype and in carbohydrate content clearly indicates that there may also be changes in other metabolite syntheses. In this paper we present the data on contents of compounds, occurring in transgenic potato tubers from field trial, known to be important for the human diet. We also determine which of the ARF-antisense plant features resulted from ARF repression. This determination was accomplished by the analysis of ARF-antisense plants transformed with cDNA encoding 14-3-3 protein in reverse orientation. The sucrose accumulation and the decrease in glycoalkaloids level were found to be characteristic features of all transgenic plants. The increase in antioxidant capacity of transgenic potato tubers should also be pointed out. The analysis of fat from modified potato tubers revealed a nutritionally valuable composition of fatty acids, including the significant increase of linoleic acid level.

14-3-3 Proteins↗

Changes in compositional parameters of tubers of potato (Solanum tuberosum) during low-temperature storage and their relationship to chip processing quality.

A four-year study of a number of compositional parameters of potato tubers during low-temperature storage was conducted to examine the compositional differences between cold-tolerant (low sugar-accumulating) and cold-sensitive (high sugar-accumulating) tubers in relation to potato chip processing quality. Compositional parameters analyzed included sucrose, reducing sugars, nitrogen, protein, ascorbic acid, and dry matter content. Pearson correlation analysis of the data illustrated that chip color was most closely correlated with reducing sugar concentration. Multiple regression analysis revealed that the relative contribution of each parameter to chip color varied greatly among the cultivars and selections evaluated and from season to season. This analysis demonstrates that the quantitative relationships between the measured compositional parameters and chip color were not sufficient to provide a general predictive index of chip color quality for tubers processed directly from low-temperature storage.

Ascorbic Acid↗

Influence of storage upon light-induced chlorogenic acid accumulation in potato tubers (Solanum tuberosum L.).

The influence of 2 weeks and 3 months of dark storage upon light-induced chlorogenic acid accumulation within tuber tissue of four potato cultivars and upon 5-, 4-, and 3-caffeoylquinic acid concentrations within cv. King Edward was determined. Storage period significantly affected (P < 0.05) the magnitude of the light-induced chlorogenic acid response with accumulation rates 3-4 times higher in tubers exposed to light after 2 weeks compared with those placed under light after 3 months. Comparison of chlorogenic acid concentrations in controls after 2 and 3 months of dark storage indicated that tuber chlorogenic concentrations decline during prolonged cold store at 5 degrees C. Rates of accumulation in response to light were cultivar-dependent with cv. Fianna the most light-sensitive and cv. Maris Piper relatively light-insensitive. In virtually all cases exposure to sodium and fluorescent light promoted higher rates of accumulation than did exposure to high-pressure mercury light sources. Chlorogenic acid values steadily increased over 15 days of illumination with, in the majority of cases, no indication of cessation. Light exposure increased 5-, 4-, and 3-caffeoylquinic acid accumulation rates in cv. King Edward. Irrespective of storage period and light source, ratios of 5-:4-:3-caffeoylquinic acid were ca. 85:15:0 at day 0 and 52:42:6 by day 15.

Chlorogenic Acid↗

Cloning and expression analysis of a gene that shows developmental regulation upon tuberization in potato.

Differential screening of a potato leaf cDNA library with cDNA probes made from tuberizing and non-tuberizing Solanum demissum plants led to the identification of a clone that is upregulated in leaves and other tissues upon tuberization. This clone was also shown to have a high level of expression in green tomato fruit, its expression falling off as the fruit turns red. No sucrose or hormonal regulation of the expression of this clone was observed and it did not respond to wounding or heat stress. Clone 32B is 532 bp long and contains an open reading frame encoding a small protein of 98 amino acids. The deduced protein sequence has a putative signal peptide for ER transport and a 10 amino acid domain in the C-terminal region of the protein, both of which are also found in the cotton LEA5, Arabidopsis Di21 and the mungbean Arg2 proteins.

Amino Acid Sequence↗

Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers.

Twenty transformed Solanum tuberosum plants issued from five different varieties and carrying a chimeric tobacco nitrate reductase gene (a truncated tobacco Nia2 coding sequence fused to the CaMV 35S promoter) were cultivated in field conditions at INRA Ploudaniel in 1999 and 2000. In 60% of the transgenic plants, the presence of the tobacco Nia2 transcript was detected by RT-PCR. These clones exhibited a drastic decrease in the nitrate content in tubers. Indeed the nitrate content decreased by about 95% in the tubers of transformed plants compared to nontransformed potato plants from the same variety. This decrease was correlated with a modified regulation of NR expression as revealed by a higher chlorate sensitivity of these transgenic lines. Two methods of nitrate content determination in tubers were also compared and were found to give similar results.

Base Sequence↗

Alpha-amylase inhibitor changes during processing of sweet potato and taro tubers.

Alpha-amylase inhibitor changes during processing of sweet potatoes (Ipomoea batatas) and taro (Colocasia esculenta) indicated that varietal differences profoundly influence the thermal inactivation profile. The alpha-amylase inhibitors of taro were almost totally inactivated during oven drying of the chips at 90 degrees C and 100 degrees C for 24 h, while 0.8-10% activity was retained in sweet potato chips under the same conditions. Relatively better thermal stability was exhibited by the sweet potato amylase inhibitors at lower temperatures (70 and 80 degrees C) as well. Cooking by boiling the tuber pieces in water resulted in retention of 29-59% amylase inhibitor in sweet potato and 11-16% in taro. Microwave baking was a better method for inactivation of amylase inhibitors in these tubers. Flour prepared from the tubers retained only trivial amounts of the inhibitor.

Colocasia↗

Inactivation of trypsin inhibitors in sweet potato and taro tubers during processing.

In order to understand the extent of elimination of trypsin inhibitors during processing of sweet potato (Ipomoea batatas) and taro (Colocasia esculenta) tubers, a detailed study was conducted using tubers processed by oven drying, cooking, and microwave baking. Between 80 and 90% trypsin inhibitor (TI) activity was retained in sweet potato chips up to 2h at 70 degrees C. Among the four cultivars of sweet potatoes, RS-III-2 trypsin inhibitors were more heat labile. Heating at 100 degrees C led to rapid inactivation of TI of sweet potatoes. Varietal differences in thermal stability were more pronounced for the trypsin inhibitors of taro than sweet potatoes. Taro inhibitors were also more rapidly inactivated than sweet potato TI. Between 17 and 31% TI activity was retained in cooked tuber pieces of sweet potatoes, while only 3-10% were retained in taro cultivars. Very effective inactivation of trypsin inhibitors of sweet potatoes and taro could be obtained through microwave baking. Flour prepared from taro was devoid of TI activity, while 5-12% TI activity was retained in the flour prepared from sweet potatoes. The study clearly established that among the four techniques used, microwave baking and flour preparation were the best methods to eliminate TI from sweet potatoes and taro.

Colocasia↗

Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers.

We have transformed potato with Nt-inhh cDNA, encoding a putative vacuolar homolog of a tobacco cell wall invertase inhibitor, under the control of the CaMV 35S promoter. In transgenic tubers, cold-induced hexose accumulation was reduced by up to 75%, without any effect on potato tuber yield. Processing quality of tubers was greatly improved without changing starch quantity or quality, an important prerequisite for the biotechnological use of Nt-inhh for potato transformation.

Amino Acid Sequence↗

Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants.

Cyclodextrins (CDs) are cyclic oligosaccharides containing six (alpha), seven (beta), or eight (gamma) glucose molecules, respectively. The cyclodextrin glycosyltransferases (CGT), which produce CDs from starch, are found only in bacteria and are used in batch fermentors with hydrolyzed starch to produce CDs commercially. Using a CGT gene from Klebsiella, we attempted to engineer the tubers of developing potatoes to produce these novel, high-value carbohydrates. A chimeric gene, consisting of (1) the patatin promoter for tuber-specific expression, (2) the small subunit of ribulose bisphosphate carboxylase (SSU) transit peptide for plastid targeting, (3) the CGT structural gene from Klebsiella and (4) the nopaline synthase 3' region, was introduced into potatoes. Both alpha and beta CDs were produced in tubers of transgenic potatoes at levels corresponding to 0.001-0.01% of the starch being converted to CDs.

Base Sequence↗

Metabolic Control Analysis of glycolysis in tuber tissue of potato (Solanum tuberosum): explanation for the low control coefficient of phosphofructokinase over respiratory flux.

We have applied Metabolic Control Analysis (MCA) in an attempt to determine the distribution of glycolytic flux control between the steps of glycolysis in aged disks of potato tuber under aerobic conditions, using concentrations of glycolytic metabolites in tuber tissue from a range of transgenic potato plants and published enzyme kinetic data. We modelled the substrate and effector kinetics of potato tuber phosphofructokinase (PFK) by reanalysing published results. Despite the scarcity of reliable kinetic data, our results are in agreement with experimental findings namely that, under the conditions described, PFK has little control over glycolytic flux. Furthermore our analysis predicts that under these conditions far more control lies in the dephosphorylation of phosphoenolpyruvate and/or in the steps beyond. We have validated the results of our analysis in two ways. First, predictions based on calculated concentration control coefficients from the analysis show generally good agreement with observed metabolite deviation indices discussed in the preceding paper [Thomas, Mooney, Burrell, and Fell (1997) Biochem. J. 322, 111-117]. Second, sensitivity analysis of our results shows that the calculated control coefficients are robust to errors in the elasticities used in the analysis, of which relatively few need to be known accurately. Experimental and control analysis results agree with previous predictions of MCA that strong co-operative feedback inhibition of enzymes serves to move flux control downstream of the inhibiting metabolite. We conclude that MCA can successfully model the outcome of experiments in the genetic manipulation of enzyme amounts.

Electron Transport↗

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.

Amino Acid Sequence↗

Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry.

A new method is presented in which gas chromatography coupled to mass spectrometry (GC-MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC-MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70-140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil- and in vitro-grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.

Gas Chromatography-Mass Spectrometry↗

Diurnal changes in sucrose, nucleotides, starch synthesis and AGPS transcript in growing potato tubers that are suppressed by decreased expression of sucrose phosphate synthase.

Sucrose export from leaves is high during the day and lower at night, when it depends on starch remobilisation. We have investigated the consequences of diurnal changes of photoassimilate supply for starch synthesis and other metabolic processes in growing potato tubers. Sucrose, the levels of the transcripts for SUS and AGPS, the levels of key metabolites, and the rate of synthesis of starch and other major end products, including protein and cell wall polysaccharides, increased twofold or more between the start and end of the light period. The stimulation of starch synthesis was accompanied by an increase of UDPglucose and ADPglucose, whereas glycolytic intermediates remained unaltered, revealing that sucrose synthase and ADP-glucose pyrophosphorylase are being co-ordinately regulated. Sucrose synthase is stimulated via an increase of its substrates, UDP and sucrose. UDP increases due to an increase of the overall uridine nucleotide pool, and a decrease of the UTP/UDP ratio that occurs in parallel with a decrease of the ATP/ADP ratio and adenylate energy charge when biosynthetic fluxes are high at the end of the day. Within the time frame of the diurnal changes, the changes in the SUS and AGPS transcript levels do not lead to significant changes in the encoded enzymes. Transformants with a progressive decrease of sucrose phosphate synthase expression, where diurnal changes in leaf sugar levels were damped, exhibited a progressive attenuation of the diurnal changes of sucrose, nucleotide sugars and nucleotides, and fluxes in their tubers. It is concluded that metabolic processes in tubers are tightly linked to the momentary supply of sucrose.

Circadian Rhythm↗

Reduced amino acid content in transgenic potato tubers due to antisense inhibition of the leaf H+/amino acid symporter StAAP1.

Transport processes across the plasma membrane of leaf vascular tissue are essential for transport and distribution of assimilates. In potato, leaves are the predominant sites for nitrate reduction and amino acid biosynthesis. From there, assimilated amino acids are exported through the phloem to supply tubers with organic nitrogen. To study the role of amino acid transporters in long-distance transport and allocation of organic nitrogen in potato plants, a gene encoding a functional, leaf-expressed amino acid permease StAAP1 was isolated. Similar to the sucrose transporter SUT1, StAAP1 expression was induced during the sink-to-source transition, indicating a role in phloem loading. To test the role of StAAP1, expression was inhibited by an antisense approach. Transgenic plants with reduced StAAP1 expression were phenotypically indistinguishable from wild type, as were photosynthetic capacity and tuber yield. However, tubers from antisense StAAP1 plants showed up to 50% reduction in free amino acid contents. In comparison, starch content was not affected or tended to increase relative to wild type. The reduction in all amino acids except aspartate in the antisense plants is consistent with the properties of amino acid permeases (AAPs) found in heterologous systems. The results demonstrate an important role for StAAP1 in long-distance transport of amino acids and highlight the importance of plasma membrane transport for nutrient distribution in plants.

Amino Acid Transport Systems↗