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PubMed1979

Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers.

A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0--12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0--40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40--55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.

Chymotrypsin↗
PubMed1990

Gene expression during tuber development in potato plants.

Potato tubers are modified stems that have differentiated into storage organs. Factors such as day-length, nitrogen supply, and levels of the phytohormones cytokinin and gibberellic acid, are known to control tuberization. Morphological changes during tuber initiation are accompanied by the accumulation of a characteristic set of proteins, thought to be involved in N-storage (i.e. patatin) or defense against microbial or insect attack (i.e. proteinase inhibitor II). Additionally, deposition of large amounts of starch occurs during tuber formation, which is paralleled by an increase in sucrose synthase and other enzymes involved in starch biosynthesis (i.e. ADP-glucose pyrophosphorylase, starch synthases, and branching enzyme). Potential controlling mechanisms for genes expressed during tuberization are discussed.

Carboxylic Ester Hydrolases↗
PubMed1996

Starch synthesis, and tuber storage protein genes are differently expressed in Solanum tuberosum and in Solanum brevidens.

Studying in vitro stem cuttings of Solanum tuberosum induced for tuberization and those of a non-tuberizing Solanum species, differences both in morphology and in gene expression were detected. Stolon formation essentially depended on light while tuberization was triggered by the elevated level of sucrose in the medium. Genes involved in starch synthesis were induced by sucrose in both species, however, starch granules were detected only in potato. A new tuber specific cDNA clone, GM7, encoding a putative metallocarboxypeptidase inhibitor and the cDNA of a proline rich cell wall protein with S. brevidens specific expression were isolated by differential screening. Sucrose mediated transcription of the tuber storage proteins like patatin and proteinase inhibitors (Kunitz-type, winI, GM7) failed in S. brevidens.

Amino Acid Sequence↗
PubMed1995

Digoxigenin-labelled cDNA probes for the detection of potato virus Y in dormant potato tubers.

An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.

DNA Probes↗
PubMed1996

Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization.

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY(O), but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY(O)-specific probe. As little as 1 pg of purified PVY(O)-RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY(O) amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY(O) concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and did not result in reduced detection of PVY(O) by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America.

DNA, Viral↗
PubMed2002

Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria.

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.

Adaptation, Physiological↗
PubMed2001

The potato tuber transcriptome: analysis of 6077 expressed sequence tags.

This is the first report of the biosynthetic potential of a tuber storage organ investigated by expressed sequence tag sequencing. A cDNA library was generated from the mature tuber of field grown potato (Solanum tuberosum var. Kuras). Partial sequences obtained from 6077 clones were assembled into 828 clusters and 1533 singletons. The average read length was 592 bp, and 2254 clones were full length. 5717 clones showed homology to genes from other organisms. Genes involved in protein synthesis, protein destination and cell defense predominated in tuber compared to stolon, shoot and leaf organs. 1063 clones were unique to tuber. Transcripts of starch metabolizing enzymes showed similar relative levels in tuber and stolon.

Expressed Sequence Tags↗
PubMed2003

Photostimulated fos-like immunoreactivity in tuberal hypothalamus of photosensitive vs. photorefractory turkey hens.

Photorefractoriness in commercial turkey hens can be viewed as a failure of previously sexually stimulatory photoperiods to maintain egg production via activation of cGnRH I neurons, but the neural locus of photorefractoriness, i.e., where in the brain failure occurs, is not known. We used a c-fos antiserum that detects c-Fos and Fos-related antigens to characterize Fos-like immunoreactivity (FLI) as a measure of neuronal activation. FLI was measured in somatically mature, photosensitive hens (held on short photoperiods [8L:16D] for at least 10 weeks) before (non-photostimulated-photosensitive group) and after 48 h of exposure to long photoperiods (16L:8D; photostimulated-photosensitive group). We also measured FLI in hens that had become photorefractory, transferred to short photoperiods for 1 week--an insufficient time period to reverse photorefractoriness--and then exposed to long photoperiods for 48 h (photostimulated-photorefractory group). FLI was nearly absent in the tuberal hypothalamus of non-photostimulated-photosensitive hens but FLI was abundant in photostimulated-photosensitive hens. FLI was greatly reduced (P<0.01) in the rostral tuberal hypothalamus of photostimulated-photorefractory hens. All hens showed variable extra-tuberal FLI in locations associated with stress, e.g., paraventricular nucleus, lateral septal area, and nucleus taenia. Double-label fluorescence immunohistochemistry with c-fos antiserum and anti-Neu-N, a neuron-specific protein, showed that a substantial fraction of tuberal FLI-positive cells in photostimulated-photosensitive hens were neuronal. These results implicate neurons in the rostral tuberal hypothalamus as a potential neural locus of photorefractoriness, as FLI in this region appears coupled with cGnRH I activation in photostimulated-photosensitive but not photostimulated-photorefractory turkey hens.

Animals↗
PubMed2001

A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers.

A multiplex reverse transcription polymerase chain reaction (m-RT-PCR) was developed for the simultaneous detection of five potato viruses and a viroid. The synthesis of cDNAs used for amplification was primed by hexanucleotides (random primers, RP). An RNA extraction procedure employing DNase I, is routinely used to isolate potato viruses and viroid (Potato virus S, PVS; Potato leafroll virus, PLRV; Potato virus X, PVX; Potato virus A and Y, PVA, PVY; and Potato spindle tuber viroid, PSTVd) from infected tissues. This extraction method produced deoxy-oligonucleotides, which in turn were used to prime the reverse transcription of RNA templates of all the viruses and the viroid. A time-course study from 15 s to 30 min showed optimal oligonucleotide generation by DNase I occurred at 10 min, an incubation time already incorporated in the extraction protocol. The presence of oligonucleotides capable of priming cDNA synthesis was also demonstrated in RNA preparations from aphids, leaves, and tubers. In order to duplicate the priming of templates by oligonucleotides, commercially available hexanucleotides were used as RP. When fragments were amplified from 5'- and 3'-ends of the random primed cDNA of PVY genome, bands of similar intensity were observed. In contrast, when two fragments (short and long) from the P1 gene region of the PVA genome were amplified, the yield of the short fragment was significantly higher in intensity than that of the long fragment in random primed cDNA. Irrespective of the origin of the primers (generated during extraction vs. commercially purchased), single or multiple viruses or the viroid were detected by amplification of random primed cDNAs present individually in the reaction or in a cDNA pool consisting of five viruses and the viroid. The cDNA produced by RP or virus specific primers (SP) was used to detect PLRV and PVY from infected tubers in a duplex reverse transcription polymerase chain reaction (d-RT-PCR). The RP cDNA gave increased detection. Comparison of RP primed cDNAs from dormant or sprouted tubers and leaves showed that for some cultivars, such as 'Shepody', leaves were more reliable for PVY and PLRV detection than the tubers, in both the d- and m-RT-PCR.

Animals↗
PubMed2002

Response surface models to predict potato tuber infection by Fusarium sambucinum from duration of wetness and temperature, and dry rot lesion expansion from storage time and temperature.

Dry rot (Fusarium sambucinum) of potatoes causes significant yield loss in storage and may also produce mycotoxins. Disease dynamics of dry rot development in potato tubers after harvest was studied and modeled. Potato tubers were surface sterilized, wounded, inoculated with a spore suspension of F. sambucinum and incubated in mist chambers placed in growth chambers at 4, 8, 12, 16 or 20 degrees C. After 0, 3, 6, 12, 24 and 48 h of incubation five tubers from each treatment were removed, dried and stored at 16 degrees C and 95% RH for 15 days. Inoculated tubers were also maintained in mist chambers for 24 h at 16 degrees C for the establishment of initial infections, dried, and stored at 4, 8, 12, or 16 degrees C for up to 90 days at 95% RH. At 15 days intervals, tubers were sliced, diameters and depths of diseased area measured, and data transformed to proportion of maximum volume diseased (PVD). The amount of infection was least at the lowest temperature tested and at the end of a 3-h wet period, but infection increased with an increase in wetness duration and temperature. At a storage temperature of 16 degrees C, lesions expanded rapidly reaching maxima in about 45 days of storage. A cubic regression model to predict infection potential from incubation temperature and duration of wetness explained 94.2% of the variation in PVD. A cubic regression model to predict lesion expansion potential as a function of storage temperature and duration explained 99.7% of the variation in PVD. These models could be used to manage potato dry rot, after validation under commercial conditions.

Food Contamination↗
PubMed2000

Differential formation of octadecadienoic acid and octadecatrienoic acid products in control and injured/infected potato tubers.

Lipoxygenases in plants have been implicated in the activation of defense responses against injury/infection. Pathogen-derived polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and their metabolites have been shown to elicit defense responses against pathogen infection in plants. However, not much is known about the role of host-derived fatty acids and their metabolites in plant defense responses. In this study, isolation and characterisation of endogenous lipoxygenase metabolites formed in potato tubers in response to injury/infection was undertaken. While 9-hydroperoxyoctadecadienoic acid (9-HPODE), derived from octadecdienoic acid (linoleic acid) is the major lipoxygenase product formed in control potato tubers, 9-hydroperoxyoctadecatrienoic acid (9-HPOTrE), derived from octadecatrienoic acid (alpha-linolenic acid) is the major lipoxygenase product formed in potato tubers in response to injury or infection with Rhizoctonia bataticola. As a result, the relative ratio of 9-HPODE to 9-HPOTrE showed a shift from 4:1 in control to 1:2 and 1:4.5 in injured and infected potato tubers respectively. From this study, it is proposed that lipoxygenase metabolites of octadecadienoic acid may be involved in physiological responses under control conditions, while octadecatrienoic acid metabolites are mediating the defense responses. This forms the first report on the differential formation of endogenous lipoxygenase products in potato tubers under control and stress conditions.

Chromatography, High Pressure Liquid↗
PubMed2001

Residue levels of chlorpropham in individual tubers and composite samples of postharvest-treated potatoes.

Chlorpropham, a herbicide and sprout suppressant, is used on stored potatoes to prolong the storage period without deterioration of produce quality. Data for residue concentrations on an individual tuber basis are required by WHO for the estimation of the variability factor. In this study, the levels of chlorpropham in individual tubers and in composite samples were determined. The distribution of chlorpropham between the peel and the tuber flesh was examined, and the fate during the cooking process (washing, boiling, frying) was studied. The concentrations in individual tubers ranged from 1.8 to 7.6 mg/kg 10 days postapplication (mean 3.8 mg/kg, RSD 39%), from 0.7 to 4.0 mg/kg 28 days postapplication (mean 2.9 mg/kg, RSD 28%), and from 0.8 to 3.8 mg/kg 65 days postapplication (mean 2.2 mg/kg, RSD 48%). The calculated residues in composite samples 10 days postapplication ranged from 4.3 to 6.1 mg/kg (mean 4.9 mg/kg, RSD 20%). Those in samples taken 28 days postapplication ranged from 3.1 to 4.2 mg/kg (mean 3.8 mg/kg, RSD 15%). The concentrations determined in composite samples of whole tubers 65 days postapplication ranged between 2.6 and 3.2 (mean 2.9 mg/kg, RSD 11%). Peeling removed 91-98% of the total residue; washing reduced residues by 33-47%. Detectable residues were found in boiled potatoes and the boiling water, and in french fries and the frying oil. Monitoring data on commercial prefried frozen french fries are reported.

Calibration↗
PubMed2001

Antioxidant activities of dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber.

Dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber (which is different from dioscorine found in tubers of Dioscorea hirsuta), was purified to homogeneity after DE-52 ion exchange column according to the methods of Hou et al. (J. Agric. Food Chem. 1999, 47, 2168-2172). A single band of 32 kDa dioscorin was obtained on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel with 2-mercaptoethanol treatment. This purified dioscorin was shown by spectrophotometric method to have scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in a pH-dependent manner. There is a positive correlation between scavenging effects against DPPH (8-46%) and amounts of 32 kDa dioscorin (5.97-47.80 nmol) added in Tris-HCl buffer (pH 7.9), which are comparable to those of glutathione at the same concentrations. Using electron paramagnetic resonance (EPR) spectrometry for DPPH radical detection, it was found that the intensities of the EPR signal were decreased by 28.6 and 57 nmol of 32 kDa dioscorin in Tris-HCl buffer (pH 7.9) more than in distilled water compared to controls. EPR spectrometry was also used for hydroxyl radical detection. It was found that 32 kDa dioscorin could capture hydroxyl radical, and the intensities of the EPR signal were significantly decreased dose-dependently by 1.79-14.32 nmol of 32 kDa dioscorin (r = 0.975) compared to the control. It is suggested that 32 kDa dioscorin, the storage protein of yam tuber, may play a role as antioxidant in tubers and may be beneficial for health when people take it as a food additive or consume yam tubers.

Antioxidants↗
PubMed2000

Molecular tools for the identification of Tuber melanosporum in agroindustry.

Tuber melanosporum Vitt., Tuber magnatum Pico, and Tuber uncinatum Chat. can be differentiated by their morphological characters. Fraud problems have arisen recently with the importation to Europe of truffles from China. T. melanosporum is morphologically very close, but distinct from the Chinese species [Tuber indicum (Cooke and Massee) and T. himalayense BC (Zhang and Winter)]. We have optimized molecular tools to unequivocally identify T. melanosporum. DNA extraction from ascocarps of black truffles is not straightforward. Problems to obtain pure DNA are due to high contents of phenolic compounds, melanine, and various polymers (proteins, polysaccharides, etc). These compounds coprecipitate with the DNA during extraction and strongly inhibit the PCR reaction. We have developed an efficient and reliable protocol for DNA extraction from truffle ascocarps. It was used successfully for DNA extraction from mycorrhizal root tips as well as from canned preparations of T. melanosporum. Several approaches to identify T. melanosporum by PCR were developed. Two specific primers for T. melanosporum were designed after comparison of the ITS region of this species with those of three Chinese fungi. They proved to be efficient to specifically detect the presence of T. melanosporum by PCR. The mycorrhizal status of trees inoculated with T. melanosporum but unable to produce truffles was confirmed in a single-step PCR reaction. A multiplex PCR approach was also developed with three sets of primers (including a specific one for Chinese truffles) to detect, in one PCR reaction, the presence of any other Tuber species mixed with T. melanosporum ascocarps. This optimized protocol, in association with the specific primers we designed, is applicable to quality control in the truffle industry from the production stages to final commercial products.

Ascomycota↗
PubMed2001

Molecular characterization of StCDPK1, a calcium-dependent protein kinase from Solanum tuberosum that is induced at the onset of tuber development.

We isolated a full-length cDNA clone (StCDPK1) encoding a calcium-dependent protein kinase (CDPK) by screening a stolon tip cDNA library from potato plants (Solanum tuberosum L.). The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family except in the N-terminal region. As described for other CDPKs, StCDPK1 has a putative N-terminal myristoylation sequence. A coupled transcription/translation system was used to demonstrate that this post-translational modification occurs in vitro. The behaviour of the myristoylated form of StCDPK1 during its purification on a phenyl-Sepharose column mimics that of the endogenous potato enzyme suggesting that this modification occurs in vivo. In addition, a possible palmitoylation site is present in StCDPK1. Southern blot analysis suggests that more than one CDPK isoform is present in potato plants. Northern blot analysis of steady-state mRNA levels for StCDPK1 in different tissues of potato plants shows that the transcript is differentially expressed in tuberizing stolons. The transcript appears in the early steps of tuber formation before the induction of other genes, such as Pin2 and patatin. This result parallels previous data on CDPK activity in potato plants which was highest at the beginning of tuberization. Our results suggest that StCDPK1 is developmentally regulated. The early and transient expression of this CDPK isoform in the tuberization process suggests that this kinase could trigger a cascade of phosphorylation events involved in tuber induction.

Amino Acid Sequence↗
PubMed2001

Acceleration of potato tuber sprouting by the expression of a bacterial pyrophosphatase.

Potato is a globally important crop. Unfortunately, potato farming is plagued with problems associated with the sprouting behavior of seed tubers. The data presented here demonstrate that using transgenic technology can influence this behavior. Transgenic tubers cytosolically expressing an inorganic pyrophosphatase gene derived from Escherichia coli under the control of the tuber-specific patatin promoter display significantly accelerated sprouting. The period of presprouting dormancy for transgenic tubers planted immediately after harvest is reduced by six to seven weeks when compared to wild-type tubers. This study demonstrates a method with which to regulate dormancy, an important aspect of potato crop management.

Carbohydrate Metabolism↗
PubMed1997

Increased potato tuber size resulting from apoplastic expression of a yeast invertase.

The role of sucrose cleavage in determining sink strength in potato was investigated by generating transgenic potato plants that expressed a yeast invertase in either the cytosol or apoplast of tubers. Cytosolic localization gave rise to a reduction in tuber size and an increase in tuber number per plant whereas apoplastic targeting led to an increase in tuber size and a decrease in tuber number per plant. Sink organ size can be manipulated through modification of sucrose metabolism.

Chimera↗
PubMed1996

Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase.

The aim of this work was to use tubers from transgenic lines of potato (Solanum tuberosum) containing increased amounts of ADPglucose pyrophosphorylase to study the role of this enzyme in the control of starch synthesis. A 4-5-fold increase in activity of the enzyme, achieved by transformation with the Escherichia coli ADPglucose pyrophosphorylase gene glgC-16, had no detectable effect on the starch content of developing or mature tubers. No significant effects were found on the contents of ADPglucose, UDPglucose, glucose 1-phosphate, glucose 6-phosphate, PP1, ATP and ADP. Flux from [U-14C]sucrose, supplied to tubers still attached to the plant, to starch increased roughly in proportion to the increase in ADPglucose pyrophosphorylase activity. These measurements of flux gave a response coefficient close to 1 for the activity of the pyrophosphorylase in respect of starch synthesis. Pulse-chase experiments with [U-14C]sucrose showed that the increased flux into starch in the transformed tubers was accompanied by an increased rate of starch turnover. Further experiments suggested that the increased turnover was associated with an increase in the capacity of the tubers to degrade starch.

Adenosine Diphosphate↗