Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes

The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.     

Increased mitochondrial DNA and RNA polymerase activity in ethylene-treated potato tubers

A purified mitochondrial fraction was isolated from potato (Solanum tuberosum L.) tubers respiring normally at 23°C or at an accelerated rate in response to treatment with ethylene (10 microliters per liter).

A pronounced increase in various mitochondrial enzymic activities was observed in response to exposure of the whole tubers to ethylene. Cytochrome c oxidase activity increased more than 50%, DNA polymerase activity increased about 2-fold, and RNA polymerase activity increased 2.5-fold. Moreover, DNA or RNA polymerase activities of mitochondria isolated from tubers not treated with ethylene were not affected by ethylene treatment in vitro. Respiratory control ratios decreased from 2.84 to 1.50 with increasing periods of ethylene treatment from 0 to 15 hours. None of these changes were observed in untreated tubers. It is concluded that the stimulation of respiration by ethylene in potato tubers is accompanied in vivo by an enhancement of mitochondrial enzymic activity of both membrane-associated enzymes which participate in the mitochondrial oxidative electron transport as well as soluble enzymes which are not directly involved in respiration.

     

Effect of ethylene and carbon dioxide on potato metabolism: stimulation of tuber and mitochondrial respiration, and inducement of the alternative path

The respiration of potato tubers (Solanum tuberosum var. Russet Burbank) which have been kept at room temperature for 10 days is stimulated upon subsequent treatment with C₂H₄ (10 microliters per liter) and O₂. The respiratory rise reaches a peak in 24 to 30 hours and thereafter declines. Coincident with the rise in tuber respiration is an increase in the respiratory rates of fresh slices and isolated mitochondria. Slices and mitochondria from C₂H₄- and O₂-treated tubers also display substantial resistance to CN, and the resistant respiration is inhibited by hydroxamates.

The longer the tubers are stored after harvest, the less effective is C₂H₄ in causing CN resistance in slices and mitochondria from treated tubers. Addition of 10% CO₂ to the C₂H₄-O₂ mixture, however, causes extensive CN resistance to develop, even in slices and mitochondria from old tubers. The results show that C₂H₄, O₂, and CO₂ act synergistically to induce alternative path development in potatoes.

     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

Inhibition of o(2) consumption resistant to cyanide and its development by N-propyl gallate and salicylhydroxamic Acid

Kinetics of inhibition of cyanide-insensitive O₂ uptake by n-propyl gallate (PG) and salicylhydroxamic acid (SHAM) were determined in fresh slices from ethylene-treated tubers of Solanum tuberosum `Norchip' and with mitochondria and lipoxygenase (EC 1.13.11.12) isolated from these tubers. PG and SHAM appeared to be inhibiting at identical sites in mitochondria but at disparate sites in slices. The apparent K_I for SHAM was similar in mitochondria and slices. However, the apparent K_I for PG in mitochondria was about 40-fold lower than the K_I for PG inhibition of lipoxygenase activity. The amount of lipoxygenase associated with mitochondria increased when tubers were treated with ethylene. PG, but not SHAM, inhibited aging-induced development of cyanide-insensitive respiration. The latter two phenomena are in accord with the hypothesis that lipid metabolism is required for the development of the alternative pathway.     

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.     

Ethylene and wound-induced cyanide-resistant respiration climaxes in potato: The synergistic role of CO2 and the selective role of lycorine

When treated with ethylene in O₂, conditioned potato (Solanum tuberosum L. cv. Russet Burbank) tubers – that is, tubers kept at room temperature for 10 days or more – yield slices that are CN^? resistant. Ten % CO₂ in the gas mixture not only synergizes the effect of ethylene, but replaces the need for conditioning as well. The response to CO₂ is more pronounced with increasing time from harvest. By contrast fresh slices from untreated tubers are CN^? sensitive, as are slices from tubers incubated in O₂ or O₂ plus CO₂. The suggestion is made that CN^? resistance is constitutive, and that treatment with ethylene/CO₂ in O₂ confers on potato tuber tissue a resistance to the extensive degradation of membrane phospholipids that normally attends slicing and leads to the loss of CN^? resistance. In this connection respiration inhibition by imidazole, an inhibitor of fatty acid α-oxidation, is extensive in slices of untreated tubers, and sharply diminished in slices of ethylene-treated tubers in proportion to their CN^? resistance. The coextensive rise of respiration rate and CN^? resistance in aged potato slices has led to the presumption that the CN^?-resistant path mediates the respiration climax. Accordingly the alkaloid, lycorine, has been considered to inhibit the development of CN^? resistance in aging potato slices because it curtails the wound-induced respiration. A comparison was carried out on the effect of lycorine on CN^?-sensitive and CN^?-resistant fresh slices – the latter obtained from ethylene/CO₂-treated tubers. Lycorine suppressed the development of the wound-induced respiration without restricting the development of CN^? resistance.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

Biogenesis and Degradation of Starch: I. The Fate of the Amyloplast Membranes during Maturation and Storage of Potato Tubers

Storage of mature or developing potato tubers (Solanum tuberosum “Up-to-Date” variety) at 4 C causes a reduction in the starch content and the elevation in the level of free sugars. This phenomenon is not observed when the tubers are stored at 25 C. Changes in the morphology of cells from developing or mature tubers after storage at 4 or 25 C have been followed by electron microscopy. During all stages of the tuber development the starch granules are surrounded by a membrane derived from the plastid envelope. Storage in the cold induces disintegration of this membrane. A membrane fraction isolated from starch granules of tubers stored at 4 C has a lower buoyant density, and the electrophoretic pattern of its proteins is different from that of a similar membrane fraction obtained from tubers stored at 25 C. It is suggested that the cold-induced changes in the starch and sugar content during storage of potato tubers might be correlated with damage to the membranes surrounding the starch granules and changes in their permeability to degradative enzymes and substrates.     

Salt hardiness and dye reduction by potato tissue and mitochondrial fractions as influenced by previous storage of the tubers

Oxygen uptake and tetrazolium reduction occurred at higher rates in discs from potato tubers (Solanum tuberosum L.) stored at 0° than in discs from tubers stored at 12.8°. Tetrazolium reduction was at a higher rate in mitochondrial fractions from tubers stored at 0° than in mitochondrial fractions from tubers stored at 12.8°. These physiological activities were more resistant to hypertonic KCl treatments in tissue and mitochondrial fractions from tubers stored at 0° than in tissue and mitochondrial fractions from tubers stored at 12.8°. Inhibition of O₂ uptake and tetrazolium reduction progressively increased with increasing concentrations of KCl for tissue and mitochondrial fractions from tubers stored at 0 and 12.8°, but inhibition was more severe and occurred at lower concentrations of KCl for the material from tubers stored at 12.8°. Tissue from tubers stored at 0° was at the same time more sensitive to hypotonic solutions and more resistant to hypertonic solutions than corresponding tissue from tubers stored at 12.8°. Adaptive changes brought on in the tubers by the stress of cold storage were demonstrated in the discs and mitochondrial fractions prepared from cold-stored tubers.     

A Quantitative Cytochemical Study of Glutamate and Glucose-6-Phosphate Dehydrogenase Activities during Chilling Injury in Tubers of Dioscorea rotundata Poir.

Onyia, G. O. C. and Gahan, P. B. 1985. A quantitative cytochemicalstudy of glutamate and glucose-6-phosphate dehydrogenase activitiesduring chilling injury in tubers of Dioscorea rotundala Poir.—J.exp. Bot. 36: 1249–1256. The response of glucose-6-phosphate dehydrogenase and glutamatedehydrogenase activities in healthy Jamaican Dioscorea rotundalatubers and those chilled at 3 ?C for 1,2,3,4, and 7 d at 70%r.h. were assessed by quantitative cytochemical assays. Bothenzymes in chill-damaged tuber tissues showed a substantiallyhigher activity than did those of the healthy tubers. An early,sharp increase in the response of the NADP-tetrazolium reductasesystem of damaged tuber tissue was significantly higher (P =0.001) than that of healthy tubers or those chilled but ableto recover. This response may be used as an early marker ofchilling injury in the yam tuber. Key words: Dioscorea rotundata Poir, quantitative cytochemistry, yam tuber, glucose-6-phosphate, dehydrogenase, glutamate dehydrogenase, NADPitetrazolium reductase     

Potato Lectin: A Cell-Wall Glycoprotein

The activity and the amount of potato lectin were measured inpotato tuber slices (Solanum tuberosum cv. Huinkul) aeratedfor 48 h. Lectin was found in a soluble form, liberated to themedium and associated with insoluble structures. Polyacrylamidegel electrophoresis in denaturating conditions and immunologicaltechniques indicated that the lectins associated to cell wall,soluble or liberated to the medium, were identical. The cell-wallfraction was found to contain 65% of total lectin in the tuber.The possible role of potato lectin in tubers was discussed. (Received June 5, 1985; Accepted September 3, 1985)     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: III. RELATIONSHIP BETWEEN GROWTH RATE OF INDIVIDUAL TUBERS, TUBER WEIGHT AND STOLON GROWTH PRIOR TO TUBER INITIATION

Potato plants (Solanum tuberosum L.) were grown in water culture.About 14 d after tuber initiation no significant differenceswere found between apical and basal tuber parts in ¹⁴C-uptakeand partitioning into various fractions from ¹⁴C-labelled photosynthate.Thus, the fresh weight of these tubers could be used as a parameterfor the sink size. The ¹⁴C-content per tuber (sink strength)20 h after ¹⁴CO₂-supply to the foliage was significantly correlatedwith the tuber fresh weight. No correlation was found betweenthe ¹⁴C-concentration of the tuber (sink activity; ct. min^–g^– fr. wt.) and tuber fresh weight. Consequently, tuberfresh weight (sink size) per se must have been a factor whichinfluenced sink strength. Stolon parameters characterizing theirgrowth prior to tuber initiation (e.g. stolon volume) and theircapacity for photosynthate transport (diameter, length) weremeasured at the time of tuber initiation. Significant correlationswere found between these stolon parameters and subsequent growthof individual tubers. Anatomical studies on the proportion ofvarious tissues in the cross sectional area of stolons supportthe idea of a negative relation between growth of individualtubers and transport resistance in the phloem of the stolons.It is concluded that in the initial phase of tuber growth, mainlyfactors outside of the tuber determine its growth rate. In laterstages of tuber growth, when the sink strength increases, thecompeting strength of individual tubers for photosynthate isdominated mainly by factors within the tuber itself, such astheir sink size and sink activity. Key words: Potato tuber, sink size, tuber initiation, transport resistance     

Laboratory evaluation of lufenuron on immature stages of potato tuber moth (Lepidoptera: Gelechiidae)

The effects of the chitin synthesis inhibitor lufenuron against potato tuber moth, Phthorimaea operculella (Zeller), eggs were determined by topically exposing different age groups of eggs (1-4 d old) to treated potato tubers (Solanum tuberosum L.) under laboratory conditions. Larval hatch from both treated (4 and 12 g [AI]/100 liter) and untreated tubers was >95%, but mortality of first instars was high in treated tubers (>90%) compared with untreated tubers. Examination of the treated tubers showed that most of the larvae were unable to penetrate or cause any noticeable damage to the potato tubers. However, the few first instars that survived were able to penetrate the tubers and continue their development to the pupal or adult stages. At 12 g (AI)/100 liter, adult emergence was <2% and most of the emerged adults had morphological deformities such as reduced wing size and they were unable to free themselves from the pupal sacs. These data suggest that topical application of lufenuron to eggs before larval hatch would reduce the amount of damage caused by potato tuber moth as part of integrated pest management program.     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

Interrelation Between Growth Rate of Individual Potato (Solanum tuberosum L.) Tubers and Their Cell Number

In potato plants fast and slow growing tubers develop on thesame plant. A hypothetical causality between tuber growth rateand tuber cell number was investigated by determining the tubercell number with the aid of an automatic counting procedure.Our data show a close correlation between tuber size and cellnumber over the whole range of tuber volumes considered (3–28cm³). If the influence of tuber size on cell number is eliminatedby means of a partial correlation analysis, the cell numberof the entire tuber is not significantly correlated with itsgrowth rate. An exclusive consideration of the smaller cells(10–30 µm) in the apical tuber region, where thecell division rate in potato tubers is highest, reveals a loosebut significant partial correlation to tuber growth rate (r= 0.383, P < 0.05). The growth rate of the slow growing tubers of any potato plantmay be enhanced by removing the fast growing tubers. In thefirst few days this enhanced growth rate is not due to a stimulationof cell division rate, but rather due to cell expansion. Potato, Solanum tuberosum L., tuber growth rate, tuber cell number     

Cyanide-resistant Respiration in Freshly Cut Potato Slices

Treating intact white potato (Solanum tuberosum L.) tuber with ethylene in air or O₂ made it possible to obtain freshly cut slices which exhibit cyanide-resistant respiration. The cyanide-resistant path requires induction in whole tubers. The data also indicate that high O₂ concentration is necessary for the full development of cyanide-resistant respiration.     

Tuberization in Potato at High Temperatures: Interaction between Temperature and Irradiance

Potato plants (Solanum tuberosum L., cv. Sebago) responded similarlyto high temperatures and low irradiance by diverting dry matterto the shoots rather than the tubers, and changes were notedin a range of morphological characteristics. It is proposedthat the effect of both high temperature and low irradianceis brought about by the increased production of a growth substance,possibly gibberellin, which inhibits tuber formation, and thattuber yield is determined by the balance between temperatureand irradiance. Solanum tuberosum L., potato, tuberization, temperature, irradiance, gibberellin     

α and β-solamarine in kennebec Solanum tuberosum leaves and aged tuber slices

Two major steroid glycoalkaloids, in addition to α-solanine and α-chaconine, were isolated from leaves and aged tuber slices of potato, Solanum tuberosum L. var Kennebec. They are glycosides of tomatidenol and have been identified as α- and β-solamarine. The compounds were not found in tuber peel or freshly sliced Kennebec tubers or in 20 other cultivars.