Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

The regulation of respiration and growth of potato tuber callus by endogenous ethylene. Suppression of ethylene action by 2,5-norbornadiene

The growth (fresh and dry weight increase) of potato tuber ( Solanum tuberosum L. cv. Bintje) callus discs was stimulated by incubation in air with 500 ppm 2,5-norbornadiene (NBD, a competitive inhibitor of ethylene action) and inhibited by incubation in air with 4 000 ppm NBD. Ethylene formation by the callus was stimulated by NBD. The development of the alternative pathway, measured in isolated mitochondria was inhibited by NBD in a concentration-dependent way. The alternative pathway capacity, measured in vivo, was inhibited by 4 000 ppm NBD, but not by 500 ppm. Uninhibited in vivo respiration, which consists of cytochrome path activity and alternative path activity, was stimulated by the treatment with 500 ppm NBD. The main contribution to this stimulation was made by the cytochrome pathway. In 4 000 ppm NBD-treated callus, uninhibited respiration seemed to be unaffected as a consequence of an inhibited cytochrome path activity, which was compensated by a stimulated alternative path activity. Both in 500 and 4 OIK) ppm NBD-treated callus the alternative path activity in vivo was stimulated.
The regulatory role for endogenous ethylene in potato tuber callus is discussed in relation to: 1) The induction of respiratory pathways, 2) the supply of reduction equivalents in vivo and 3) growth.     

Effect of a granulovirus on mortality and dispersal of potato tuber worm (Lepidoptera: Gelechiidae) in refrigerated storage warehouse conditions

Potato tuber worm (PTW), Phthorimaea operculella (Zeller), is a world-wide pest of potato. In rustic stores, PTW larvae can infest 100% of stored tubers. Treatment of tubers in rustic stores with the PTW granulovirus (PoGV) has been demonstrated to protect stored tubers. This is the first study to show the effects of PoGV for protection of tubers stored in refrigerated warehouse conditions. Tubers were treated by dipping in aqueous suspensions of PoGV or water. An estimated 0.0819 larval equivalents of virus or 1.88×10⁹ viral occlusion bodies were deposited on each kilogram of tubers. They were held at 16°C for 11 days before lowering the temperature by 0.5°C per day until 10°C was reached. The tubers were stored at this temperature for 53 days. Mean numbers of infested tubers at the end of the assay was affected by both pre-infestation rate and virus treatment. Mean numbers of infested tubers in the control treatment was 3 tubers per chamber higher than in the virus treatment providing strong evidence that PoGV controlled larvae and minimized spread into un-infested tubers. Of the larvae that were retrieved in virus-treated infested tubers, the mean mortality was 87% compared to 37% in controls.     

Characterization of the early events of potato tuber development

The early events of potato ( Solanum tuberosum L. cv. Superior) tuberization were examined by using a model system of axillary bud tuber development from petiole-leaf single-node cuttings. Both fresh weight and starch accumulation were monitored to establish a developmental framework for morphological changes. Fresh weight and starch content began to increase in axillary buds after 2 days. Visible changes in bud morphology could be detected 4 days after the start of incubation. Substantial increases in both total protein and total RNA were observed at the onset of tuber morphology. Immunoblot analysis showed that the major tuber protein, patatin, could be initially detected in day 4 buds and that a 22-kDa proteinase inhibitor could be initially detected at day 8. Northern blot analysis corroborated this pattern of accumulation at the RNA level for both protein types. Substantial accumulation of the two proteinase inhibitor mRNAs occurred later than patatin mRNA accumulation. The results of this study showed that there is considerable accumulation of both protein and mRNA occurring during the early stages of tuber development prior to the substantial accumulation of the major tuber storage proteins.     

An integrated view of the hormonal regulation of tuber formation in potato (Solanum tuberosum)

The hormonal regulation of the consecutive steps in the formation of tubers on the potato plant ( Solanum tuberosum L.) is described and discussed. An integrated view of the complex regulation of the initiation and growth of stolons and tubers is presented, with special emphasis on the commonly observed lack of synchronization of the various steps in tuber formation within a plant.     

Effect of ageing on sterol metabolism in potato tuber slices

When fresh potato tuber slices were incubated with [1-¹⁴C]-sodium acetate, cycloartenol was heavily labelled but no radioactivity was recovered in 24-methylene cycloartanol and free sterols. If potato slices were aged for 0–24 hr before feeding with radioactive acetate, a rapid increase of the label in the sterol precursors and the free sterols was observed. The free sterol content was 5 × higher after ageing for 24 hr. Isofucosterol synthesis was especially stimulated. The synthesis of sterols during the ageing process seems to be related to the appearance of a cycloartenol C24-methylase and may be linked to a biogenesis of membranes.Nomenclature: (1) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-cholest-24-en 3β-ol; (2) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-ergost-24(28)-en 3β-ol; (3) 4α,14α-dimethyl 9β,19β-cyclo 5α-ergost 24(28)-en 3β-ol; (4) 4α, 14α-dimethyl 5α-ergosta 8.24(28)-dien 3β-ol; (5) 4α-methyl 5α-ergosta 7,24(28)-dien 3β-ol; (6) ergosta 5,24(28)-dien 3β-ol; (7) stigmasta 5,Z-24(28)-dien 3β-ol; (8) (24R)-24 methyl cholest 5-en 3β-ol; (9) (24R)-24 ethyl cholest 5-en 3β-ol; (10) (24S)-24 ethyl cholesta 5,E-22(23)-dien 3β-ol; (11) cholest 5-en 3β-ol.     

Adenine nucleotide contents in callus-forming potato tuber discs with different respiratory characteristics

ATP content (per g fresh weight) and energy charge were higher during incubation at 8°C than at 28°C in the early stages of callus induction of potato tuber discs ( Solanum luberosum L. cv. Bintje). After a transfer from 28°C to 8°C, ATP content increased while a sharp decline in ATP content was observed after a transfer from 8°C to 28°C. ADP and AMP pools did not increase correspondingly. When the callus discs had entered the logarithmic growth phase, the energy charge was maintained within relatively narrow limits (0.77–0.80) at all culture temperatures.     

Change in phosphorylase isoenzymes during dedifferentiation of potato tuber cells

The phosphorylase isoenzyme composition of soluble preparations isolated from potato ( Solanum tuberosum L. cv. Spunta) tuber-derived callus has been studied by polyacrylamide gel electrophoresis and affinity electrophoresis. Native electrophoretic profiles indicate that dedifferentiated callus tissue contains a single form of phosphorylase that differs in primer requirement, charge and affinity towards branched α-1,4/1,6-glucans from the major phosphorylase form (phosphorylase II) in potato tuber. This latter molecular form is missing in dedifferentiated callus. However, callus phosphorylase appears to be closely related to tuber phosphorylase I, a minor form found in the original explant tissue.     

Characterization of potato genotypes by chlorophyll fluorescence during plant aging in a Mediterranean environment

Field experiments were conducted in Sicily (south Italy) during two seasons to characterize by chlorophyll (Chl) fluorescence four genotypes (Spunta, Sieglinde, Daytona, and Ninfa) of potato (Solanum tuberosum L.) for off-season production during plant aging and to analyse the possible relation between Chl parameters and tuber yield. Chl fluorescence parameters [initial fluorescence (F₀), maximum fluorescence (F_m), F_v/F_m, time in which maximal fluorescence occurs (T_max)] gained from Kautsky kinetics and Chl content were measured weekly, from 5^th to 6^th leaf appearance to beginning of plant senescence in the first season and to full plant senescence in the second season. F₀ and F_v/F_m were the most reliable Chl fluorescence parameters for the definition of genotypic differences while Chl content and T_max were the most reliable Chl parameters to predict plant aging. Tuber yield was highly correlated with Chl content, T_max, F₀, and F_m.     

H_2O_2和水杨酸对陈化马铃薯切片交替呼吸途径影响的比较(英文)

外源 5 .0mmol/LH2 O2 和 0 .1mmol/L水杨酸 (salicylicacid ,SA)处理均可明显提高陈化 2 4h的马铃薯切片的交替呼吸途径容量 (Valt)及其与总呼吸的比值 (Valt/Vt)。应用交替氧化酶的单克隆抗体进行Western杂交的结果表明 ,H2 O2 和SA处理均可明显提高陈化马铃薯切片中交替氧化酶的表达水平。用氧同位素分辨法研究 ,结果表明 :H2 O2 处理对陈化马铃薯切片中交替呼吸途径的实际运行没有影响 ,而SA处理对交替呼吸途径的实际运行具有明显的促进作用。上述结果表明 ,H2 O2 和SA对植物组织交替呼吸途径的影响存在差异 ,二者均可促进交替氧化酶的表达从而诱导交替呼吸途径容量的发生 ,但H2 O2 不影响其实际运行 ,而SA还可同时诱导其实际运行。     

用于测定抗氰呼吸途径活性的氧同位素分馏法和氧肟酸抑制法的比较

氧同位素分馏法和氧肟酸抑制法的测定结果都表明,马铃薯切片在１２ ～２４ ｈ 陈化期间抗氰呼吸途径的实际活性基本保持恒定,尽管前者的测定结果是后者的２ 倍。此外,用上述２ 种不同方法进行研究的结果都表明,内源乙烯在诱导陈化切片抗氰呼吸途径容量产生的同时并不调控其实际运行。上述结果表明尽管氧肟酸抑制法不能准确测定抗氰呼吸途径的实际活性,但用于一些比较研究仍是可行的     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

Memristors: Memory elements in potato tubers

A memristor is a nonlinear element because its current-voltage characteristic is similar to that of a Lissajous pattern for nonlinear systems. This element was postulated recently and researchers are looking for it in different biosystems. We investigated electrical circuitry of red Irish potato tubers (Solanum tuberosum L.). The goal was to discover if potato tubers might have a new electrical component - a resistor with memory. The analysis was based on a cyclic current-voltage characteristic where the resistor with memory should manifest itself. We found that the electrostimulation by bipolar sinusoidal or triangle periodic waves induces electrical responses in the potato tubers with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K⁺ channels, transforms a memristor to a resistor in potato tubers. Our results demonstrate that a voltage gated K⁺ channel in the excitable tissue of potato tubers has properties of a memristor. Uncoupler carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decreases the amplitude of electrical responses at low and high frequencies of bipolar periodic sinusoidal or triangle electrostimulating waves. The discovery of memristors in plants creates a new direction in the understanding of electrical phenomena in plants.     

Lipolytic and peroxidative enzyme expressions in cultured potato tuber cells

Potato cells (cv. Norchip) were cultured from tuber parenchymal tissue and subcultured to dissociate and habituate the despecialized cells. After several subculturings on a minimal nutrient media, this line of cells demonstrated repeatable physical growth profiles for dry weight (DW), fresh weight (FW) and protein. Two enzymes of plant lipid metabolism were investigated, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX), which respectively liberate and peroxidize fatty acids from lipid in cellular membranes. LAH, measured as p-nitrophenyl palmitate hydrolase, was present in this line of cells in easily detectable amounts (317 units g^-1 DW) albeit much lower than that found in mother tuber (9878 units g^-1 DW). The presence of LAH in this line is significant because LAH isozymes are often described as storage proteins, yet activity per gram fresh weight in these unorganized cells is reasonably constant until culture growth exits the linear phase. However, LOX, the most active free fatty acid metabolizing enzyme in potato tubers (89,800 units g^-1 DW), was not detectable in this line of callus or suspension cultured cells. The absence of LOX activity in this line of cells was verified by a number of assay approaches and was confirmed by activity staining of extracted enzymes separated in polyacrylamide gels. The absence of LOX in these cultured cells is especially important in determining the functions of this lipid peroxidation system and how it may be genetically regulated.Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.A laboratory cooperatively operated by the Midwest Area, Agricultural Research Service, U.S. Department of Agriculture, The Minnesota Agricultural Experiment Station, the North Dakota Agrcultural Experiment Station, and the Red River Valley Potato Grower's Association.     

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase

The CO₂ evolution of intact potato tubers (Solanum tuberosum, L., var. Bintje) was analyzed during a 10-day period of their warm (25 ± 2°C) or cold (5 ± 1°C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO₂ evolution rates were analyzed at 20°C, to reflect their possible capacities. The 20°C CO₂ production declined from 13 to 8 mg kg^–1 h^–1 after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg^–1 h^–1. In contrast, 20°C CO₂ evolution did not change after the first day of cold storage, increased up to 14.5 mg kg^–1 h^–1 after 2 days, and decreased to about 12 mg kg^–1 h^–1 after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O₂ min^–1 mg^–1 attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.     

Alternative pathways of photosystem I-dependent electron transport in two genetically different potato cultivars in vitro

Alternative pathways of electron transport involving photosystem I (PSI) only were studied in leaves of potato plants (Solanum tuberosum L., cv. Desiree), modified by yeast invertase gene, controlled by tuber-specific class I patatin B33 promoter with proteinase II signal peptide for apoplastic localization of the enzyme. Nontransformed (wild-type) potato cultivar Desiree was used as a source of control plants. Phototrophic cultures grown in vitro on the sucrose-free Murashige and Skoog medium, as well as plants grown on the medium with 4% sucrose were examined. Various PSI-dependent alternative pathways of electron transport were discriminated by quantitative analysis of kinetic curves of dark reduction of P700⁺, the primary electron donor of PSI, oxidized by far-red light known to excite selectively PSI. In potato plants with two different genotypes, four exponentially decaying kinetic components were found, which suggests the existence of multiple alternative routes for electron input to PSI. Inhibitor analysis (with diuron and antimycin A) allowed identification of each route. A minor ultra-fast component originated from weak residual excitation of PSII by far-red light and represented electron flow from PSII to PSI. Ferredoxin-dependent cyclic electron flow around PSI accounted for the middle component, and two slower components were assigned to donation of electrons to PSI from reductants localized in the chloroplast stroma. The rates of all components were somewhat higher in leaves of the transformed plants than in the wild-type plants. However, relative contributions of separate components to the kinetics of dark P700⁺ reduction in leaves of both potato genotypes were similar. Growing plants on the medium with sucrose dramatically increased the amplitude of absorbance change at 830 nm in the transformed (but not in wild type) plants, which indicated a drastic increase in P700 concentration in their leaves.     

Fatty acid hydroperoxides biotransformation by potato tuber cell-free extracts

Cleavage of 13-HPOD, 13-HPOT, 9-HPOD and 9-HPOT by potato tuber cell-free extracts was investigated. 13-HPOD and 13-HPOT enzymes were degraded almost completely while 9-HPOD and 9-HPOT were partially transformed. GC-MS analysis of the volatile compounds formed during the reactions revealed that (Z)-3 hexenal, (E)-2-hexenal, pentenols and dimers of pentene were obtained from 13-HPOT while from 13-HPOD hexanal and pentan-1-ol were formed. No volatile was found when 9-HPO isomers were used as substrate, but colneleic acid was produced. When Triton X-100 was omitted in the extraction buffer, only pentenols and dimers of pentene were identified from 13-HPOT and pentan-1-ol from 13-HPOD. Our results reveal that potato tubers that contain Lox, which forms mainly 9-HPO, are able to metabolise the four HPO isomers. Moreover, 13-HPO cleaving activities are due to two distinct enzymatic systems based on, respectively, homolytic and heterolytic mechanisms. The fact that oxygenation of reaction medium dramatically decreases the amount of product resulting from homolytic cleavage strengthens the hypothesis of an anaerobic reaction due to Lox.     

Growth pattern, tuber formation and hormonal balance in in vitro potato plants carrying ipt gene

Nodal cuttings of in vitro grown potato plants (Solanum tuberosum, cv. Miranda) were transformed by a vector plasmid carrying ipt gene of Agrobacterium tumefaciens. From the initial teratoma stage 5 clones of transgenic plants (1, 2, 11, 13 and 15) were obtained, which displayed in varying degree shortening of the internodes, decrease of the leaf size, decrease of apical dominance and poor rooting. In addition, two of the clones (11 and 13) showed increased stolon and tuber formation. In all these clones the endogenous level of free cytokinins (CKs) was increased: from 40% in clone 11 to almost 300% in clone 1. Also free indole-3-acetic acid (IAA) level was increased, but to a lower degree; the maximal increase was 160% (clone 13). Applied kinetin or IAA (1 mg·l^-1) strongly suppressed root and tuber formation in clones 11 and 13, although they did not affect or even stimulated these processes in control plants. For control plants the minimal medium sucrose concentration necessary for tuber initiation was 6% whereas in clone 11 plants 2% was sufficient. Different distribution of endogenous CKs and IAA was observed in clone 11 and control plants. The highest CK content was found in transgenic plants in stems and in controls in leaves. In clone 11 plants abscisic acid (ABA) level was significantly increased in comparison to the control throughout the cultivation period. Ethylene formation was strongly increased the first week after the subcultivation and later on the difference between transgenic and control plants rapidly diminished. Reactions of clone 11 plants to red (RL) and blue light (BL) were similar to reactions of control plants. In RL clone 11 plants were tall and thin with stunted leaves; in BL they had a teratoma-like appearance and formed a very high number of tubers. The role of hormones in these changes in growth and tuber formation is discussed.