Increased Chorismate Mutase Levels as a Response to Wounding in Solanum tuberosum L. Tubers

Discs excised from Solanum tuberosum L. cv White Rose tubers demonstrated a 4.5-fold increase in chorismate mutase activity 48 hours after excision. Incubation in the presence of cycloheximide (25 micromolar) or actinomycin D (100 micromolar) completely inhibited the wound response suggesting de novo synthesis of chorismate mutase. Ratios of activity in the presence of the activator tryptophan to that in the absence of tryptophan remained relatively constant during the induction period. This indicated either a constant ratio of tryptophan sensitive to tryptophan insensitive isozymes, or that only one form of chorismate mutase was present. Chromatography of crude extracts on three different columns yielded only one peak of chorismate mutase activity, activated by tryptophan in each case. Incubation under white light had no effect on chorismate mutase activity when compared to dark controls.     

Purification and Properties of Fructokinase from Developing Tubers of Potato (Solanum tuberosum L.)

Fructokinase has been purified from developing potato (Solanum tuberosum L.) tubers by a combination of hydrophobic interaction, affinity chromatography, and gel filtration. The protein has a native molecular mass of approximately 70 kD but is apparently a dimer. Ion-exchange chromatography and two-dimensional western blots resolved three major fructokinases, designated FK-I, FK-II, and FK-III in order of their elution from a Mono-Q column. Fructokinase activity proved labile when proteins were purified in the absence of fructose. Kinetically, FKs I, II, and III all have broad pH optima with peaks at about pH 8.5. The enzymes have a high specificity for fructose (K_m values ranging from 0.041 to 0.128 mm), and can utilize a range of nucleoside triphosphates. Unlike FKs I and II, FK-III is not inhibited by fructose concentrations in excess of 1 mm. MgADP inhibited activity of the three FKs (between 68 and 75% inhibition at 1.0 mm), whereas fructose 6-P caused inhibition at concentrations of 10 mm. There were no regulatory effects observed with a range of other metabolites. K⁺ (10 mm) activated FK-I by 4-fold and FKs II and III by only about 50%.     

Factors Affecting the Formation of In vitro Tubers of Potato (Solanum tuberosum L.)

In vitro (mini) tubers were induced within 6–8 weeks inserially propagated potato shoot cultures by subculturing tomedium containing 2.0 mg 1^–1 benzylaminopurine (BAP) and6 per cent sucrose in 8- and 24-h days. The effect of BAP inpromoting tubering was greater in short than in long days. Inshort days most of the tubers were formed above the agar, inlong days within the agar. Tubering was promoted less effectivelyby the addition of (2-chloroethyl)-trimethylammonium chloride(CCC) to the medium, but CCC reinforced the effect of BAP leadingto earlier tubering above the agar. Tubering eventually tookplace after 4—5 months on medium without hormones, soonerin short than in long days. Periods of short days and low temperaturesgiven to long-day cultures did not accelerate tubering. Abscisicacid had little effect on, and GA2 strongly inhibited, tubering.Tubering was also inhibited by sealing the culture vessels butnot if ethylene-absorbing agents were included. Solanum tuberosum L, potato, tissue culture, tubers, cytokinin, ethylene, daylength, propagation     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

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     

Allosteric Properties of Chorismate Mutase from Quercus pedunculata

Chorismate mutase from Quercus pedunculata Ehrh. leaves has been purified by ammonium sulfate precipitation, molecular sieving and hydroxyapatite chromatography. Some results obtained during the purification suggest the presence, in oak, of two isofunctional forms of the enzyme, the one sensitive, the other insensitive to the action of aromatic amino acids. The regulable form exhibits a molecular weight of about 45,000. It is inhibited by tyrosine and by phenylalanine and is activated by tryptophan. In addition to its activating properties, this latter compound, endowed with a great affinity for the enzyme, reverses the inhibition due to the two other amino acids. The H⁺ concentration of the medium plays an important role in the sensitivity of the enzyme with regard to its effectors. Inhibition by tyrosine and by phenylalanine is maximal at pH 6.5, at which value the two ligands present an identical effect. At alkaline pH values, the rate of inhibition decreases regularly, tyrosine becoming the most effective inhibitor. Activation by tryptophan is particularly acute in a mildly acid medium; at pH 6.5, this effector increases the enzymatic activity threefold. Its action is weak (20% activation) in the optimum pH zone (pH 7.8) and increases towards the more basic pH values. As a result, tryptophan maintains a constant level of enzymatic activity throughout a large pH zone (between 6.5 and pH 9.0). The physiological significance of these results is discussed.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Cytokinin Activity in Stolons and Tubers of Solanum tuberosum during the Period of Tuberization

In water-culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), changes in cytokinin activity in the stolon tips and newly formed tubers during tuberization were studied. Tuberization was induced by withdrawing nitrogen from the nutrient solution. — The cytokinin activity was low in the stolon tips prior to tuberization, but increased considerably in both stolon tips and young tubers during tuberization. At the same time qualitative changes in the cytokinin spectrum occurred. These qualitative changes are reversible if ‘regrowth’ of young tubers is brought about by a sudden high supply of nitrogen. — Despite the close correlation between tuberization and cytokinin activity, it is assumed that cytokinins are not directly responsible for the onset of tuberization, although they play an important role in tuber growth.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Effects of Gamma-Irradiation on the Activity of Tonoplast H+-ATPase from Potato Tubers (Solanum tuberosum L.)

Tonoplast vesicles were prepared from potato tubers (Solariumtuberosum L.) on a step gradient (0% and 6%, w/w) of dextranT-70 to clarify the mechanism by which the tonoplast H⁺-ATPaseis inactivated by gamma-irradiation. H⁺-ATPase activity andH⁺ -pumping were examined after irradiation of tubers (in vivoirradiation) and of isolated tonoplast vesicles (in vitro irradiation)at doses up to 1.0 kGy. Both in vivo irradiation and in vitroirradiation resulted in significant decreases in ATPase andH⁺-pumping activities. The ATPase and H⁺-pumping activities12 h after irradiation were much lower than those 2 h afterirradiation. Solubilized H⁺-ATPase was inactivated, in a dose-dependentmanner, by irradiation (enzyme irradiation) to a greater extentthan was observed after in vitro irradiation or in vivo irradiation.The activity of ATPase 12 h after enzyme irradiation was almostthe same as it was 2 h after enzyme irradiation. The free fattyacid content of vacuolar membranes was increased by in vivoirradiation and by in vitro irradiation with an accompanyingdecrease in tonoplast H⁺-ATPase activity. Lipids from irradiatedtonoplasts had a considerable inhibitory effect on the activityof solubilized H⁺-ATPase. This result suggests that the directinactivation of H⁺-ATPase in potato tonoplast by gamma-irradiationis augmented by the effects of deterioration of membrane lipidsthat is induced by the irradiation. (Received December 21, 1994; Accepted May 16, 1994)     

Microtuberization in potato (Solanum tuberosum L.)

Twenty-two genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under six in vitro culture conditions. Cultures maintained under a short photoperiod (10 h of 6–12 μmol m^–2 s^–1) and low temperatures (day 20°±2°C and night 18°±2°C) had both a higher yield (255 mg/plantlet) and a greater number (2/plantlet) of microtubers than those maintained under long days (16 h of 38–50 μmol m^–2 s^–1) combined with high temperatures (day 28°±2°C and night 25°±2°C) (yield 207 mg/plantlet; microtuber number, 0.9/plantlet), over a wide range of genotypes. After the plantlets had been cultured under long days for an initial period of 60 days, continuous darkness advanced microtuberization by 2–3 months in various genotypes. Under short-day and low-temperature conditions the addition of 6-benzylaminopurine increased microtuber yield from 255 mg/plantlet to 645 mg/plantlet and average microtuber weight from 115 mg to 364 mg. A similar pattern was observed under conditions of long days and high temperature, and continuous darkness and low-temperature. Microtubers produced under light had a greater number of eyes (maximum average: 5.96/microtuber) than those produced in the dark (maximum average: 3.50/plantlet). The genotype × cultural conditions interactions were significant indicating the importance of developing genotype-specific protocols to maximize microtuberization. Received: 17 September 1997 / Revision received: 12 December 1997 / Accepted: 1 January 1998     

Energy Metabolism in Rhizomes of Acorus calamus (L.) and in Tubers of Solanum tuberosum (L.) With Regard to their Anoxia Tolerance

Rhizomes of the marsh plant Acorus calamus (L.) and tubers of the flooding-intolerant Solanum tuberosum (L.) var. Bintje, both kept under strict anoxia, differ markedly in their fermentation properties. The fermentation capacities as measured by ADH and LDH activities and their respective product concentrations were estimated. While rhizomes of Acorus calamus, having high ADH and low LDH activities, accumulate mainly ethanol, tubers of Solanum tuberosum tend towards lactic acid fermentation. The total amount of adenine nucleotides is quite stable in Acorus calamus, whereas they show a sharp decline in S. tuberosum during the first 6h of anoxia. The adenylate energy charge of A. calamus recovers after a short initial drop (AEC > 0.8). AEC values of S. tuberosum decrease rapidly and remain at very low values (AEC ～ 0.3). Tuber tissues became soft and lost viability after about 48–72 h of anoxia at 25 °C. This might be due to tissue acidification and impaired energy metabolism, but not to the lack of energy reserves. Energy metabolism of A. calamus is well adapted to anoxia.     

Sucrose Metabolism in Tubers of Potato (Solanum tuberosum L.): Effects of Sink Removal and Sucrose Flux on Sucrose-Degrading Enzymes

Excision of developing potato (Solanum tuberosum L.) tubers from the mother plant, followed by storage at 10°C, resulted in a rapid, substantial decrease in sucrose synthase activity and considerable increases in hexose content and acid invertase activity. A comparison of the response of three genotypes, known to accumulate different quantities of hexoses in storage, showed that both sucrose synthase activity and the extent to which activity declined following excision were similar in all cases. However, there was significant genotypic variation in the extent to which acid invertase activity developed, with tubers accumulating the highest hexose content also developing the highest extractable activity of invertase. Similar effects were found in nondetached tubers when growing plants were maintained in total darkness for a prolonged period. Furthermore, supplying sucrose to detached tubers through the cut stolon surface prevented the decline in sucrose synthase activity. Maltose proved to be ineffective. Western blots using antibodies raised against maize sucrose synthase showed that the decline in sucrose synthase activity was associated with the loss of protein rather than the effect of endogenous inhibitors. Although there were indications that maintaining a flux of sucrose into isolated tubers could prevent the increase in acid invertase activity, the results were not conclusive.     

The Relation Between the Nitrogen Concentration and Photosynthetic Capacity of Potato (Solanum tuberosum L.) Leaves

Measurements of the rate of light-saturated photosynthesis (P_max)were made on terminal leaflets of potato plants growing in cropssupplied with 0, 3, 6, 12, 24 and 36 g N m^–2. Measurementswere made between 100 and 154 d after planting. Two types ofleaf were selected—the fourth leaf on the second-levelbranch (L₄, _B1) and the youngest terminal leaflet that was measurable(L_YM). Later, the total nitrogen concentration of each leaflet(N_L) was measured. A linear regression between P_max and N_L,common to both leaf positions, explained 68.5% of the totalvariation. With L₄, _B1 leaves there was a significant improvementin the proportion of variation explained when regressions withseparate intercepts and a common slope were fitted to individualfertilizer treatments. These results suggest that an increasingproportion of leaf nitrogen was not associated with the performanceof the photosynthetic system with increasing nitrogen supply.This separation between nitrogen treatments was not as clearfor L_YM leaves. Stomatal conductance to transfer of water vapourwas neither influenced by leaf position nor directly by nitrogensupply. Rather conductance declined in parallel with the declinein photosynthetic capacity. Solanum tuberosum, potato, nitrogen, photosynthesis, stomatal conductance, leaf     

Wound-Induced Metabolism in Potato (Solanum tuberosum) Tubers: Biosynthesis of Aliphatic Domain Monomers

Suberin, a cell specific, wall-associated biopolymer, is formed during normal plant growth and development as well as in response to stress conditions such as wounding. It is characterized by the deposition of both a poly(phenolic) domain (SPPD) in the cell wall and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. Although the monomeric components that comprise the SPPD and SPAD are well known, the biosynthesis and deposition of suberin is poorly understood. Using wound healing potato tubers as a model system, we have tracked the flux of carbon into the aliphatic monomers of the SPAD in a time course fashion. From these analyses, we demonstrate that newly formed fatty acids undergo one of two main metabolic fates during wound-induced suberization: (1) desaturation followed by oxidation to form the 18:1 ω-hydroxy and dioic acids characteristic of potato suberin, and (2) elongation to very long chain fatty acids (C20 to C28), associated with reduction to 1-alkanols, decarboxylation to n-alkanes and minor amounts of hydroxylation. The partitioning of carbon between these two metabolic fates illustrates metabolic regulation during wound healing, and provides insight into the organization of fatty acid metabolism.Key Words: suberin, potato, Solanum tuberosum, carbon flux analysis, abiotic stress     

橡胶树白粉菌分支酸变位酶基因克隆及蛋白表达纯化

由橡胶树白粉菌引起的橡胶树白粉病是橡胶树的重要的叶部病害之一,严重影响橡胶的产量。然而目前对橡胶树白粉菌的致病机理研究匮乏。分支酸变位酶是莽草酸途径的关键酶,能够将分支酸转化为预苯酸,为植物提供氨基酸及大量代谢产物,在植物抗病中起到非常重要的作用。而植物病原物在致病过程当中能够分泌分支酸变位酶影响植物莽草酸途径,从而抑制植物的防卫反应。因此研究橡胶树白粉菌分支酸变位酶在其致病过程中的功能具有一定的意义。试验利用同源比对分析在橡胶树白粉菌基因组中获得一个分支酸变位酶同源蛋白,并用PCR克隆获得橡胶树白粉菌分支酸变位酶基因,命名为OHCmu。后续构建GST-OHCmu融合原核表达载体,筛选最优诱导条件,并利用GST亲和层析柱对蛋白进行纯化。结果表明橡胶树白粉菌OHCmu基因大小843 bp,具有1个内含子,编码263个氨基酸;具有d5csma_结构域,属于Chorismate mutaseⅡ蛋白家族;GST-OHCmu融合蛋白外源诱导表达在供试条件下(IPTG:0.8 mmol/L, 16℃)可以有较好的表达,获得融合蛋白大小约为56 kD。经过GST亲和层析柱纯化、切割GST标签后,顺利获得浓度较高、较纯的橡胶树白粉菌OHCmu蛋白。研究结果为后续OHCmu蛋白的特性及致病机理研究提供参考。     

Temperature-Dependent Modulation of the Photoinhibition-Sensitivity of Photosystem II in Solanum tuberosum Leaves

High temperature (38°C) dramatically exacerbated the photoinhibitionof photosystem II in 23°C-grown potato leaves. This workshows the existence of an adaptive mechanism that cancels thesynergism between heat and strong light and markedly enhancesthe resistance of photosystem II to photodamage. Photoinhibition-resistanceof photosystem II was triggered by exposing leaves in weak lightto moderately elevated, non-injurious temperatures in the range32–38°C and was established within about 20 min, thusproviding a rapid process of photosynthetic adaptation to simultaneousincrease in temperature and light intensity. The reversal at23°C occurred on a much longer time scale of several hours.Temperature-induced increase in PSII photoresistance did notrequire de novo protein synthesis, as judged from its insensitivityto cycloheximide and chloramphenicol, and was not accompaniedwith any apparent change in the antenna size of photosystemII. From the presented chlorophyll fluorescence data, one canpostulate that temperature-dependent regulation of the photoinhibition-sensitivityof photosystem II could primarily involve a change in the PSIIreaction center, the nature of which remains to be established. (Received January 27, 1994; Accepted April 18, 1994)     

Differential expression of invertase genes in internal and external phloem tissues of potato (Solanum tuberosum L.)

The cloning of promoter sequences of two invertase genes from potato (Solanum tuberosum L.) is described. Histochemical analysis of series of reporter transgenic lines reveals phloem-specific expression from both promoters, with one expressed preferentially in internal phloem and the other in external phloem of stem vascular bundles.