The amylolytic activity of Ca-deficient pumpkin plants (Cucurbita pepo L.)

We studied the significance of actual accessibility of ions for presence of α-amylase in roots and cotyledons of young pumpkin plants. We considered α-amylases to be—in agreement with published data—amylolytic enzymes which lose their activity after being dialysed against EDTA or a dilute solution of hydrochloric acid and which can be reactivated by addition of Ca²⁺. N connection with activity and Ca²⁺ nutrition appeared in cotyledons; it did, however, in roots: Ca-deficient plants revealed after preparation either completely inactive enzymes or enzymes with slight activity. It was, however, possible to reactivate them by addition of Ca²⁺ during preparation. So, using polyacrylamide disc electrophoresis we present evidence for the appearence of amylase with typical characteristics for α-amylases of other sources. Its relative mobility was the same for isolates from both roots and cotyledons. Its biosynthesis was not dependent upon metal which is associated with enzyme. No conclusion can be drawn as to whether Ca-deficient α-amylases are activein vivo; therefore it was impossible to judge whether α-amylase activity is subject to feed-back regulation.     

Inheritance of parthenocarpy in summer squash (Cucurbita pepo L.)

The inheritance of the tendency to set parthenocarpic fruit in the summer squash (Cucurbita pepo L.) line Whitaker was studied. Two parental lines, Whitaker (parthenocarpic) and Caserta (non-parthenocarpic), and the F1 and F2 generations and backcrosses to both parents were tested. The parthenocarpic tendency of individual plants was scored on a scale from 1 (non-parthenocarpic fruit) to 5 (parthenocarpic fruit). The Whitaker line produced parthenocarpic fruit and had a mean score of 4.2, whereas Caserta did not set parthenocarpic fruit and had a score of 1.55. The heritability estimates indicated that genetic gains from selection were feasible. The additive-dominant model showed a good fit, with epistasis being negligible or nonexistent. The hypothesis of monogenic inheritance with incomplete dominance was not rejected within the degree of dominance range from 0.2 to 0.5. These results indicate that parthenocarpy is controlled by a single locus, with incomplete dominance in the direction of parthenocarpic expression.     

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.)

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall “loosening.”     

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH₄⁺) as the sole source of nitrogen. Growth of NH₄⁺-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH₄⁺ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH₄⁺ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH₄⁺ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH₄⁺ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.     

Peroxidases of different parts of the pumpkin plant (Cucurbita pepo L.)

We compared the occurrence of peroxidase isozymes in protein extract from roots, hypocotyls and cotyledons of 10 dayCucurbita pepo plants and of adult leaves of older plants by means of starch gel and polyacryl amide gel electrophoresis. We reached maximum discrimination by means of starch gel electrophoresis: 11 zones were ascertained on the cathode side and about 2 on the anode side at pH 3.1. Two zones occurred regularly:A and (the latter having a more complicated structure). ZoneD is characteristic for roots, but is it suppressed and seldom found with leaves. On the other hand zonesC ₁ andC ₂ are clearly discernible with leaves but are substantially less evident with roots. The character of anodic zoneZ is discussed later in this paper.     

Influence of a variation potential on photosynthesis in pumpkin seedlings (Cucurbita pepo L.)

The influence of a variation potential on photosynthesis in pumpkin seedlings (Cucurbita pepo L.) was investigated in our work. It was shown that the variation potential induced by cotyledon burning propagates into a leaf. It decreases CO₂ assimilation and transpiration as well as increases nonphotochemical quenching. Investigation of isolated chloroplasts showed that lowering of the pH in incubation medium from 6.9–7.2 to 6.5 increases nonphotochemical quenching. It was proposed that lowering of the cytoplasmic pH induced by the variation potential takes place in the photosynthetic response development.     

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

The influence of plant growth regulators on callus induction in pumpkin (Cucurbita pepo L.) hairy roots

Following in vitro infection with Agrobacterium rhizogenes wild strain (mannopine, 8196) and two A. tumefaciens transconjugant strains (C58C1 pArA4abc and C58C1 pArA4b) transformed (hairy) roots were induced in pumpkin (C. pepo L.) cotyledons. The presence of pRi T-DNA in pumpkin long-term hairy root cultures was determined by Southern hybridization. The influence of plant growth regulators on callus induction in root explants from hairy root lines, which differed mutually in morphology and growth rate, was tested by the addition of growth regulators to basal nutrient medium; while 2.4-D inhibited root proliferation in all hairy root lines tested, callus induction depended both on plant growth regulators and the root line.     

Pumpkin (Cucurbita sp.) seed globulin II. Alterations during germination

Alterations in pumpkin seed globulin during germination wereexamined mainly by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The total protein content of etiolated cotyledonsdecreased to 32% of the initial content 14 days after germination,and the globulin was rapidly degraded to produce new componentswith molecular weights of about 40,000 (major component) and30,000 daltons, which were soluble in a solution with relativelylow concentrations of salt at neutral pHs in contrast to insolublecrystalline globulin. The proportion of these soluble proteinsto the total amount of protein extracted in 2 M NaCl solutionincreased rapidly during the period of 2 to 4 days after planting.During this period, the globulin was rapidly degraded with progressiveincrease of the component of 40,000 daltons. This species wasseparated into two polypeptide chains with molecular weightof about 20,000 daltons by treatment with sulfhydryl reducingreagent. The soluble fraction preserved oxaloacetate decarboxylaseactivity. These changes in solubility and subunit structure,and the preservation of the decarboxylase activity of heat stableproteins obtained from etiolated cotyledons are discussed. (Received April 9, 1976; )     

Virus-induced changes in the subcellular distribution of glutathione precursors in Cucurbita pepo (L.)

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.     

Sulfite Inhibition of Photosystem II in Illuminated Spinach Leaves

PS II activity (dichlorophenolindophenol photoreduction) inchloroplasts isolated from sulfite-treated spinach leaves inlight was inhibited but not in darkness. Sulfite treatment decreasedthe variable part of fluorescence induction and the fluorescenceintensities of emissions at 685 and 694 nm at 77K, but it hadno effect when sulfite was administered together with DCMU.These results indicate that sulfite inactivates the PS II reactioncenter when electron transport takes place. (Received August 5, 1983; Accepted November 25, 1983)     

Changes in the subcellular distribution of glutathione during virus infection in Cucurbita pepo (L.)

Changes in the subcellular distribution and quantification of glutathione were studied with electron microscopic immunogold cytochemistry in Zucchini yellow mosaic virus (ZYMV)-infected Styrian pumpkin plants (Cucurbita pepo L. ssp. pepo var. styriaca Greb.) two weeks after inoculation. The amount of gold particles bound to glutathione was statistically evaluated for different cell structures, including mitochondria, plastids, nuclei, peroxisomes, and cytosol. In general, ZYMV-infected plants showed higher gold labelling density in intact mesophyll cells of the 5th (older leaves) and the youngest fully developed leaves (younger leaves), and decreased levels of glutathione within root tip cells when compared to the control. In general, within older and younger leaves the highest amount of gold particles was found in mitochondria and the lowest amount in plastids. In ZYMV-infected older leaves, an increase in glutathione was found in peroxisomes (1.7-fold), the cytosol (1.6-fold), mitochondria (1.4-fold), and nuclei (1.2-fold), whereas glutathione levels in plastids did not differ significantly when compared to control cells. In ZYMV-infected younger leaves elevated glutathione contents were found in the cytosol (3-fold), nuclei (2.1-fold), peroxisomes (1.8-fold), and plastids (1.5-fold), whereas mitochondria showed an insignificant decrease in glutathione levels in comparison to the control. In root tip cells of ZYMV-infected plants the amount of gold particles bound to glutathione was decreased in all investigated cell structures by between 0.7- to 0.8-fold. Additionally, total glutathione contents were determined in older and younger leaves using high-performance liquid chromatography (HPLC), which revealed no significant differences between control and ZYMV-infected leaves. The relevance of the results of both methods were compared and are discussed.     

Developmental instability: measures of resistance and resilience using pumpkin (Cucurbita pepo L.)

Fluctuating asymmetry measures random deviations from bilateral symmetry, and thus estimates developmental instability, the loss of ability by an organism to regulate its development. There have been few rigorous tests of this proposition. Regulation of bilateral symmetry must involve either feedback between the sides or independent regulation toward a symmetric set point. Either kind of regulation should decrease asymmetry over time, but only right–left feedback produces compensatory growth across sides, seen as antipersistent growth following perturbation. Here, we describe the developmental trajectories of perturbed and unperturbed leaves of pumpkin, Cucurbita pepo L., grown at three densities. Covering one side of a leaf with aluminium foil for 24 h perturbed leaf growth. Reduced growth on the perturbed side caused leaves to become more asymmetrical than unperturbed controls. After the treatment the size-corrected asymmetry decreased over time. In addition, rescaled range analysis showed that asymmetry was antipersistent rather than random, i.e. fluctuation in one direction was likely to be followed by fluctuations in the opposite direction. Development involves right–left feedback. This feedback reduced size-corrected asymmetry over time most strongly in the lowest density treatment suggesting that developmental instability results from a lack of resilience rather than resistance.   © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78, 27–41.     

Comparison of effects of calcium deficiency and IAA on the pumpkin plant (Cucurbita pepo L.)

In pumpkin plants suffering from calcium deficiency growth of the root system is characteristically suppressed. A biochemical symptom of calcium deficiency is a decrease or even complete disappearance of activity of one of the isoperoxidases (isoperoxidaseD). In the present study the two characteristics were used to compare the effects of calcium deficiency and of IAA in the supraoptimal concentration of 10^?5 M. Peroxidases were separated by centrifugal fractionation: peroxidasesA andB were found in the sediment corresponding to the mitochondrial fraction, peroxidaseD in the supernatant. It was not possible, however, to obtain the sediment completely free of peroxidaseD. A negative correlation between the activity of isoperoxidaseD and the logarithm of concentration of IAA in nutritive solution was found and similarities in the effects of IAA in supraoptimal concentration and of calcium deficiency on the development of root system were demonstrated. Still, it would be difficult to imagine that the mechanism of the action of the two factors is the same. For this reason, a scheme of possible interactions was suggested with the aim to explain how similar changes in the content of isoperoxidases are brought about by the two factors. In this scheme, calcium is considered chiefly as a substance regulating membrane permeability.     

Nectar resorption and translocation in Cucurbita pepo L. and Platanthera chlorantha Custer (Rchb.)

Nectar resorption and sugar translocation were studied in Cucurbita pepo (Cucurbitaceae) and Platanthera chlorantha (Orchidaceae) by micro-autoradiography. In both species, nectar was resorbed in pollinated and unpollinated flowers and ovules developing into seeds were found to be the main sugar sink. In C. pepo, the mobility of resorbed sugars in pollinated female flowers was higher than in unpollinated ones; male flowers showed lower mobility of resorbed sugar. In P. chlorantha, radioactivity was detected in pollinated flowers below and above labelled unpollinated ones: the nearer the flower, the stronger the accumulation of label in developing fruits.     

籽用美洲南瓜EMS诱变突变体库的构建及表型分析

【目的】通过构建籽用美洲南瓜突变体库,从而加快籽用美洲南瓜种质资源创新进程,突变体库的构建对品种选育、品种改良以及遗传基础的拓宽具有重要的意义。【方法】利用1.8%诱变剂甲基磺酸乙酯（EMS）处理籽用美洲南瓜ZHL4种子15h,然后对M1和M2代群体单株进行表型变异观察,同时对M2群体变异株系ZHL4-33进行显微组织结构观察。【结果】（1）M2群体中共筛选到242个突变植株,45种表型变异,变异类型涵盖了突变株的各个生长时期以及各植物器官,总的突变频率达到了25.17%。（2）叶片显微结构显示,突变体栅栏组织厚度显著高于野生型,排列紧凑,维管形成层痕迹明显;茎显微结构显示,突变体维管束多而密集,导管直径小于野生型,髓部发达,细胞间隙较小,细胞数目有所增加。【结论】研究初步构建了由425个M2家系所组成的籽用美洲南瓜突变体库,为籽用美洲南瓜功能基因组研究和新品种选育奠定了材料基础。     

光周期对西葫芦185品系顶芽和叶片衰老的调控

在短日照下 ,西葫芦 (CucurbitapepoLinn .) 185品系的植株发生衰老。结构学、基因表达与系列生化分析证实 :短日照启动了顶端分生组织由营养生长锥向花芽的转化 ,进而其组成细胞发生编程性死亡 (PCD) ,导致顶端生长势的丧失 ;与长日照处理相比 ,短日照处理在发育晚期也引起大量叶肉细胞发生PCD ,进而叶片出现衰老。核酸酶活性的高度表达是PCD过程中一个非常重要的分子事件。实验证实 ,西葫芦 185品系植株衰老进程的发生与顶端分生组织和叶肉细胞中发生PCD密切相关。     

Qualitative differences of pumpkin (Cucurbita pepo L.) proteins influenced by Ca2+ nutrition

The effect of Ca-deficiency on changes in the protein content of cotyledons and roots of germinating pumpkin (Cucurbita pepo L.) plants was studied by means of immunoelectrophoresis. Antiserum against seed proteins gave 11 clear precipitation zones in homological reactions and 7 zones with proteins from cotyledons of 10 day-old plants. Several less mobile zones in the area of start appeared in variant “?Ca”. Using antiserum against root proteins of 10 day-old control plants, 8 precipitation zones in the homological reaction and 6 zones (two cathodic zones are missing) in the heterological reaction were obtained. The patterns of proteins from the root and those arising from the protein of seed are both changed under Ca-deficiency.     

Structure of Protochlorophyll-containing Plastids in the Inner Seed Coat of Pumpkin Seeds (Cucurbita pepo)

The inner seed coat of seeds of Cucurbita pepo L. cv. Ohlsens Enke Köks was used to study the development of protochlorophyll-containing plastids with an abnormal ultrastructural composition. The pumpkins were harvested at different stages during fruit development and they thus contained seeds with different developmental stages. The dry weight of seeds of the developmental stages used varied from 0.04 g to 0.3 g. Such a series of seeds with decreasing water content indicating increasing maturity contained different amounts of protochlorophyll, from 0.20 μg/g fresh weight to 500 μg/g fresh weight. The ultrastructure of the protochlorophyll containing plastids changed greatly during development. In young seeds with a low content of protochlorophyll, regular prolamellar bodies were found and starch grains filled most of the plastids. During development the starch content decreased and the prolamellar bodies increased in size and lost their regularity. During maturation the plastids accumulated plastoglobuli, probably containing protochlorophyll, and finally the internal structure of the prolamellar body tubular complex was lost.     

Carbohydrate metabolism before and after dehiscence in the recalcitrant pollen of pumpkin (Cucurbita pepo L.)

Pumpkin (Cucurbita pepo L.) pollen is starchy, sucrose‐poor and recalcitrant, features opposite to those of several model species; therefore, some differences in carbohydrate metabolism could be expected in this species. By studying pumpkin recalcitrant pollen, the objective was to provide new biochemical evidence to improve understanding of how carbohydrate metabolism might be involved in pollen functioning in advanced stages. Four stages were analysed: immature pollen from 1 day before anthesis, mature pollen, mature pollen exposed to the environment for 7 h, and pollen rehydrated in a culture medium. Pollen viability, water and carbohydrate content and activity of enzymes involved in carbohydrate metabolism were quantified in each stage. Pollen viability and water content dropped quickly after dehiscence, as expected. The slight changes in carbohydrate concentration and enzyme activity during pollen maturation contrast with major changes recorded with ageing and rehydration. Pumpkin pollen seems highly active and closely related to its surrounding environment in all the stages analysed; the latter is particularly evident among insoluble sucrolytic enzymes, mainly wall‐bound acid invertase, which would be the most relevant for sucrose cleavage. Each stage was characterised by a particular metabolic/enzymatic profile; some particular features, such as the minor changes during maturation, fast sucrolysis upon rehydration or sharp decrease in insoluble sucrolytic activity with ageing seem to be related to the lack of dormancy and recalcitrant nature of pumpkin pollen.