Inhibition of Water Melon (Citrullus vulgaris) Urease by Fluoride

Effect of fluoride on the activity of purified urease from seeds of watermelon (Citrullus vulgaris) was studied. Fluoride exhibited a concentration dependent inhibition both in presenceand absence ofthe substrate. The inhibition was non-competitive. Addition of 8mM β-mercaptoethanol gradually abolished the fluoride inhibition. β-mnercaptoethanol, in presence of fluoride, also exhibited a concentration dependent suppression of inhibition caused by fluoride. The significance of these observations is discussed.     

Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

Inhibition of Urease from Seeds of Water-melon (Citrullus vulgaris) by Heavy Metal Ions

Urease from the seeds of water melon was found to be inhibited by heavy metal ions like copper, lead, nickel and cobalt. The order of effectiveness of these metals as inhibitors was Cu²⁺ > Pb²⁺ > Ni²⁺ > Co²⁺. The inhibition by these ions was noncompetitive. Time — dependent interaction of urease with nickel and cobalt exhibited a biphasic inhibition behaviour in which approximately half of the initial activity was lost rapidly (within 2 min) and remainder in a slow phase. The inhibition was largely irreversible, hence could not be reversed by dialysis. These observations are suggestive of half-and-half distribution of — SH groups on the native enzyme resulting urease into asymmetric oligomeric molecule.     

Molecular Asymmetry in Pigeonpea Urease: pH Inactivation Studies

Pigeonpea (Cajanus cajan) urease was inactivated by incubating it in buffer of low pHs i.e., 4.8 and 4.5. The pattern of inactivation at both pHs was found to be biphasic, in which half of the activity was destroyed more rapidly than the remaining half. This distribution of active site into two categories is suggestive of site-site heterogeneity, or more specifically, the half-site reactivity of the enzyme moiety. Our pH studies on the rate of reaction showed the presence of two ionizable groups of pK _a values 6.2 ± 0.1 and 8.8 ± 0.1, respectively (Srivastava PK & Kayastha AM, J Mol Catal B: Enz, 16 (2001) 81-89). The later group corresponds to the pK _a value of cysteine group. Here we correlate the loss of urease activity by low pH treatment is due to the effect on essential thiol residues.     

Volatile Flavor Components of Watermelon (Citrullus vulgaris)

Volatile flavor components of watermelon fruit (Citrullus vulgaris) were obtained by distilling juice under reduced pressure and extracting the resulting distillate with Freon-11. The volatiles were separated into acidic and neutral fractions and then analyzed by gas chromatography and gas chromatography-mass spectrometry.

Fifty-two compounds were found for the first time as flavor components of watermelon. Among them, the following compounds have not been previously reported as naturally occurring flavor components: 4-oxononanal and 2-hydroxy-, 2-pentyloxy-, 2-hexyloxy-, 2-octyloxy-, 2-nonyloxy-, 2-[(Z)-3-noneyloxy]-, 2-[(Z)-2-nonenyloxy]-, 2-[(Z,Z)-3,6-nonadienyloxy]-, 2-benzyl-oxy-, and 2-phenetyloxy-5-pentyltetrahydrofurans.     

The Function of Urease in Citrullus Seeds

Urease is present in considerable quantity in the cotyledonsof Citrullus, though elsewhere in the plant it is present onlyin traces or is absent; urease activity in the cotyledons changesduring growth, showing an initial rise followed by an abruptdrop almost to zero. These changes, under a wide variety ofconditions, are not correlated with those in the major nitrogenfractions; they are, however, closely correlated with cell extensionand the associated changes in water content and respiration.A connexion with chlorophyll formation is possible but unlikely.It is suggested that the changes in cotyledonary urease constitutemerely one aspect of the ‘protoplasmic differentiation’that takes place as a cell matures.     

西瓜甜瓜质量性状的分子标记与定位研究进展

分子标记在作物育种、资源遗传多样性分析、遗传图谱构建等方面有着广泛的应用,常见的分子标记方法主要有RFLP、RAPD、SSR、AFLP、SCAR、CAPS、SNP等.迄今为止,国内外学者已经利用分子标记技术对西瓜和甜瓜的20多个质量性状进行了标记和定位.本文主要对西瓜和甜瓜质量性状的分子标记和定位研究进展进行了综述,发现RAPD标记使用最多,而更具优势的SSR标记使用较少;对抗病虫等育种相关的基因研究较多,且多数标记遗传距离偏大,达不到图位克隆的要求,而对西瓜和甜瓜遗传基础相关的基因研究较少.     

Fat Metabolism in Germinating Citrullus vulgaris

A column chromatographic technique, enabling identificationand quantitative estimation of fatty acids, has been employedto study fat metabolism in Citrullus during germination in thelight. This plant is characterized by an unusually rapid disappearanceof storage fat as the cotyledons expand and turn green. In spiteof the high catabolic activity there is no evidence for accumulationof free fatty acids or short-chain fatty acids at this stage.Information on this point derived from acid value or saponificationvalue of the oil is shown to be untrustworthy. Citrullus seed fat contains the following percentages of acids:linoleic 70·6, oleic 7·2, palmitic 10·1,stearic 11·2, and arachidic 0·6, and careful analysishas also revealed small amounts of octadecatrienoic acids, bothconjugated and non-conjugated. All the major acids are brokendown at rates proportionate to the quantities originally present,with the exception of oleic acid which is metabolized somewhatmore rapidly. ‘Linolenic’ acid is synthesized in the expandinggreen cotyledons and the fatty acid composition of the latter,in the late germination stages, resembles that of a green leafand is very different from that of the seed. The results suggest a rapid removal of storage fat from thecotyledons and concomitant formation in small quantity of atypial leaf fat as the new photo-synthetic function develops.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

The Effect of Dew on Plant Water Balance in Citrullus vulgaris and Cucumis melo

A temperature gradient air-leaf can be created purely by radiation loss with a sky-radiator. Loss of radiation from leaf to an artificial sky causes water vapor condensation from a saturated atmosphere as dew on the leaves of plants. The condensed water was found to be taken up by the leaves of melon (Cucumis melo) and water melon (Citrullus vulgaris) bringing about a favorable water balance by reduction in the water saturation deficit and by increase of the water potential. In a number of individual experiments with matched plants a minimum 31% and maximum 117% increase of leaf fresh weight due to uptake of condensed water was noted.     

Kinetics of Thermal Inactivation and Molecular Asymmetry of Urease from Dehusked Pigeongea (Cajanus cajan L.) Seeds

The purified urease from pigeonpea was moderately stable at ?10°C. The shelf-life of the enzyme on storage in 0.1 M Tris-acetate buffer, pH 6.5, at ?10°C showed a single exponential decay with a t_1/2 of approx. 30 days. In the presence of additives like 5mM dithiothreitol the t_1/2 increased to 223 days at the same temperature, in a single exponential decay. The Arrhenius plot of the kinetics of the pigeonpea urease catalysed urea hydrolysis over the temperature range 27 to 77°C, was linear. The Q₁₀ value was found to be 1.46. The energy of activation calculated from the Arrhenius equation was found to be 5.1 kcal/mole active site. The thermal denaturation of pigeopea urease at 65 and 70°C was found to obey biphasic kinetics in which half of the activity is destroyed faster than the remaining half. The time course of thermal inactivation can be described by the following equation, consisting of two first order terms: A_t = A_fast.e^-k _fast + A_slow.e ^-kslow^.t where, A_t is the residual activity at time t, A_fast and A_slow, k_fast and k_slow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. The data suggests the existence of site-site heterogeneity in oligomeric urease molecule from pigeonpea.     

杉木－籽西瓜经营模式研究

对杉木－籽西瓜经营模式的研究表明,杉木连栽林地采用籽西瓜套种经营模式,可提高土壤肥力,防止林地水土流失,促进杉木幼林生长,套种林分的生产力平均比对照高１４１．８５％．材积生产力高１９１．２２％,实现了林地的持续生产力和幼林的速生丰产．     

Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys− auxotroph

We have isolated cDNA clones encoding cysteine synthase (CSase, EC 4.2.99.8), which catalyzes the terminal step in cysteine biosynthesis, by direct genetic complementation of a Cys^? mutation in Escherichia coli with an expression library of Citrullus vulgaris (watermelon) cDNA. The library was constructed from 8-day-old etiolated seedlings of C. vulgaris in the λZAPII vector, converted to a plasmid library by in vivo excision, and then used for transformation of cysteine auxotroph E. coli NK3, which lacks the cysK and cysM loci. The complementing cDNA containing a 560 by 5′-untranslated region encodes a polypeptide of 325 amino acids of M_r 34342. The translational product reacted with an antibody raised against CSase A of Spinacia oleracea. CSase and β-pyrazolealanine synthase activities were demonstrated in vitro in extracts from E. coli cells expressing the cDNA. Genomic DNA blot analysis indicated the presence of a single copy of the gene, designated cysA, in the C. vulgaris genome. RNA blot hybridization indicated constitutive expression of cysA in cotyledons, hypocotyls and radicles of green and etiolated seedlings. These data suggested that this cDNA clone encodes CSase A the homolog of which in spinach is localized in the cytoplasm. The molecular phylogenetic tree of the amino acid sequences of CSaes from plants and bacteria suggested that there are three families in the CSase superfamily; the plant CSase A family, the plant CSase B family and the bacterial CSase family. The proteins in the plant CSase A family are the most conserved relative to the ancestral CSase protein.     

Fat Metabolism in Germinating Citrullus vulgaris. Part II

Breakdown of fat in watermelon seedlings germinated in the darkoccurs in essentially the same way as in the light. Free fattyacids do not accumulate and the composition of the lipid remainsalmost unchanged during its rapid utilization. The low weight of ‘protoplasmic lipid’ remainingin the etiolated cotyledons after the storage fat has disappearedcontains a lower proportion of linolenic acid than that fromgreen cotyledons of a corresponding age. The weight of phosphatide (lipid P) per cotyledon increases both in light- anddark-grown seedlings until 8 days, thereafter declining in thedark but continuing to rise in the light. Phosphatide and glyceridefractions from the same fat resemble each other fairly closelyin fatty acid composition.     

Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad)

A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases.     

Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski)

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.Abbreviations BAP 6 Benzylaminopurine - NAA -Naphthalene acetic acid - MS Murashige and Skoog's medium - IAA Indole acetic acid - KN Kinetin