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Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits.

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (alpha-subunit) and 65 (beta-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in beta-subunit and twofold increase in alpha-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total beta-subunit to alpha-subunit content. The beta- to alpha-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.

Journal Article↗

Immunocytochemical localization of copper,zinc superoxide dismutase in peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons.

In previous works using cell fractionation methods we demonstrated the presence of a Cu,Zn-containing superoxide dismutase in peroxisomes from watermelon cotyledons. In this work, this intracellular localization was evaluated by using western blot and EM immunocytochemical analysis with a polyclonal antibody against peroxisomal Cu,Zn-SOD II from watermelon cotyledons. In crude extracts from 6-day old cotyledons, analysis by western blot showed two cross-reactivity bands which belonged to the isozymes Cu,Zn-SOD I and Cu,Zn-SOD II. In peroxisomes purified by sucrose density-gradient centrifugation only one cross-reactivity band was found in the peroxisomal matrix which corresponded to the isozyme Cu,Zn-SOD II. When SOD activity was assayed in purified peroxisomes two isozymes were detected, Cu,Zn-SOD II in the matrix, and a Mn-SOD in the membrane fraction which was removed by sodium carbonate washing. EM immunocytochemistry of Cu,Zn-SOD on sections of 6-day old cotyledons, showed that gold label was mainly localized over plastids and also in peroxisomes and the cytosol, whereas mitochondria did not label for Cu,Zn-SOD.

Blotting, Western↗

A study of inhibition of urease from seeds of the water melon (Citrullus vulgaris).

Urease from seeds of the water melon was found to be inhibited by various salts of sodium. Sodium fluoride strongly inhibited the activity in the low urea concentration range. The enzyme was also inhibited by a high concentration of urea which was completely abolished in the presence of 10 mM sodium fluoride. Time-dependent inactivation of urease with iodoacetic acid, N-ethylmaleimide and p-hydroxymercuribenzoate exhibited biphasic kinetics in which half of the initial activity was lost in the fast phase and the remainder in a slow phase. Each phase exhibited first-order kinetics. These observations are suggestive of the existence of half-and-half distribution of sites.

Enzyme Activation↗

[Studies of molecular marker-assisted-selection for resistance to Fusarium wilt in watermelon (Citrullus lanatus) breeding].

The RAPD marker OPP01/700 proved to be linked to the Fusarium wilt resistant gene in watermelon wild germplasm PI296341 was cloned and sequenced, and transferred into a SCAR marker. The linked marker was tested to be one copy by Southern blotting. The technique of detection of the SCAR-amplified products was simplified. These techniques were applied in selecting resistant plants in F3 of the introgression of the disease resistant gene. It is the first report in this field of transfer ring a RAPD marker linked to disease resistance gene into SCAR marker, and establishing the technique of molecular marker-assisted-selection for breeding Fusarium wilt resistance cultivars.

Breeding↗

Toxicity of five Sudanese plants to young ruminants.

The toxicity of 5 Sudanese plants credited with medicinal value for man, i.e. Citrullus colocynthis, Jatropha aceroides, J. glauca, Solanum dubium and Lagenaria siceraria, was studied by giving the dried or minced plants to Nubian goats, Desert sheep or Zebu calves by mouth or stomach tube. The clinical, haematological and pathological changes indicated that all five plants reduced the ability of the liver to synthesize protein, although there was no evidence of interference with the excretion of bilirubin. Kidney dysfunction and haemoconcentration also occurred. Citrullus colocynthis and Jatropha species in doses of 0.5 to 10 g per kg per day killed goats after dosing for periods ranging from 1 day to 2 weeks. Calves were less susceptible. The fruits and leaves of L. siceraria, in doses of 1 to 5 g per kg per day, caused death after a similar period but with less regularity. The seeds were less toxic. The fruits of S. dubium in doses of 2.5 to 10 g per kg per day killed goats in 2 to 5 days. Similar doses of the leaves caused deaths in 8 to 36 days. In sheep, both fruits and leaves required a longer period of dosing to cause death.

Animals↗

Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes.

A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.

Alleles↗

Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon.

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C3-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 x 10(11) M(-1) s(-1), demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions.

Amino Acid Sequence↗