Biochemical Genetic Studies of Maize Peroxidases (Zea May L.)

Nagai, Chifumi
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Genetic and biochemical properties of principal peroxidases of maize, Px3 and Px7, were determined and their changes during development of morphological mutants were examined. Horizontal gel electrophoresis was applied for resolution of peroxidase isozyme bands. An unusual Px3 allele, Px3-6, produced two bands electrophoresing similar to those produced by alleles Px3-1 and Px3-2. A hypothesis was tested that Px3-6 is a gene duplication. Densitometric methods identified the S and F bands of Px3-6 to be electrophoretically identical to those of Px3-1(S) and Px3-2(F). Electrophoresis under varying gel concentrations showed no evidence of differences in mobility and size between S and F bands from Px3-6(SF), Px3-1(S) and PX3-2(F) alleles. Genetic results suggested that Px3-6 (SF) allele is a tandem duplication of Px3 genes. Chromosomal location of Px3 locus previously assigned to Chromosome 7, was determined using gene markers o2, Pn, s1 and Tp, and waxy translocation 7-9a. The most probable location of Px3 was assigned between 96.6 and 100.32 on the long arm of Chromosome 7. Partial purification of enzymes Px3 and Px7 was achieved by (NH4)2S04 fractionation. Con A chromatography and Blue Sepharose chromatography. Anodal and catho peroxidases could be separated by Con A chromatography, and Px3 and Px7 were separated by Blue Sepharose. Michaelis constant (Km) values of Px3 and Px7 were determined for six allelic enzymes of the two loci. No significant differences were found in Km values among allozymes at each locus on these substrates— ferulic acid, caffeic acid and o-dianisidine. However, Km values of Px3 and Px7 allozymes on ferulic acid were significantly different. Px7 showed higher affinity to ferulic acid, a lignin precursor, evidently utilize ferulic acid more efficiently than Px3 and make a greater contribution of lignin formation in maize plants. The molecular size of Px7 was shown to be 72,000, about twice as much as large as Px3 (MW35,000). Genetic studies, however, provided no evidence that Px7 produces dimeric or polymeric proteins. Studies were conducted on peroxidase variations during the pro-flowering development of 14 isogenic, morphological mutants of maize. Leaf peroxidase activity increased during development (4-7 weeks) of five non-GA responding dwarf mutants—br, br2, na, and py—, while two GA-responding dwarfs, d and d-tn had the same level of peroxidase activity as control. Except for na2, no change in band pattern was found. Other types of morphological mutants. Knotted (Kn), slashed leaf (s1) and Ragged leaf (Rg) also showed high peroxidase activity relative to control at the later stages of development (5-7 weeks). Increasing peroxidase activity in mutant ^ was characterized by an isozyme previously unreported, and designated Px-Rg. Increased peroxidase activity may relate to elevated lignin formation or phenol oxidation in 'diseased-like' mutant plant Rg. Wind effects on young maize plants were examined under continuous wind (1.8m/s - 4.2m/s velocity) in greenhouse experiments. After 3 days of treatment, retardation in plant growth (plant height, fresh weight) of 4.2m/s wind treated plants was observed. After 7 days of treatment, peroxidase activity was significantly higher than that of control. It was clear that wind (4.2m/s) affected plants both in retardation in growth and elevated peroxidase activity.
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