Defect of phosphofructose kinase activity in a sugary2 mutant --Pan, D Several genes are known in maize which affect the quality and quantity of various carbohydrates found in the endoperms. The su2 mutant was originally described by Eyster in 1943, and the starch extracted from endosperms of this mutant is 10 to 15% higher in apparent amylose than in normal endosperms. Although the starch compositions of the su2 mutant are altered, purified su2 amylopectin and amylose have properties similar to those of normal amylose and amylopectin, suggesting that the direct mechanism of polyoligosaccharide synthesis is probably not altered, but likely the pathway relevant to the starch synthesis pathway has been regulated. In this report we would like to demonstrate that the biochemical lesion of su2 is likely due to the defect of phosphofructose kinase (PFK) enzyme activity (Fruc-6-P + ATP Fruc-1,6-bisphosphate + ADP). Table 1 shows the PFK activity of enzyme preparations obtained from nonmutant and mutant maize endosperms at 23 DAP.

Table 1.
 
Genotypes
Phosphofructose Kinase Activity umole/mg/min
Su2Su2Su2
0.172
Su2Su2su2
0.142
Su2su2su2
0.119
su2su2su2
0.063

Protein precipitate between 20% and 45% (NH4)2SO4 saturation was applied onto DEAE-Cellulose column. After washing the column with 50 mM Tris-HCl buffer, pH 7.0; the enzyme was eluted with 0.15 M KCl in 50 mM Tris-HCl buffer.  The fractions containing enzyme activity were pooled and collected by 60% (NH4)2SO4 precipitation. After dialysis, the enzyme preparation was assayed for PFK activity.

The results in Table 1 demonstrate the proportionality of the PFK activity with the copy number of the Su2 gene, suggesting that the su2 locus could be a structural gene for the fructose-1,6-bisphosphate kinase enzyme. If the mutation of su2 is the result of the defect of PFK activity, one might expect that there will be an alteration of the metabolites accumulated in the amyloplast of the su2 mutant as compared to the nonmutant. The study of the distribution of metabolites in the amyloplast isolated by a nonaqueous technique was therefore carried out. As expected, the results in Table 2 show a lower activity of PFK activity in the su2 mutant, causing the reduction of fruc-1,6-bisphosphate synthesis, and therefore a reduction of 3,P-glyceraldehyde. As a result, the accumulations of glucose-6-phosphate and fructose-6-phosphate are increased in the mutant amyloplast. Based on these results, we consider that the gene encoding PFK activity had been affected in the su2 mutant. However, we do not rule out the possibility that other protein components in su2 have also been modified as a result of mutation, since quite often in maize, mutants with aberrant starch synthesis are usually linked to multiple defects on biochemical pathways linked to the synthesis of polysaccharides. Details of this study have been submitted for publication elsewhere.

Table 2. Levels of soluble fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate and 3,P-glyceraldehyde in su2 mutant and nonmutant amyloplasts isolated by a nonaqueous technique.
 
Metabolites
Nonmutant
su2 Mutant
 
nano mole/gram of endosperms (wet weight)
Fructose-1,6-bisphosphate
23.00
7.68
Glucose-6-phosphate
960.00
1790.00
Fructose-6-phosphate
450.00
750.00
3-P-Glyceraldehyde
77.00
20.00

 
 
 


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