Endosperm Mutants of Maize

Sugar metabolism in kernels of starch-deficient endosperm mutants of maize (Zea mays L.) was examined to determine how single locus mutations of carbohydrate metabolism affect carbohydrate metabolism as a whole. Activities of 14 enyzmes were measured in extracts from endosperms from isogenic lines of normal, shrunken, shrunken-2, shrunken-4, brittle-1, and brittle-2 maize in an OH43 background. Nearly every enzyme activity examined was affected in some or all of the mutants. Sucrose synthase and aldolase activities were lower in all mutants compared to normal. ADP-Glc pyrophosphorylase activity in immature kernels was much higher in brittle endosperms than in normal, but absent in brittle-2 and shrunken-2 endosperms. The activity in those genotypes exhibiting activity was positively correlated with sucrose concentration in the kernels. Sucrose may be modulating the coarse control of ADP-Glc pyrophosphorylase activity by affecting the genetic transcription of message for this enzyme. Sorbitol dehydrogenase activity was negatively correlated with its substrate, fructose, supporting the hypothesis that sorbitol dehydrogenase converts fructose produced during sucrose degradation into sorbitol. Glucokinase activity was positively correlated with mature kernel dry weight. This supports the hypothesis that glucokinase activity may limit sucrose utilization. Shrunken4 extracts had lower activities for a number of enzymes, supporting the view that this mutant may have an impediment to protein synthesis. Elevated sucrose levels were evenly distributed throughout 20-day postpollination shrunken-2 kernels, whereas a sucrose concentration gradient existed in normal kernels between the basal region and the upper endosperm. This gradient is apparently generated by the utilization of sugars and may facilitate the movement of sugars into developing corn kernels. The metabolic characterization of starch-deficient endosperm mutants can provide valuable information about metabolism in normal kernels. For example, the substantial decrease in starch accumulation in bt-2 and sh-2 mutants indicates the importance of the enzyme ADP-Glc pyrophosphorylase in the synthesis of starch. The small amount of starch found in these mutants is attributed to a low residual activity of ADP-Glc pyrophosphorylase (8). The observation that the sh mutation results in only a 30% or less decrease in dry weight (5, 13) indicates that the residual sucrose synthase activity and the unaffected invertase activity ( 11) are capable of partially compensating for the deficiency in the sucrose synthase1 activity. Previous studies (2, 6, 13) have shown that the starchdeficient mutations result in profound changes in sugar, starch, and protein accumulation in developing kernels. In this study, a selected set of enzyme activities were measured in endosperm extracts from normal and starch-deficient maize kernels in order to evaluate how the genetic lesions affect the overall carbohydrate metabolism ofmutant kernels. Some correlations between enzyme activities and kernel characteristics are presented that provide evidence for the roles and regulation of certain enzymes in carbohydrate metabolism. Finally, the distributions of sugars in the tissues of normal kernels and the sh-2 mutant were evaluated in order to observe the effect of sucrose utilization on sugar concentrations in the different tissues of developing corn kernels. MATERIALS AND METHODS A number of mutants of maize are known that accumulate less starch and storage protein in the endosperms of their kernels than do normal kernels (2, 6, 13). The genetic lesion of several of the starch-deficient mutants are known. The sh' mutant is known to be deficient in sucrose synthase1 (5), the endosperm specific or inducible form of sucrose synthase (4, 15). Both sh-2 and bt-2 mutants are deficient in ADP-Glc pyrophosphorylase activity (17). The sh-4 mutant reportedly affects pyridoxyl phosphate metabolism (3). The nature of the bt mutant is not known (2). Abbreviations: s/i, shrunken; bt, brittle; bt-2, brittle-2; sh-2, shrunken-2; sh-4, shrunken-4; DPP, days postpollination; r, correlation coefficient for linear regression; P, probability. Normal OH43 maize (Zea mays L.) and isogenic sh, sh-2, sh-4, bt, and bt-2 mutants in an OH43 background were grown in the field in the summer of 1986 and 1988. Enzyme activities were measured in kernels grown in 1986. The distribution of sugars in normal and sh-2 kernels were determined on kernels grown in 1988. Ears were pollinated by hand and harvested at designated times after pollination. Whole endosperms were dissected by hand from freshly harvested kernels, frozen immediately on dry ice, and lyophilized. Lyophilized endosperm tissue was ground to a powder and stored at -80OC until used for enzyme extractions. Designated samples of freshly harvested kernels were dissected into base, pericarp, embryo, lower endosperm, middle endosperm, and upper endosperm by hand as described previously (10), frozen, and lyophilized as described above. Enzymes were extracted from lyophilized endosperms by the procedure described earlier (10). The activities of sucrose