The opaque-2 gene (o2) in maize has been the subject of a great deal of study in relation to its effect on the improvement of protein quality in maize endosperm. Researchers regard the reduction of zein synthesis as the primary effect of this gene.
The mechanism by which the reduction of zein is brought about in the mutant has not yet been established. Most of the current research on the mechanism of zein synthesis in opaque-2 seems to be based on the assumption that the primary effect of this gene is to reduce the production of zein. While this lower zein synthesis is the most striking effect of the opaque-2 gene, this is only one of the most obvious alterations caused by this mutation. Evidence indicates that the opaque-2 locus is regulatory and that, besides regulating the synthesis of zein, it has a wide range of pleiotropic effects on the plant. For instance, the opaque-2 mutant changes the following characteristics of a normal plant: (1) reduces the synthesis of zein and yield (probably due to a lower rate of starch synthesis); (2) delays germination (about 2 days) and pollen shedding; (3) accumulates more water in the developing endosperm; (4) increases the production of albumins and globulins and ribonuclease activity in immature endosperms. Hence, the opaque-2 gene alters several traits in the maize plant. In addition to these abnormalities conditioned by the opaque-2 gene, we have detected an alteration not previously described, which affects the production of pollen. We found lower yield of pollen and, as other authors have shown, reduction in the weight and circumference of the cob. The development of pollen grains occurs before the beginning of zein synthesis. The data presented in this report indicate that the opaque-2 gene (o2) begins to exert its effect before the synthesis of zein.
Because of all these changes caused by the opaque-2 gene, we wondered at which level this gene exerts its primary effect. For instance, could the opaque-2 gene affect the structures (the tassels and the ears) where both the male and female gametophytes are formed? This question originated as a result of our research with the opaque-2 mutant, and our observation that opaque-2 tassels do not produce as much pollen as their normal counterparts. To verify our previous experiences, we planted normal and opaque-2 seeds (both in the W64A background) at the University of California, Davis (where plants are grown under irrigated conditions) at two different dates. Pollen was collected from these plants by placing paper bags on the tassels at around 6:30 A.M. and collecting the pollen at around 1:00 P.M. These collections were made for several days, until no further pollen shedding was noticed. The collected pollen was immediately weighed.
After the tassels had finished shedding pollen, they were removed from the plant at the height of the last leaf on the stem. The tassels were dried for 6 weeks at room temperature. The total weight and the moisture content of each tassel were determined. The secondary spikes were separated from the main spike (continuation of the stem); their respective weights were also determined, and the numbers and lengths of the secondary spikes found in each tassel were recorded.
The ears of the plants from which pollen collections and tassel measurements were made were harvested 65 days after pollination. The kernels were shelled and the cobs were dried for 8 weeks at room temperature, and moisture determinations were made at the end of this drying period. The weight and the circumference of each cob were recorded. The data were analyzed using the t test for difference between two means with unequal sample sizes.
The results of the pollen collections were as follows:
The differences in the amounts of pollen collected between normal and opaque-2 are significant : there is a drastic reduction in the pollen grains produced by opaque-2. For each mg of pollen produced by opaque-2, its normal counterpart yielded 3 mg. The results of the second planting verify this observation; the reduction of pollen in opaque-2 was the same as in the first planting.
To determine the cause for the lower production of pollen in opaque-2, several measurements of weight and length of the tassel structures were made. The results presented in Table 1 are the means of 30 tassel measurements for normal and 35 for opaque-2. When the means of the total tassel weight of normal and opaque-2 are compared, the difference is highly significant. On the average, the weight of a normal dried tassel exceeds an opaque-2 tassel by 487 mg. A similar analysis of the weights of the main spike also shows highly significant differences, however, opaque-2 was 139 mg heavier. A comparison of the weights of the secondary spikes reveals that normal exceeds opaque-2 by 656 mg. This difference is also highly significant, P < 0.001, and the difference in this category is even greater than in the two previous comparisons (the variances for each of the three categories are homogeneous). The normal secondary spikes weigh more because there are more than in opaque-2. In normal maize an average of 18 secondary spikes were present, while in opaque-2 only 8 secondary spikes were found. Pollen grains are produced in the spikelets, most of which are located on the secondary spikes, and the drastic reduction of pollen in opaque-2 seems to be due to the lower number of secondary spikes in the mutant.
The frequency distribution of the lengths of the secondary spikes is shown in Figure 1. In both genotypes, the lengths of the secondary spike have a normal distribution and their variances are homogeneous. However, they have different means; opaque-2 has fewer and longer secondary spikes than normal. On the average, the length of a secondary spike in normal maize is 10.8 cm and in opaque-2 is 13.3 cm. The difference between these means is also highly significant. The increase in length of the secondary spikes of opaque-2 may have occurred as a result of the fewer secondary spikes produced by opaque-2 tassels. In relation to pollen production, however, the increase in the length of the secondary spikes of opaque-2 did not compensate for the fewer numbers of secondary spikes found in the mutant tassels. The reduction in the numbers of secondary spikes (from 18 in normal to 8 in opaque-2) seems to be one of the main causes for the severe reduction in pollen yield in opaque-2.
The differences between normal and opaque-2 cobs are highly significant for circumference and weight. Previous studies have detected similar reduction in cob weight in hybrids carrying the opaque-2 gene (P. S. Baenzinger and D. V. Glover, Agron. Abstr. 1975; D. Makonnen, and L. F. Bauman, Euphytica 25, 1976).
The reduction in the number of secondary spikes and in the circumference and weight of the opaque-2 mutant cobs implies that the opaque-2 gene (o2) affects the development of the structures where the male and female gametophytes are formed. This development takes place before the formation of the endosperm tissue and much before the synthesis of zein (zein synthesis has not been detected until about 10 days after fertilization). From the data presented in this paper, we must conclude that the primary effect of the opaque-2 gene (o2) is not in the reduction of zein synthesis.
Microscopic observations of normal and opaque-2 pollen grains do not reveal any obvious differences in size or shape. Therefore, we suggest that the lower production of pollen by opaque-2 tassels is not due to the reduction in size of the pollen grain, but to a reduction in the number of pollen grains. A study is underway to determine the number of pollen grains produced per floret, which should give us a better understanding of the reduction of pollen grain yield in opaque-2.
Last summer (1981), pollen was collected from normal and opaque-2 plants in Illinois High Protein (IHP) background. IHP normal plants yielded 3.5 times more pollen than IHP opaque-2, which leads us to believe that our original observation of lower pollen production in W64A02 is correct, and that the reduction is not due to the genetic background but due to the opaque-2 gene itself.
Jorge R. Jiménez and Nancy H. Jiménez
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