When does paramutation take place?
--Bernard C. Mikula and Beth Besaw

In MNL 1993 I reported that significant, heritable differences in the level of paramutation could be related to controlled conditions in which seedlings were grown for the first two or three weeks before being transplanted to field conditions for maturity. If seedlings were grown for 15 days under 32 C in LL (constant light) before being transplanted to field conditions for maturation, then paramutant R expression in the aleurone was essentially colorless. If seedlings were grown for 10 days at 32 C LL then shifted to LD (12 hr. light:12 hr. dark) conditions for days 11-15, then the paramutant R gene showed significantly more aleurone cells with dark pigmentation. During this first two weeks of somatic development, in these R/R-st heterozygotes undergoing paramutation, what is the functional status of the R gene? Is it on or off? The paramutant R-gene phenotype, observed in the aleurone of testcross kernels at the end of the life cycle, is expressed as a mosaic of cells in which the gene is on or off. The data reported in MNL, 1993, can be interpreted as a turning off of the R gene just prior to or during tassel determination under 32 C and constant light. This "off condition" was accomplished by the 15th day when no tassel primordia were yet visible. If in the last five days of this 15-day period the seedlings were given LD cycles, significantly more pigment could be observed in the testcrosses at maturity. This can be interpreted as meaning the R gene was on until turned off by the 32 C LL conditions. Additional evidence which can be interpreted to support this conclusion comes from a comparison of R gene expressions of the pollen sampled from the upper and lower tassel branches of the same plant. If seedlings started at 32 C were switched to 22 C for the last five days of the first 15 days of seedling development, the greatest reduction in paramutated R-gene expression occurred in samples from the lower tassel branches. R-gene expression of pollen from upper tassel branches of plants which as seedlings received treatments of 22 C or 32 C did not differ. This is interpreted to mean that in 32 C-LL conditions the R gene undergoes more repression in pollen tested from the lower tassel branches. The amount of repression, represented in the pigment scores of kernels from testcross ears, is dependent on the number of days the environmental conditions are applied. This raises another question. For an individual plant, is the entire five-day period essential to bring about the high degree of repression or is a five-day period essential simply to ensure that all treated plants will respond equally? To restate the question, for how long must the signal to repress the R gene be sustained to achieve a given level of paramutant R-gene expression in the treatment of a single meristem? How can this environmental signal, reported in the kernels as degrees of variegation, be accounted for as quantitatively stored "genetic memory" whose phenotype can be incremented in the positive or negative direction? What accounts for the clonal sectoring pattern reported in the aleurone where the R gene is highly paramutated and no transposable element has yet been implicated for the R locus?

If it is assumed the R gene is "on" throughout development, what assumptions must be made about the ear-shoot meristem as well as tiller meristem? Under the 15 days of controlled conditions on which I have reported, these two lower meristems are farther behind in development than the terminal meristem. If as reported, the terminal meristem is sensitive to environmental conditions, then it should not be surprising that the lateral meristems would differ in R-gene expression, since under our conditions control of seedling environment is terminated on the 15th day or the 21st day, depending on temperature. The lateral meristems are, subsequently, controlled by field conditions which in early spring vary over a wide range of temperatures; periodicity of light conditions will depend on season and latitude. Where the terminal meristem was initiated under controlled conditions to generate repression of the R gene, testcrosses of tillers of these same plants show significantly more pigmented cells than those from the main tassel. That the level of paramutation in the ear is different from the tassel was found in some preliminary evidence where a strong paramutation expression was induced in the terminal meristem. A bimodal distribution of R-gene expression was found among the R/R-lst heterozygotes which were selfed the previous year. Seeds from the self-pollinated heterozygote could be separated, phenotypically, according to whether the paramutant R gene came through the tassel or through the ear. Because of the high degree of paramutation, the R gene from the male was nearly colorless and the R-lst phenotype in the same kernel, contributed by the female, could be identified. The paramutated R gene inherited through the female, because of endosperm dosage, was strongly pigmented. The two classes of seeds from the self-pollinated heterozygote, one showing the R-lst phenotype, the other darkly pigmented, were planted. A second round of paramutation with R-lst showed the R genes from male gametes in the F1 were significantly lighter than those from the female gametes of the same plant. More extensive testing of this phenomenon is planned now that it is possible to influence the terminal meristem unequivocally. 

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