Initial conditions during tassel determination control the level of R-allele silencing (paramutation)
--Mikula, BC; Kappen, T

Paramutation, gene silencing for the r locus, has been reported, over the past 40 years, as pigment scores determined by sampling kernels from testcross ears. Kernels were matched against standard kernels representing varying grades of pigment from colorless to completely pigmented.

Score variations from year to year and experiment to experiment have been attributed to environmental conditions or stochastic variation. In 1967 I showed a relationship betweeen pigment scores and environmental conditions which plants received during the third and fourth week of seedling development, the time of tassel determination. Experiments since 1967 have been directed toward exploring developmental intervals during which paramutation could be influenced, in seedlings, by temperature and light conditions.

It was found that maximum silencing, as judged by the lowest kernel pigment scores, was achieved for seedlings started in continuous light, days 1-10, and maintained at 32 C LL (continuous light), days 11-15. Seedlings started at 22 C LL, days 1-15, then switched to 32 C LL, days 16 to 21, showed similar reduced pigmentation.

The data of Table 1 explore the developmental interval affecting paramutation within days 11-15 in 32 C conditions and the days 16-21 at 22 C. Seeds of a heterozygote R-g/r-m (r-m weakly paramutagenic mutant from R-st ) from a single ear were used in all the experiments. 50 kernels from six to eight testcross ears, representative of latest pollen samples, were scored by matching each seed against a set of standard kernels ranging from one, colorless, to 21, completely pigmented. Seedlings were held in continuous light except for the days given LD (12hr. light/12hr dark) conditions. At the end of each experiment at 15 days or at 21 days seedlings were transferred to field conditions for maturity to be testcrossed at anthesis.

Most silencing occurred in seedlings given 32 C LL conditions days 11-15, lines 1-3, Table 1. Similar scores were found following 15 days at 22 C if followed for six days, days 26-21, by 32 C LL conditions, line 5. In lines 2 and 3, LD conditions applied after 32 C LL had no effect on silencing. In line 3 a single LD cycle, day 11, had no effect on the pigment score, however, two LD cycles applied days 11-12 showed nearly four scoring units of reduced silencing, line 4, compared to line 2.

Plants started in 22 C LL conditions for days 1-15 showed least silencing (highest pigment scores) when LD cycles were applied days 16-21, line 6. If seedlings were given 6LL cycles at 22 C, days 16-21, a reduction of nearly two scoring units was found, line 7. The LL 32 C cycles applied within the 16-2l-day interval in line 5, showed no increased silencing when applied after 4 or 3 LD 22 C cycles, lines 8 and 9. When the number of LD cycles was reduced to two, days 16-17, and the number of 32 C LL cycles increased to four, increased silencing was found, line 10. Line 11 shows that 4 LL 32 C cycles following 4LD 22 C cycles produced the same pigment scores reported for lines 6, 8 and 9.

It is inferred from the table that developmental conditions favoring increased R -allele silencing from a paramutagenic allele are available, following 32 C LL conditions, days 11-15 or a week later following 22 C LL conditions, from day 16-21. The table also shows that two LD 22 C cycles can influence silencing if applied days 11-12, line 4, or a week later, days 16-17, line 10. It is inferred the epigenetic conditions determining paramutation are temperature related and are separated by a week at the two temperatures of 32 C and 22 C. The source of variation in pigment scores from year to year becomes apparent when considering that much of the work in paramutation has depended on pollinations from field grown material. Given early spring growing conditions, variations in levels of gene silencing are to be expected from the data of the table since two-day intervals at the critical stage of tassel determination can bring about a change in silencing of four scoring units.

In MNL 70 we reported that seedlings from 22 C LD conditions, similar to line 6, showed nearly the same extremes of score variation as reported in the above table when pollen for testcrosses was sampled from a single plant. The earliest pollen collections from a single plant showed testcross scores significantly lighter than the last pollen collections made from the lowest tassel branches, seven days later. This means that under certain early spring temperature conditions considerable variation in pigmentation can be expected from the pollen samples of tassels of single plants over the seven days of anthesis.

Given this variation for the r -locus, under paramutagenic conditions, it is not surprising that geneticists in the middle of this century considered unstable genes "sick". The system shows a response to early temperature conditions resulting in pigment patterns in testcrosses that show gradients of R -allele expression when pollen is sampled and tested over the seven days of anthesis. The variation in pigment expression from single pollen samples was easily assumed to be stochastic rather than epigenetically determined. The stability of the Mendelian gene may undergo some revision as more unstable genes are explored in a developmental context.

The instability of the aleurone pigment expression in paramutation appears to be a product of developmental processes sensitive to the "initial conditions" of tassel determination (before tassel initiation). From the data above this period could involve an experimental window of two to three days. It would be interesting if the aleurone pigment expression controlled by Spm, Mu and Ac, reported by Walbot (Science 248:1534-1537) and Fedoroff (Genetics 121:591-608), might also be related to this specific period of tassel determination.

Because the r-allele can be silenced, incrementally, from generation to generation, genetic systems that show incremental silencing provide model systems for investigating how the environment can change Mendelian gene expressions. The role of the environment in genetics has been neglected throughout this century as pointed out by Jablonka and Lamb in their book, Epigenetic Inheritance and Evolution, 1995, Oxford Univ. Pr. Perhaps maize can provide systems where the role of the environment in gene stability can be examined. The longer developmental periods of maize provide larger "windows" for the dissection of developmental processes and their relationship to temperature and light. It may be considered that using a weakly paramutagenic allele, as well as the lower temperature of 22 C, attenuates the developmental process involved in paramutation making it possible to see results in pollen samples as recorded in Table 1.

Table 1. The role of temperature and light/dark cycles in the control of R-allele silencing under paramutagenic conditions following germination under 32 C and 22 C LL conditions.
 

Days 1-10	Days 11-15	Score	Line
LL 32 C	5LL 32 C	8.1	1
LL 32 C	5LL 32 C 6LD 22 C	7.6	2
10LL 32 C	1LD 22 C 4LL 32 C	7.2	3
10LL 32 C	2LD 22 C 3LL 32 C	11.5	4
Days 1-15	Days 16-21	Score	Line
LL 22 C	6LL 32 C	8.8	5
	6LD 22 C	13.4	6
	6LL 22 C	11.8	7
	4LD 22 C 2LL 32 C	13.5	8
	3LD 22 C 3LL 32 C	13.4	9
	2LD 22 C 4LL 32 C	11.2	10
	4LD 22 C 4LL 32 C	13.8	11

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