Environmental programming of paramutation in meristems of two and three week old seedlings
--Bernard C. Mikula

In MNL, 1992, I reported, with pictures, that temperature and light conditions applied to two and three week-old seedlings could significantly alter the level of paramutation of the R gene when testcrossed at maturity. This report follows up with testcross scores of 80 plants which received light and temperature treatments as two and three week-old seedlings. Growth chamber conditions were those reported in MNL 1992. The seeds used in all lines of Table 1 came from a single ear of an R R-lst heterozygote. Scoring was done by matching 50 kernels from each testcross ear against a set of standard kernels ranging from 0 to 20, colorless to fully pigmented, respectively. Colorless kernels were excluded from computed scores since a colorless paramutated R phenotype could overlap colorless R-lst or r expressions. Scores of testcross ears from pollen sampled from plants in each line are reported as pooled ear means for the earliest and latest pollinations of the plants in that line. The fifth column of Table 1 shows the preliminary conditions in which the seedlings were maintained prior to the tassel inductive conditions (last column) applied in the second or third week depending on temperature. Earlier work (MNL42) had shown that seedlings can be ready for tassel induction a week earlier at 32 C than for those seedlings raised at 22 C. The last column shows the variety of treatment combinations applied during the period tassels are determined. The LL symbol represents constant light; LD represents 12 hr. light:12 hr. dark photoperiod. The number of cycles is represented by a single digit before the LL or LD symbol.

Table 1. Testcross scores reflecting changes in paramutant R-gene expression following early temperature and daylength treatments of two and three week-old seedlings. LL=constant light, LD=12 hr. light:12 hr. dark.
 

		Pooled Means		Growth Chamber Environment
Line	n	Earliest Pollinations	Latest Pollinations	da. 1-15	da. 16-21
48	8	8.0±3.4	9.3±5.0	LL 22 C	2 LD 22 C-4 LL 32 C
47	7	10.3±2.0	13.5±3.2	LL 22 C	2 LD 22 C-4 LL 22 C
46	6	8.7±3.1	10.9±2.9	LL 22 C	6 LL 22 C
49	9	9.3±1.7	12.5±1.1	LL 22 C	4 LD 22 C-2 LL 22 C
50	6	9.9±4.0	14.4±3.3	LL 22 C	4 LD 22 C-2 LL 32 C
45	8	9.8±2.6	13.4±2.1	LL 22 C	6 LD 22 C
				da.1-10	da.11-15
30	6	3.3±.6	3.2±1.5	LL 32 C	5 LL 32 C
31	7	8.2±2.3	9.6±3.8	LL 32 C	5 LL 22 C
26	5	8.7±3.5	9.2±3.2	LL 32 C	5 LD 32 C
27	7	9.3±3.2	10.7±2.5	LL 32 C	5 LD 22 C
28	7	9.2±3.8	9.2±3.6	LL 32 C	2 LD 32 C-3 LL 22 C
29	4	8.8±1.2	10.8±3.2	LL 32 C	2 LD 22 C-3 LL 22 C

When pollen of single plants was sampled over the seven day period that pollen was shed and testcross scores of the earliest and latest pollen samples are compared in Table 1, the earliest pollen samples produced testcross scores consistently lighter than the latest. A statistically significant difference exists between the scores of the earliest and last pollinations of lines 49 and 45, P= .001 and .02 respectively, the two lines with the smallest standard errors. A wider range of difference is observed in testcross scores between individual plants in lines which were started in 22 C temperatures and were given light or temperature perturbations in the third week; this is reflected in larger standard errors for each pooled mean. A significant difference (P = .001) exists between the scores of the 30 plants which were started in 22 C and subjected only to 22 C treatments in the third week (lines 45, 46, 47, 49) and those scores of the thirty plants of the last four lines of Table 1 (lines 26, 27, 28, 29, 3l). The greatest range of variation within and between the scores of each line is found in the latest pollinations representing the pollen from the lowest branches of the inflorescence. The gametes from these lowest branches are, therefore, the most likely to undergo a heritable change in paramutant R-gene expression following environmental treatment. Testcross scores from pollen sampled from the upper branches, the earliest pollinations, are essentially alike for both temperature conditions.

One of the objectives of this work was to discover experimental operations where short treatment periods could result in significant changes in the testcross scores for R-gene expression. Line 48, treated with LL conditions at 32 C for the last four days, shows the largest standard error as well as the lowest score for seedlings started in 22 C LL conditions. Because of the small sample sizes required by limited growth chamber space, inferences regarding other mixed treatments will require further study. Line 30 shows that seedlings held in constant light for 32 C throughout the first 15 days yielded plants at maturity whose testcross R-gene pigment scores approached the colorless condition. It is these nearly colorless testcross ears that make the strong case for the paramutant R-gene being influenced by the environment. With these same environmental treatments no change has been noted in the R-gene expression in the absence of the paramutagenic allele R-lst. Nor was it possible to produce this extreme change in paramutant R-gene expression maintained in the Wisconsin inbred W22 background. It may be concluded the paramutation process, which involves transposable elements, is responsive to temperature and light conditions. That transposable elements in somatic tissue respond to temperature has been reported for many years for a number of different plants. This is the first report where the variegation associated with transposable elements expressing in gametogenic tissues of maize can be placed under environmental control so that binary (on-off) change in gene expression can be followed in later generations.

That the R gene is responding to developmental cues seems likely since floral determination is brought about by the application of light-dark conditions between days 11 and 15 at 32 C or between days 16 and 21 at 22 C. Inbred W22 seedlings held in LL conditions during either of these periods will not be florally induced until removed to the photoperiods of field conditions at the end of their respective LL treatment periods. Plants which received LL conditions have two more nodes when compared with those plants which received LD conditions at 22 C. Increased node number has been consistently associated with lower paramutant R-gene scores.

The late R. A. Brink, RA considered that the tassel gave rise to a mosaic of paramutant R-gene expressions. Tassel branch mosaics for paramutant R-gene expression was confirmed in reports of testcrosses of pollen samples, all made the same day, from five tassel branches of single plants (Cooper, HB, H. B., U. Wis. Thesis, 1964). The Wisconsin experiments used inbred W22 into which an R gene, highly selected for "stability", had been backcrossed and maintained through many generations of selection. Using the R gene in the inbred W22 background I have found only small differences between early and late pollen collections. In inbred W22 small but statistically significant differences in paramutant R-gene expression were found between testcrosses of plants matured from environmentally treated seedlings (Mikula, B, Genetics 56: 733-742). The small differences required a skeptical attitude until much more control of experimental operations could be found. I feel the data above permit me to relax my skepticism about whether the results are real. My dependence on an R gene, considered metastable (mottled), but highly inbred and selected for "stability" of expression, may have been responsible for the lack of responsiveness to early environmental conditions. Logically, why should one use a highly inbred, stabilized, "unstable" gene if instability is what is to be studied? Why was I so blind for so many years? I failed to consider the genetic operations of selection for uniformity of expression which shaped the R gene stock in the W22 background with which I was dealing. Stadler, LJ was aware and warned of this operational trap long ago.

The work reported in Table 1 makes it possible to consider different conclusions from those proposed by the Wisconsin Laboratory for the paramutant R gene in inbred W22 (Genetics 52:407-424). 1) Paramutation changes are not random but depend on environmental conditions of seedlings at the time of tassel determination. 2) A polarity of paramutant R-gene expression results from certain environmental treatments: the lower branches produce darker paramutant expressions than the earlier upper branches. 3) The time course of paramutation in the terminal meristem of the main stalk can be fixed within a few days at a developmental stage prior to tassel differentiation. 4) Because paramutation takes place late, just prior to terminal meristem differentiation into tassels, one could expect paramutation from the ears to be different from those of the tassel. 5) Uniformity or variation of paramutation is an expression of the environmental conditions affecting the meristem when seedlings are two or three weeks old depending on temperature and perhaps light.

With increased attention being focussed on methylation of transposable elements a greater emphasis must be placed on experimental operations which can explore developmental events throughout the life history of the maize plant. The extended life cycle of maize compared with shorter cycles of "certain" other plants can be regarded as an asset providing longer developmental windows for experimentally influencing epigenetic events. The early work in plant physiology dealing with biological rhythms and floral induction provides a rich literature worth attention for methods which may influence developmental processes of genetic interest, especially methylation. Since the paramutant R gene has been associated with changes in methylation and probably has associated transposed segments of transposed elements, it seems likely this transposed material could provide the transducer which responds to temperature and light. A simple germination test of an R gene with plant pigment will show that roots and coleoptiles fail to produce pigment at higher temperatures or when certain colors are filtered out of white light. Are these light and temperature responses the function of transposed material controlling transcription of the R gene through DNA modification?

The seeds scored in lines 30, 45 and 47, Table 1, were grown out the following year with no further treatment as seedlings. Figure 1 shows that the differences recorded in the scores of 1991 are maintained the following year, 1992. The four ears on the left of Figure 1 came from a plant represented in line 30, the four ears to the right are representative of plants from lines 45 and 47.

Figure 1. Testcross seeds from Table 1 of Line 30 (left column of ears) and 45 (right column) were grown and testcrossed in 1992. Differences observed in testcrosses the previous year persist in above testcrosses made a year later without further treatment as seedlings.

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