Copyright 8 1995 by the Genetics Society of America Environmental Programming of Heritable Epigenetic Changes in Paramutant r-gene Expression Using Temperatureand Light at a Specific Stage of Early Development in Maize Seedlings Bernard C. Mikula Defiance College, Defiance, Ohio 43512 Manuscript received April 21, 1994 Accepted for publication May 12, 1995 ABSTRACT Different heritable expression-states wereprogrammed into R alleles fromR/R-lst heterozygotes under different temperature conditions applied during a developmental period in which flowering is induced. At maturity, R-allele expressions in test crosses of male gametes derivedfrom R/R-lst seedlings raised 15 days in 32" and continuous light conditions differed significantly from those of sib seedlings raisedfor 15 days in 22" and continuous light conditions and shifted to six 12-hr light-dark cycles, days 16-21. This experiment provides the first evidencein higher organisms that environmental conditions, applied at a specific stage of development cause a heritable change in a specific allele expression. My earlier evidence required a statistical analysis for demonstrating heritable change; I present photographic evidence of this environmental effect on four R alleles. ARAMUTATION (BRINK1956) provides a sensitive controlled before or during tassel differentiation. I re- P genetic system where heritable quantitative varia- ported in a previous paper (MIKULA1967) that when tion in the expression of a single gene can be followed R is exposed to a paramutagenic allele (R-st),the envi- from generation to generation. BRINK showed that ronmental conditions canpromote significant heritable when the R allele (dark kernel pigmentation) is made change inR-allele expression. R alleles from plantsthat heterozygous with its allele, R-st (stippled pigment pat- as seedlings were raisedat 21" and LL conditions (con- tern), the R allele produces significantly less pigment tinuous light) during the third and fourth week of seed- after removal from a heterozygote withR-st. The change ling development, whencompared in testcross progeny, in R-allele expression is heritable; furthermore, all R conditioned significantly less pigment in test crossesat alleles (100%) upon removal from this R/R-st heterozy- maturity than Ralleles of plants matured from seedlings gous combination are changed, and the change is di- grown at the same temperature in LD (12-hr lightdark rected at the R locus only. Later studies(MIKULA 1961; cycles) conditions for this same period. The identifica- MCWHIRTERand BRINK 1962; COOPER 1964; STYLESand tion of acritical developmental period leading to BRINK1966) showedthat if the R allelewas maintained changes in R expression suggested that experimental with R-st alleles for several generations, paramutation control of paramutation was possible. Three other ob of the R allele was additive. This additive effecton theR servations reinforced this inference: the level of para- allele was followed in male gametesfor five generations mutation could be correlated with the number of LD (STYLESand BRINK 1966). FEDOROFF (1989), while .kycles in the third week of seedling development; pre- studying the ontogenetic expression of the transposon liminary tests showed a polarity ofparamutant R allele Spm (SupP-essm-Mutatm),reported evidence that an in- expression in which early pollen samples from the up active Spm, by association over severalgenerations with per branches of single tassels showed more paramuta- an active Spm, can become progressively more active. tion than samples taken from lower branches of the The R alleles and cryptic Spm elements in maize repre- same tassel; and incremental changes in paramutant sent genetic systems whose phenotypes can be incre- R expression from generation to generation (MIKULA mentallyprogrammed by previousassociation with 1961) showedthat small changes inthe level ofparamu- other genetic elementsduring development (FEDOROFF tation couldbe detected and amplified fromgeneration and BANKS 1988). to generation. Paramutation (BRINKet aL 1968; DOONERet al. 1991) Further, it was found that tassel initiation in the was considered to be the result of changes accumulated W22 inbred could be delayed under continuous light in the R gene throughout somatic development. Coo conditions. It then became possible to askthe following PER (1964) and SASTRY et al. (1965) reported variation questions: how early in the life of the seedling could in paramutation from gametes sampledfrom different tassel determination take place; did temperature and tassel branches reflected a mosaicism developmentally light play a role; how many lightdark cycles were re- Genetics 140 1379-1387 (August, 1995) 1380 Mikula B. C. quired fortassel determination; could thelevel ofpara- Scoring procedures: A sample of 50 kernels from each test- mutation be correlatedwith programmed environmen- cross ear was scored by matching each kernel against a set of standard kernels ranging from 0 (colorless) to20 (completely tal conditions applied in early seedling development; pigmented). Pooled means and standard deviations for each could paramutation be inhibited or driven toward the environmental treatment are reported in Figure 6. completely colorless level; when during early develop- ment is the seedling most responsive to environmental RESULTS variables of temperature and light; and could the level of paramutation in four new R accessions be correlated Extensive preliminary testing showed no tassels were with environmental conditions administered to young initiated in inbred W22 under LL conditions. This seedlings? made it possible to determine the number of LD cycles essential for tassel initiation. Tassel initiation was deter- MATERIALS AND METHODS mined 1 week after LD treatments, however plants re- mained in the inhibitory LL conditions until six to eight Seed stocks: Environmental programming strategy for the first 3 weeks of seedling development was based on work with plants could be assayed for the presence of tassel pri- the Rand R-st alleles in inbred W22 background provided by mordia. The presence of tassel primordia was deter- JERRY KERMICLE, University of Wisconsin. The effect of the mined by dissecting away the leaves surrounding the environment on paramutant R-allele expression,first re- terminal meristem, under magnification. The presence ported in inbred W22 (MIKULA1967), required a statistical of an elongated terminal meristem and the presence analysis for its evaluation. In an effort to find more responsive R alleles, new R allele accessions that had not undergone a of branch primordia were observed when tassel initia- high degree of inbreeding and selection were screened for tion had taken place. Figure l shows that tassels could dark kernel pigment expression. The four newR-g alleles be induced with six LD cycles applied days 16-21 at from New Mexico and Arizona, 221, 229, Z1, 269, supplied 22" or a week earlier with only four LD cycles applied by Native Seeds/SEARCH, Tucson, AZ, are reported as GI, days 11- 15 at 26". The results in Figure 1, based on the G3, G4, and G5, respectively in this document. The new R-g accessions condition, in r/r/R endosperm, a uniformly dark presence or absence of tassel primordia in seedlings of pigmentation, comparable to that of the W22 R-r gene. All the W22 inbred foreach combination of environmental four R-g lines were crossed to a R-kt (light stipple with few conditions, provided a model system that made it possi- very fine spots) known to causeintermediate levels of paramu- ble to test whether paramutation could be influenced tation of the R allele. For convenience, the R-g genes will be by combinations of temperature and light variables ap referred to simply as R, eliminating the lower case designa- tion,-g, signifjmg lackof plant color. The R-kt allele, in W22 plied at a time seedlings were undergoing thetransition background, was supplied by the Maize Genetics Coop., Uni- from vegetative to floral phase of meristematic activity. versity of Illinois. The testcross ears of RR-lst heterozygotes of alleles Growth chamber conditions: Seeds from single ears of the G1, G3, G4, and G5, in Figure 2, A and B, show that a R/R-kt heterozygotes, representing the four different R al- difference in the amount of pigment for all four new leles, were germinated on white, moist facial tissue pads in stainless steel, glasscovered pans. Continuous light was sup R alleles can be observed in response to early environ- plied by 14 200-W VHO cool white fluorescent tubes supple- mental conditions. Seedlings raised in 31" LL condi- mented by 12 60-W incandescent bulbs. In 3 or 4 days, when tions for the first 3 weeks in the left column of both coleoptiles were largeenough to handle safely, seedlings were photographs show kernels with less pigment than those transplanted to soil conditions in plastic pots. Seedlings were raised in 22" LD conditions during the same period. started 50-60 cm distant from the light source and grew to- The paramutagenic allele, R-lst, present in half of the ward the light for the remaining 2- or %week periods they were held in the controlled environment conditions. Average kernels on each ear, has a phenotype too light to be light intensiq during this early stageof seedling development visible in the photographs; thus, it is possible to com- was 35 W/m . Dark conditions were obtained by placing the pare, visually, the environmental contribution to the seedlings into light-tight boxes. For eachenvironmental treat- paramutation of