This report presents our initial observations on the effect of B chromosomes on induced mutation frequencies at the yellow-green-2 (yg2) locus in maize. Inbred W22 plants (Yg2/Yg2) which contained 0 or 8 B chromosomes were each crossed as female parents by a second inbred which was yg2/yg2 to produce Yg2/yg2 heterozygotes which contained 0 or primarily 4 B chromosomes. These two kernel types will be designated as O-B and 4-B. Because these two types are the result of a cross between two highly inbred lines, they should be isogenic except for the presence of 8 chromosomes.
The O-B and 4-B kernels were mutagenized and grown under rigorously controlled conditions. The kernels were soaked in aerated water at 20 C for 72 h. After this treatment, the primordia of leaves four and five have been estimated to consist of 3,000 and 500 cells, respectively (Plewa et al., MNL 57:147-149, 1983). The kernels were then treated with 10 mM ethylmethane sulfonate (EMS) in a 0.1 M phosphate buffer (pH 7.4) for 8 h at 20 C under aeration, or with 250 rads of 137 gamma irradiation. A concurrent control was included. Following the treatment, the kernels were rinsed in running tap water for 30 min and were placed in soil in 10 cm plastic pots. The pots were coded and placed in a plant growth chamber at 20 C with a 17 h photoperiod. After 20 to 25 days of growth, mature (with ligules present) fourth and fifth leaves were analyzed. The number of yg2 sectors per leaf was determined using a fluorescent light box to illuminate the leaves. Only sectors greater than 1 mm in length were counted. The data were analyzed using a 2 x 3 completely randomized factorial ANOVA test, and pair-wise comparisons (t-test) of relevant comparisons are presented.
Table 1 presents the results from an experiment of this type. These populations are inadequate to accurately estimate spontaneous mutation frequencies in untreated material of control populations. However, the frequency of yg2 sectors in EMS-treated plants of the 4-B group was significantly higher (P < 0.01) than the O-B group in both leaves four and five. The mutation frequency in gamma-radiated material was significantly higher in leaf five, and higher, but not significantly different in leaf four. These results indicate that the presence of B chromosomes in the maize genome makes the yg2 locus more susceptible to induced mutation by EMS, and possibly by ionizing radiation.
The results of a second experiment are presented in Table 2. The male parent crossed onto O-B and 8-B females in the W22 inbred genetic background to produce this experimental material was of a different origin from the male used to produce the material analyzed in Table 1. Significantly higher (P < 0.025) frequencies of yg2 sectors were present in the 4-B group than in the 0-B group in both gamma-radiation and EMS-treated material.
B chromosomes in maize are relatively genetically inert because maize plants that contain low numbers of B chromosomes are phenotypically indistinguishable from plants without B chromosomes. However, they are not completely inert because certain processes have been identified which are controlled by factors located on B chromosomes (reviewed by Carlson, Ann. Rev. Genet. 16:2-23, 1978). One known effect of B chromosomes is that recombination is increased in certain segments of the A chromosomes when B chromosomes are present (Ayonoadu and Rees, Genetica 39:759 1968; Hanson, Genetics 63:601, 1969; and others). We speculate that the B chromosome-mediated increases in recombination may be related to increased mutation rates. It may simply be that the presence of additional chromatin due to the presence of B chromosomes imposes stress on the DNA replicative and/or repair mechanisms, which causes the cells to be more susceptible to mutagens.
These observations are also relevant to the "bodyguard" hypothesis of Hsu (Genetics 79:Suppl. 137, 1975). Hsu proposed that because heterochromatin is located adjacent to the nuclear membrane in interphase, it may function as a bodyguard to absorb environmental insults (mutagens) to protect the centrally-located euchromatin. This hypothesis specifically refers to protection against chemical mutagens and not to ionizing radiation. B chromosomes of maize are largely composed of heterochromatin. Hsu suggested that an excellent material to test the "bodyguard" hypothesis would be maize, and he stated, "if the bodyguard hypothesis is correct, then the mutation rate induced by chemical mutagens should be inversely correlated with the number of B chromosomes." The experimental results in these experiments with EMS are exactly the opposite of what would be expected if the bodyguard hypothesis is valid; thus, the current experiments do not support the "bodyguard hypothesis." The experimental results with gamma irradiation are also in disagreement with this hypothesis because the bodyguard hypothesis predicts that ionizing radiation would efficiently penetrate the cell and not be influenced by the presence of heterochromatin.
David Weber and Michael Plewa
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