Mol Gen Genet (1992) 234:353-360 © Springer-Verlag 1992

Reactivation of Mutator transposable elements of maize by ultraviolet light Virginia Walbot Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA Received February 11, 1992 / Accepted April 14, 1992

Summary. After epigenetic loss of Mutator activity, the family of Mu elements in Zea mays becomes immobile and highly methylated; in addition, Mu9, the presumptive autonomous regulatory element, is transcriptionally silent and its copy number decreases in successive crosses to non-Mutator lines. Spontaneous reactivation, scored as restoration of somatic instability of potentially mutable alleles of Bronze-2, of such cryptic Mutator lines is rare, occurring with a frequency of about 10 -4. Irradiation of pollen with 254 nm ultraviolet light increases reactivation rate in the progeny kernels by up to 40-fold. Accompanying reactivation, the copy number of Mu9 elements increased, two-fold in one line and 20 to 40-fold in a second line. Reactivation may involve direct D N A damage or immediate physiological stress in the treated pollen. Key words: Zea mays - Somatic instability - Bronze-2 Genomic shock - Pollen

Introduction Monitoring of somatic excision of transposable elements from reporter alleles is the primary assay for transposon activity in maize. With this assay epigenetic phenomena have been shown to play an important role in the regulation of transposable element activities. Three features of element excision during plant development can be readily quantified: frequency (number of revertant sectors), timing (size of sectors) and pattern (distribution of sectors within organs). A reversible alteration in a feature is termed a "change of phase" (reviewed in Fedoroff 1983). One of the most dramatic changes of phase is a switch from active to inactive status, observed as the complete loss of somatic excision ability, i.e. a non-spotted kernel. This epigenetic switch occurs without loss or mutation of the regulatory (autonomous) elements encoding transposase. Activity loss is correlated with an epigenetic change in the genome, namely increased

methylation of the family of Mu elements (Chandler and Walbot 1986; Bennetzen 1987), of the autonomous En/ Spm (Enhaneer / Suppressor-Mutator ) element (Cone et al. 1986; Banks et al. 1988), and of the autonomous Ac element (Schwartz and Dennis 1986; Chomet et al. 1987). It is not known whether methylation is the primary cause or is a consequence of inactivation. Once established, however, methylation is likely to maintain the inactive phase by suppressing transcription of regulatory elements and/or by affecting recognition of transposon termini by transposase (Gierl et al. 1988; Kunze and Starlinger 1989). Mutator lines of maize are widely used for transposon mutagenesis, because they cause a high forward mutation rate of ~ 10 -4 to 10 -5 per locus (Walbot 1991). Mutator lines contain up to several hundred copies of a diverse family of Mu elements defined by their shared terminal inverted repeats (Walbot 1991). In most Mutator lines, the genetic element that regulates Mu element mobility is also multicopy, because Mutator activity is transmitted to > 90% of the progeny of a cross between a non-Mutator and a Mutator line (Robertson 1978). In contrast, lines containing active Ac and En/Spm elements typically show Mendelian segregation of only one or two elements (reviewed in Fedoroff 1983). Using rare lines that transmit Mutator activity as if only a single regulator were present, several laboratories have recently cloned a new type of Mu element that hybridizes with the sequence that segregates with Mutator activity (MuA2, Qin et al. 1991 ; MuR1, Chomet et al. 1991 ; Mu9, Hershberger et al. 1991). The restriction maps of the three elements identified by this criterion are congruent and most probably represent the same 4.9 kb element. We have cloned and sequenced Mu9, a representative of this new element type (Hershberger et al. 1991). Most non-Mutator lines lack Mu9 elements; inactive Mutator lines contain methylated forms of Mu9 that are apparently transcriptionally silent (Hershberger et al. 1991 and unpublished data). Inactive Mutator lines with a potentially mutable reporter allele provide excellent material for assessing the

354 impact of mutagens and environmental stress on the reactivation of transposon activities. When a transposon family is reactived, somatic instability is restored for a readily scored phenotype. Using inactive lines containing bz2: :mul, a Bz2 allele with a M u l element in the second exon, I previously reported that gamma irradiation of seed can reactivate somatic instability in the subsequent generation of progeny kernels (Walbot 1988). It was surprising, however, that when reactivation occurred only a few progeny per treated plant had the spotted kernel phenotype typical of an active Mutator line. Cell lineage studies indicate that in maize seed the apical meristem contains one group of two to four cells that will form the tassel, and additional cell pairs that will form each ear (McDaniel and Poethig 1988). Consequently, reactivation of Mutator in one of these meristem cells should result in a large fraction of gametes with an active Mutator system. The assay is very indirect, however, because about 45 cell divisions intervene between treatment and final scoring: ear or tassel development requires about 26 cell divisions (Otto and Walbot 1990), gamete formation requires 2 mitotic divisions for sperm and 3 for the egg, and aleurone development in the progeny requires 17 cell divisions (Levy and Walbot 1990). It is clear that reactivation was stimulated by gamma irradiation, but it is likely that Mutator activity was epigenetically lost again in most cell lineages during this long developmental history. To assess more directly the ability of DNA-damaging agents and environmental stress to reactivate Mutator, I have utilized a well-established protocol for irradiating pollen with UV-C (Neuffer 1957). Radiation with a maximum fluence rate at 254 nm is efficiently absorbed by DNA and has been used as a mutagen in maize pollen (Stadler and Sprague 1936; Stadler and Uber 1942) and in many other organisms. I report here that UV-C radiation can induce reactivation of cryptic Mutator activities to about 10-fold above the spontaneous frequency. In addition, I report that this radiation treatment has a physiological impact on pollen surface structure that may contribute to its effectiveness in transposon reactivation. Materials and methods Maize lines. Two inactivate Mutator lines with different Bz2 reporter alleles were utilized. One line was homozygous for bz2: .'Mul ; this reporter allele has been sequenced and contains a 1376 bp M u l element inserted near the end of the second exon (Nash et al. 1990). This line has been maintained by selfing for several generations; it is the line used in a previous study with gamma irradiation (Walbot 1988). The second line was derived by self-pollination of plants heterozygous for bz2-mu4 (Nash et al. 1990) by recovering individual ears that failed to show somatic instability; thus, the population is segregating for the reporter gene activity and one-quarter of the sample will be bz2. By sequence analysis bz2mu4 is now known to contain a 4.9 kb Mu9 insertion near the beginning of exon 2 (Hershberger et al. 1991), and this allele has been renamed bz2: .'Mu9.

Pollen treatment and pollination. Approximately 100 inactive Mutator plants were used in each experiment. Mature tassels were collected from about one third of this population by cutting the main stem at the topmost leaf; the tassels were placed in a glass of tap water mixed with lemon-lime soda as a source of sugar (10% v/v) and kept indoors for use on several days. To collect fresh pollen for irradiation, two to three anthers that had just begun to shed pollen were used per treatment. The pollen in these anthers was dispersed as a monolayer onto a 10 cm square quartz glass plate about 1.5 mm thick (GM Associates, Oakland, Calif.); each treated sample contained 4000-5000 pollen grains. The quartz plates were carefully cleaned with ethanol and dried before use; to exclude pollen from the edges of the plate, a frame of glassine paper was used to mask approximately 1 cm along the edge of the plate prior to dispersing the pollen. After removing the paper frame, the loaded plate was placed on a platform inside a ventilated wooden box. The platform had a 9 cm square window cut into it, so that the quartz plate was irradiated from above and below by two pairs of lamps (15 W germicidal lamps, G15T8, Ultraviolet Products) mounted on the top and bottom of the box. The lamps were 12 cm from the quartz plate. Prior to use, the lamps were allowed to warm up for 15 rain to reach a steady irradiance of 60 J/m 2. To insert the quartz plate without opening the box, a small hinged door was built at the level of the interior platform. After irradiation, viable pollen was collected by tilting and tapping the quartz glass over a piece of glassine paper. Each irradiated sample was used to pollinate one ear of the bz2 tester stock. To reduce contamination, the plants in reactivation tests were separated spatially and temporally from active Mutator lines carrying a mutable bz2 allele. To assess spontaneous reactivation, untreated pollen from the cut tassels of inactive Mutator plants was crossed onto one or more bz2 tester ears each day pollen was shed. Ears of the detasseled inactive Mutator plants were crossed with bz2 tester or selfed to test for spontaneous reactivation through the ear. The remaining inactive plants were self-pollinated as a third check for spontaneous reactivation. Somatic excision in kernels was assessed by scanning them on the ear using a Nikon stereozoom microscope at 1.5X magnification; kernel numbers are rounded off to the nearest multiple of 50. Pollen viability. Pollen germination was assessed by dispersing control or treated pollen grains onto sterile, clear media (5 mM CaC12, 1 mM boric acid, 5% sucrose, 1 mM MgClz) solidified with 0.4% Gelrite (Kelco) in a plastic petri dish. After 24 h incubation in the dark at 25 ° C, intact pollen tubes were counted as a fraction of total pollen grains in three fields of 100 grains each in different areas of the plate. Hybridization analysis. DNA purification and Southern blot hybridization conditions were as previously described (Walbot and Warren 1988). The BX3 probe is a 1.0 kb internal fragment of Mu9 (Hershberger et al. 1991); as an internal control for DNA loading, we measured hybridization to a 2.3 kb internal HindIII fragment of Alcohol dehydrogenase-1 (Walbot and Warren 1990).

355 Based on Southern blotting analysis, the bz2 tester in the W23/K55 hybrid background lacks M u 9 but does contain cross-hybridizing fragments of a different size (Hershberger et al. 1991).

30co

[]

, m

•~-

Results

Pollen irradiation and germination in vitro

E

20-

10[]

Because of the eccentric placement of the two sperm nuclei, irradiation from two sides or agitation of pollen is required for efficient UV treatment (Pfahler 1973 ; Coe et al. 1988). In these experiments a quartz glass plate, transparent to UV-C light, was used to hold a monolayer of pollen grains during irradiation from above and below. Both inactive M u t a t o r lines, b z 2 : : M u l and bz2: : M u 9 , have been introgressed into the same hybrid W23/K55 background. They showed similar in vitro pollen germination rates of about 30% after, and similar sensitivities o f germination to, UV-C radiation (Fig. 1). During the first 10 s (600 J/m 2 of exposure) there is little impact on pollen tube growth, but with progressively longer exposure times tube growth decreased and more exploded pollen tubes were seen. Following 30 sec o f irradiation almost all treated pollen failed to produce a normal tube. The primary variable during irradiation was exposure to UV-C, as pollen was exposed to a temperature ~ 2 ° C above ambient for 30 s or less. In addition to decreasing viability, UV-C treatment altered the pollen surface. In samples irradiated for < 10 s, virtually all grains could be tapped off the quartz glass plate. With increasing irradiation, progressively more grains become attached to the plate by a sticky exudate; these grains could not be dislodged easily. After a 30 sec exposure to UV-C, up to one-third of the pollen grains werre attached to the plate (data not shown). This reaction to UV-C radiation was unexpected but indicates that rapid physiological changes occur after exposure to UV-C.

Pollination with UV~C~treated pollen

In 1989, a 20 s (1200 J/m 2) exposure time was used for all pollination experiments (Table 1), because at this dosage about half o f the treated pollen germinated in vitro relative to the untreated control. With 20% of the pollen germinating in a sample of 4000-5000 grains and ~ 250 silks per bz2 tester ear, on average only a few viable pollen grains should be delivered to each silk. Under these pollen-limited conditions there should be little competition between grains but sufficient viable pollen to result in a full ear. Pollen given the 20 s UV-C dosage, however, yielded ears with 0-20% seed set (data not shown). These results indicate that the in vitro germination test for pollen viability seriously overestimates biological function, namely the more stringent test of growth through a silk and effective fertilization. Consequently, shorter exposure times were used in subsequent experiments.

0

I

5

i

10

I

15

i

20

215

0

Treatment Time (s)

Fig. 1. Pollen viability in an in vitro germination test. As described in Materials and methods, freshly shed pollen was bidirectionally exposed to UV-C for the times indicated and germination was scored 24 h later. Open symbols indicate the inactive bz2: : Mul line, closed symbols, the inactive bz2 : : Mu9 line

Table 1. Reactivation of somatic mutability in bz2:.'Mul after a

20 sec treatment of pollen with UV-C Crosses performed

UV

Number of kernels Total Spotted

Reactivation frequency

bz2: :Mul (X) bz2: :Mul x bz2 bz2xbz2::Mul bz2 x bz2::Mul

No No No Yes

9500 3300 26100 3400

7.6x 10-5 2.1 x 10-3

0 0 2 7

This experiment was conducted in 1989 using a homozygous bz2: :Mul, inactive Mutator line that had been maintained by selfing for several generations. The UV-C treated pollen was used on 118 tester ears, some of which set no seed (X), self cross

Spontaneous and UV~induced reactivation o f somatic instability

Spontaneous reactivation events typically involved one or two kernels on a single scored ear, resulting in a frequency o f spontaneous activation in the range of 10- 5 to 10 -4 for both bz2: : M u l and b z 2 : : M u 9 (Tables 1, 2, 3). This observation demonstrates that reactivation typically occurs late in plant development, such that only single kernels or a small sector of anthers contains an active M u t a t o r system. There was one exception to this pattern (Table 3) in which a single self-pollinated bz2: : M u 9 ear of an inactive M u t a t o r plant had 26 revertant kernels (of 144 total). Consequently, spontaneous reactivation, at least in the ear, can also occur early in organ development. In a previous study of spontaneous reactivation of bz2: : M u l , a few ear sectors encompassing multiple kernels were also found (Walbot 1986). In the first two experiments, treatment with UV-C for 20 s or 12 s resulted in about a 10-fold increase in reactivation over the spontaneous frequency for both bz2 : : M u l and bz2 : : M u 9 . There was a marginally higher reactivation frequency with the shorter treatment time, and a large increase in seed set per ear (data not shown).

356 To examine the relationship between U V - C dosage and reactivation, a third experiment was conducted (Table 3), varying dosage in 3 s increments. In the shortest time tested, 3 s (180 j/m2), reactivation frequency approached 10- 2; this represents a 40-fold enhancement of the spontaneous reactivation rate. Restoration o f somatic excision decreased progressively with longer exposure times, and kernel yield followed the same pattern (Table 3). The heritability o f transmission of the reactivated M u t a t o r system was confirmed by planting a subset of the spotted kernels f r o m each experiment. These plants were crossed to bz2 tester and somatic mutability scored; 21/23 individuals transmitted the spotted kernel phenotype to their progeny. This result indicates that in m o s t cases reactivation occurred in both sperm in the pollen grain.

Mu9 methylation and copy number Characteristically, inactive M u t a t o r lines contain M u l elements in which the H i n f l sites in the terminal inverted repeats and other internal sites are methylated (Chandler and W a l b o t 1986; Bennetzen 1987); this was true for the inactive lines used here (data not shown). A second characteristic o f inactive lines is stabilization o f M u l element copy n u m b e r (Walbot and W a r r e n 1988). N o w that M u 9 , the candidate regulatory element has been identified, its copy n u m b e r is of greater interest and was therefore measured in the inactive and reactivated lines. To assess M u 9 copy number, genomic D N A was digested with SstI and DraI to release a diagnostic 4.2 kb fragment f r o m the 4.9 M u 9 element (Hershberger et al. 1991). Southern blots were prepared and first hybridized to an Alcohol dehydrogenase-1 gene-specific probe; the intensity of hybridization to A d h l was used to normalize for loading differences a m o n g the samples. Lanes containing amounts o f D N A equivalent to 1, 10 and 50 copies o f M u 9 were set up, using a cloned M u 9 plasmid, and used for comparison with the genomic samples (data not shown). The M u t a t o r lines have been introgressed with the the bz2 tester line; the tester lacks the diagnostic 4.2 kb fragment, but it contributes single-copy amounts of several larger cross-hybridizing fragments seen in all lanes (Fig. 2). The D N A sample prepared f r o m pooled seedlings o f the active bz2: : M u l line contained > 50 copies of the 4.2 kb M u 9 fragment (Fig. 2, lane 1). This high copy n u m b e r o f M u 9 is typical of our M u t a t o r lines (Hershberger et al. 1991). In contrast, the inactive bz2: . ' M u l line contained the equivalent of 11 copies of M u 9 (Table 4; Figure 2A, lane 2). In kernels in which somatic instability was restored, however, M u 9 copy n u m b e r typically increased to over 20 copies per plant, approximately doubling M u 9 copy n u m b e r c o m p a r e d to the inactive line (Table 4; Figure 2A, lanes 3-7). A qualitatively similar pattern was found with the b z 2 : : M u 9 line: a copy n u m b e r > 50 in the active line, a very low copy n u m b e r in the inactive line, and an increased copy n u m b e r in reactivated individuals. There

Table 2. Reactivation of bz2: :Mul and bz2::Mu9 after UV-C irradiation of pollen for 12 sec Crosses performed

UV

Number of kernels Total Spotted

Reactivation frequency

bz2 x bz2::Mul bz2: :Mul (X) bz2 x bz2: :Mul bz2 x bz2.':Mu9 bz2::Mu9 (X) bz2 x bz2::Mu9

No No Yes No No Yes

14750 8250 7500 10250 13250 4600

1.4 x 10-'~ 1.2 x 10-3 9.8 x 10-5 1.5 x 10 -4 2.2 x 10-3

2 0 9 1 2 10

This experiment was conducted in 1990 by pollinating approximately 120 ears with UV-C treated pollen and a lesser number of control ears with untreated pollen. The bz2::Mul source was the same as described for Table 1. The bz2::Mu9 seed was pooled from a self-pollinated population of heterozygous parents; one-quarter of the plants used in this experiment lacked the reporter allele but no correction has been made in the calculated frequencies Table 3. Reactivation of bz2." :Mu9 after UV-C irradiation of pollen for varying time periods UV-C (sec)/Cross

O/bz2::Mu9 (X) O/bz2 x bz2 : :Mu9 3/bz2xbz2::Mu9 6/bz2 x bz2::Mu9 9/bz2 x bz2 : :Mu9 12/bz2 x bz2::Mu9 15/bz2 x bz2.":Mu9

Number of kernels" Total Spotted

Reactivation frequency

6600

27

11050

2

3.0 x 10 -4b 1.8 x 1O- 4

13670 10860 4580 1640 360

108 63 11 3 0

7.9x 10-3 5.8 x 10-3 2.4 x 10 - 3 1.8 x 10-3 -

a Plants segregating for bz2::Mu9 were used in this 1991 experiment. Approximately 40 crosses of each treatment type were performed. The selfed Mutator ears had a low yield of 20-200 kernels per ear. The yield on bz2 tester ears using pollen treated with UV-C decreased progressively with dosage b There were only two ears that contained spotted kernels; one ear had a single kernel and a second ear had 26 spotted kernels. The multiple kernels probably arose from a single reactivation event during somatic development. Consequently, reactivation frequency has been calculated based on two events; if all 27 spotted kernels represented independent reactivation events, the frequency would be 4.1 x 10 -3 are several striking quantitative differences (Table 4; Figure 2B). First, the pooled seedling sample of the inactive b z 2 : : M u 9 line had only a faint signal at the diagnostic 4.2 kb fragment size. The signal represented approximately 1.5 copies o f M u 9 per haploid genome. This result suggests that the only copy o f M u 9 in some inactive individuals is the element present in the b z 2 : . ' M u 9 reporter allele. Because this inactive stock is derived f r o m selfed progeny of a b z 2 : : M u 9 / b z 2 heterozygote, one-fourth of the individuals lack the reporter allele and some of these m a y be missing M u 9 altogether. A second quantitative difference is that reactivated b z 2 : : M u 9 individuals showed a dramatic increase in M u 9 copy number, f r o m 1.5 to 37-62 copies. This amplification of copy n u m b e r far exceeds the twofold increase observed in the reactivated bz2." : M u l line. In addition to the diagnostic 4.2 kb M u 9 internal fragment, the inactive lines contained novel, higher m o -

357

bz2::Mu9

bz2::Mul

~ @u

Reactivated

A

\~

Reactivated

B

4--

Adhl

Adhl

Table 4. Mu9 copy number in reactivation experiments

Reporter allele

Mu9 copy number"

Active line

Inactive line

Reactivated individuals

bz2::Mul bz2::Mu9

55 59

11 1.5

9, 22, 24, 26, 28 38, 39, 53, 55, 62

a With the exception of the inactive bz2::Mu9 line, all values are rounded off to the nearest integer. The values represent the calculated Mu9 copy number based on densitometric scanning of the autoradiogram shown in Fig. 2B

lecular weight fragments (marked with open arrows in Fig. 2) that were not found in the reactivated progeny. Both SstI ( 5 ' - G A G C T C ) and DraI ( T T T A A A ) are reported to be insensitive to 5-methylcytosine modification at sites that can be methylated in plant D N A , namely 5 ' - C G and 5 ' - C N G (Nelson and McClelland 1991). SstI is methylation sensitive, but only with complete methylation of the internal C or hemimethylation of both C residues. The actual SstI digestion sites in M u 9 elements are in a sequence in which the terminal C should not be methylated; hence although the impact of hemi-methylation at the terminal C alone has not yet been tested

Fig. 2A, B. Mu9 copy number in active, inactive and reactivated lines measured by the abundance of a diagnostic SstI-DraI fragment. Panel A shows bz2: :Mul samples and panel B bz2::Mu9 samples. The sample types are indicated along the top of the figure. DNA was prepared from five germinating seedlings of the active and inactive lines ~0 determine an average copy number in each source. DNA was also prepared from individual reactivated seedlings. Although lane loading was similar based on absorbance units, a normalized loading factor was calculated from .the hybridization signal of the Adhl internal control (bottom section of each panel); note that the lines are segregating for two alleles of Adhl and these signals were summed to determine loading. A series of Mu9 copy number reconstructions of 1, 10, and 50 copies (not shown) was also present on each blot. Multiple autoradiographic exposures were utilized to obtain densitometry information in the linear range for each lane; the blot shown is overexposed for the lanes with the highest Mu9 copy number. The open arrowheads indicate novel high molecular weight fragments in the inactive lines that hybridize to Mu9; the solid arrow in the right margin of the figure indicates the position of the diagnostic 4.2 kb fragment

(Nelson and McClelland 1991) this C should not be methylated in M u 9 . It is possible that one or both o f these enzymes is inhibited by increased cytosine methylation within M u 9 in the regions near the restriction sites. Such sensitivity has not been extensively studied, but has been uncovered in several cases in which highly purified, specifically methylated D N A substrates were employed. Nelson and McClelland (1991) conclude their recent compilation with the statement "...methylation-induced 'action at a distance' m a y be more c o m m o n than has previously appreciated. We have tested only a few enzymes for sensitivity to base modification outside their canonical recognition sequences." Discussion

Ultraviolet light was one of the first agents used to induce mutations in higher eukaryotes. In maize, efficient mutagenesis of pollen was achieved with b o t h U V - C and UV-B, utilizing wavelengths f r o m 240-310 nm (Stadler and Sprague 1936; Stadler and U b e r 1942). Unlike Xrays, which often cause c h r o m o s o m e breaks and multiple marker loss, at least some UV-induced phenotypic changes affect single genes (Neuffer 1957). Using a 30 s exposure of pollen in a chamber similar to that described

358 here, the frequency of mutation of A1 to al was 1.1 x 10 -3. Here I report UV-mediated reactivation of cryptic transposons. I assayed for restoration of somatic instability in potentially mutable reporter alleles of Bz2, a gene encoding a late step in anthocyanin biosynthesis. One allele contains a non-autonomous M u l insert, and the second contains Mu9, the candidate for the regulatory element of the Mutator family. In both lines the frequency of reactivation is increased by UV-C treatment to about 10 -3, a ten-fold increase over the spontaneous frequency. Radiation at 254 nm, the peak frequency for absorption by DNA, has been previously tested for its ability to stimulate mutability at two recessive alleles of al: al-m, containing the receptor of Dt, and al-rnl, containing a dSpm element (Neuffer 1966). The test lines lacked genetically active copies of the respective autonomous elements, Dt or En/Spm. After pollen irradiation, mutability was restored in approximately 10 .3 progeny - there were three mutables among 2868 al-m kernels and two mutables among 1958 kernels carrying al-ml. Considering all of the reported cases, it appears that transcriptionally silent copies of regulatory elements exist in many stocks and that UV-C treatment can activate these heretofore cryptic copies. With the growing ozone depletion and concomitant increase in UV-B fluence on earth, radiation-induced activation of cryptic transposons may lead to a biological amplification of the initial D N A damage. There is substantial support for the hypothesis that Mu9 (Hershberger et al. 1991) and related elements (MuR1 and MuA2 from work by Chomet et al. (1991) and Qin et al. (1991), respectively) are the transposaseencoding elements regulating Mutator activities in maize. Our active Mutator lines contain many copies of Mu9, > 50 in the two examples examined in this study. In parallel with the decrease in M u l copy number observed in lines that have epigenetically lost Mutator activity (Walbot and Warren 1988), such inactive lines contain fewer copies of Mu9. Indeed, in the inactive bz2::Mu9 line there is an average of only 1.5 copies of Mu9 per plant. Upon reactivation, however, Mu9 copy number increased 20 to 40-fold. A dramatic increase in M u l copy number was previously reported for an active but low copy-number Mutator line (Hardeman and Chandler 1989) and was also seen after gamma irradiation-induced reactivation of bz2: .'Mul (Walbot 1988). Amplification of Mu copy number is unusual; copy number maintenance during outcrossing of active lines (Alleman and Freeling 1986; Walbot and Warren 1988) requires only a two-fold increase late in the sporophyte to ensure copy number maintenance in all of the progeny and the reactivated bz2: :Mul line also showed only a two-fold increase. When loss of Mutator activity occurs during the life cycle of a single plant, one occasionally finds one ear with an active Mutator system and a second ear showing loss of activity (Walbot 1986). The simplest expectation would be that the number of Mu9 elements is stabilized in the inactive lineage and then halved relative to the active ear by outcrossing. The very low Mu9 copy num-

ber of the inactive bz2.:Mu9 line compared to a sister active line, suggests that rapid loss of Mu9 elements can occur. It is possible that excision of Mu9 copies can occur without concomitant insertion, effectively reducing copy number. Massive loss of Mu9 may predispose cells to epigenetic loss of activity. A wider survey of Mu9 copy number in inactive lines will be required to address this question. McClintock (1984) coined the term "genomic shock" to describe stress responses that stimulate activation of transposable elements in an otherwise stable stock. The initial example of this phenomenon - cycles of chromosome breakage and repair in maize - resulted in activation of Ac (reviewed in Fedoroff 1983). Subsequently, transposable element activities have been activated following plant disease (Dellaporta et al. 1984; Johns et al. 1985), tissue culture (Peschke et al. 1985), ultraviolet light treatment (Neuffer 1966; this report) and gamma irradiation of seed (Walbot 1988). In addition, cases of spontaneous reactivation of cryptic transposable element activities also occur (Fedoroff 1989; Hardeman and Chandler 1989; Pan and Peterson 1991; Walbot 1986; this report). Although transposable element insertions generally reduce gene activity, it has been argued that excision of transposons may generate the allelic variation required for evolution, the raw material upon which selection acts (Schwarz-Sommer et al. 1985). Activation of transposons during stress could be one mechanism for accelerating the production of new alleles, some of which may positively affect the fitness of the individual or its progeny. The mechanism of "genomic shock" is not clear. If activation of regulatory element transcription is the important factor in reactivation, then at present it appears equally plausible that direct D N A damage and associated repair activity or physiological stress per se could be responsible. Because UV-C is a mutagen, some direct DNA damage undoubtedly occurs. Furthermore, D N A repair following D N A damage can result in demethylation of elements and hence may indirectly activate transcription. On the other hand, germinating maize seeds in 5-azacytidine, an agent that blocks maintenance methylation, is ineffective in reactivating somatic excision in Mutator lines (reviewed in Walbot 1991). This is in contrast to results with methylated, inactive copies of transgenes in tobacco that have been successfully reactivated by 5-azacytidine treatments (c.f. Weber et al. 1990 and references therein) and one report on the reactivation of a maize Uq element by this treatment (Pan and Peterson 1989). Unfortunately, studies of the correlation between methylation and Mutator activity have been based only on the indirect assay of examining the non-autonomous elements such as Mul, and this question must be re-examined using Mu9 and the related or (identical) regulatory elements identified in al-Mum2 stocks (Chomet et al. 1991; Qin et al. 1991). The possibility of direct activation of transposons by physiological stress has received little attention. It is noteworthy, that irradiated pollen became stuck to quartz glass. This demonstrates that UV-C can have a rapid impact on the physical properties of pollen; it is

359 likely t h a t U V light is a b s o r b e d b y the oily p o l l e n k i t t in the exine o f the grain, resulting in the sticky e x u d a t e ( D o b s o n 1989). P r e v e n t i o n o f U V d a m a g e is one role h y p o t h e s i z e d for p o l l e n k i t t , a c o m p l e x m i x t u r e o f m o l e cules d e p o s i t e d b y t a p e t a l cells j u s t p r i o r to a n t h e r dehiscence. T h e r e a c t i o n to U V - C m a y b e p a r t o f n o r m a l p o l l e n b i o l o g y , i.e. the a c u t e r a d i a t i o n t r e a t m e n t acc e l e r a t e d a r e a c t i o n t h a t n o r m a l l y occurs in s u n l i g h t to alter the p r o p e r t i e s o f p o l l e n k i t t to t r a n s f o r m i n d i v i d u a l , n o n - s t i c k y p o l l e n into g r a i n s t h a t will a d h e r e to c o r n silks. Thus, it r e m a i n s a p o s s i b i l i t y t h a t a l t e r e d p o l l e n p h y s i o l o g y r a t h e r t h a n direct D N A d a m a g e results in r e a c t i v a t i o n o f M u t a t o r . A s m a n y stress responses involve the a c t i v a t i o n o f o t h e r w i s e t r a n s c r i p t i o n a l l y silent genes, the p o s s i b i l i t y t h a t u l t r a v i o l e t light directly activates t r a n s c r i p t i o n o f M u 9 w a r r a n t s investigation. Acknowledgements. I thank M.G. Neuffer for his advice on pollen

treatment, Chris Warren for performing the hybridization analyses, Mary Alice Reid and Ann Stapleton for assistance with pollinations, Chaz Andre for preparing figures, Hanya Chrispeels for her comments on the manuscript, and Chester Washington for building the UV treatment box based on the prototype provided by Dr. Neuffer. This research was supported by a grant from the USDA (89-37280-4840).

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C o m m u n i c a t e d b y H. S a e d l e r