Final Report: Impact of UV-B on MaizeEPA Grant Number: R824900
Title: Impact of UV-B on Maize
Investigators: Walbot, Virginia
Institution: Stanford University
EPA Project Officer: Hahn, Intaek
Project Period: December 1, 1996 through November 30, 1999
Project Amount: $450,000
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Ecosystems
Objective:This project has two long-term goals. First, we wish to define the impact of UV-B radiation on mutation induction in maize and to determine whether new mutations result primarily from direct DNA damage or from the activation of mutagenic transposable elements. The second goal is to define the genes whose expression is increased after radiation and to determine the physiological role(s) of the gene products in shielding and repair of damage. In carrying out these objectives, we use supplemental UV-B with a spectrum and fluence typical of slight elevation of UV-B in terrestrial sunlight to examine DNA damage and its consequences in maize somatic leaf tissues and in pollen. We will develop methods for damage detection using antibodies specific to individual types of DNA adducts. We will establish appropriate genetic lines to test the hypothesis that UV-B can reactivate cryptic Mu transposable elements in maize pollen, setting in motion a mechanism to increase mutation frequency.
- Assessing DNA Damage
Using the antibody detection method we perfected in the early 1990s, we can separately quantify 6,4 photoproducts and pyrimidine dimers, which together constitute the major damage after UV-B irradiation. Maize plants were grown under field conditions, in which UV-B fluence varies over the day, and in growth chambers and greenhouses with radiation simulating solar radiation supplemented with UV-B. UV-B supplementation was restricted to the conditions that simulate depletion of no more than 50 percent of the ozone layer in the temperate zone. Whole plant studies were performed for the most part in sufficient light to ensure rapid photoreactivation.
Similarly, field-shed pollen, the male reproductive tissue, was collected from the natural environment, from greenhouse-grown plants without UV-B supplementation (UV-B depleted environment), and from samples irradiated for 10 seconds to 15 minutes, with UV-B simulating increased fluence in the natural environment. Such irradiated pollen samples did not simultaneously receive long wavelength UV-A or visible light at a high fluence, reducing the chances for near-simultaneous photoreactivation of DNA damage.
Major Conclusions From Studies of Somatic Tissues
DNA damage accumulates in sunlight but dark repair restores DNA integrity. Various maize organs were irradiated under diverse regimes, with or without photoreactivating visible light present. In many protocols, epidermal layers were peeled from the underlying tissue so that we could separately assess the amount of damage and rate of DNA repair in the outer and inner tissues. DNA damage was assessed using antibodies directed to the 6,4 photoproducts and antibodies to pyrimidine dimers. Both types of damage accumulate during the day, indicating that repair capacity is insufficient to fully reverse the damage caused by UV-B during daylight hours. During the night, however, the accumulated damage was corrected. This result suggests that "dark repair" of DNA damage is a major contributor in maize, in effect, correcting the accumulated daytime damage to return the plant to baseline by each morning.
Epidermal cells accumulate the highest amount of DNA damage. As predicted from previous work on other plants, most damage occurs in the epidermal layers (80?90% of the DNA dimers and photoproducts) with bulky underlying tissues accumulating much lower damage levels per unit of DNA. We were surprised to find, by confocal microscopy, that the nuclei of maize epidermal cells are near the outer cell surface; this observation suggests that the DNA of the epidermal cell is a major UV-B shield protecting the inner tissue layers.
Anthocyanin contributes to shielding. By manipulating plant genotype, we explored the impact of varying levels of red anthocyanin pigment on DNA damage, either by measuring damage at a specific fluence or varying fluence to achieve a specific level of damage (a test of reciprocity, important in interpreting photobiology experiments). In somatic tissues, most anthocyanin accumulates in epidermal cells, and within these cells the anthocyanin is localized to a large central vacuole that occupies most of the cytoplasmic volume. We found that dark red epidermal layers confer substantial shielding to underlying inner tissues. Because the epidermal nuclei lie above the central vacuole, epidermal damage often remained high even in red tissues. We conclude that anthocyanin (and related colorless flavonoids that absorb in UV-B) likely contribute substantially to shielding the internal cells where photosynthesis occurs. This shielding may prevent irreversible damage to photosynthetic reaction centers. This component of our study identifies one pathway (one set of genes) that contribute substantially to DNA shielding in leaves.
Major Conclusions From Studies With Pollen
Supplemental UV-B is mutagenic. Pollen of several genotypes was irradiated for 0 to 15 minutes at fluence rates expected in the natural environment after depletion of 33?50 percent of the ozone in the temperate zone. Control pollen (0 irradiation, but otherwise handling was equivalent to the treatment groups) and test pollen samples were delivered to recipient unfertilized ears of the bronze2 (bz2) genotype. Bz2 encodes a glutathione S-transferase that is required for anthocyanin pigment sequestration in the vacuole; when mutant (bz2), kernels are bronze-colored. The control and test pollen were Bz2, consequently, purple kernels were expected from the cross (Bz2/bz2). Bronze-colored kernels could indicate a mutation in the Bz2 gene in pollen or contaminating bz2 pollen.
Although no verified new bz2 mutants were recovered, this assay scores for mutation at only one locus. For a more robust assessment of mutation frequency, the F1 kernels were grown to adult plants and self-pollinated. Nearly all (199/200) new maize mutations are recessive, only visible when both copies of a gene are mutant. Consequently, we reasoned that new, recessive mutations could be present in kernels derived from irradiated pollen, and that the presence of these new mutations could be scored in the self-pollinated F2 generation. This hypothesis was verified: 7 minutes of supplemental radiation increased mutation frequency of new visible seedling mutations significantly above background, and a few mutations were also recovered after 3 minutes of supplemental radiation. As corn pollen is wind-borne and is viable for 15?30 minutes, the experimental parameters indicated that supplemental UV-B could increase the mutation frequency. After the irradiation, pollen was exposed to low fluence sunlight (through the brown paper pollinating bag) allowing some opportunity for photoreactivation or other DNA repair under normal conditions. Mutations are not permanent until DNA replication, which in sperm nucleic present in pollen does not occur until after fertilization to form the zygote and endosperm, approximately 24 hours after pollination or until after DNA repair in which a "mistake" is made in attempting to correct the DNA damage. Given the long delay between pollination and fertilization, we propose that UV-B is mutagenic to pollen because error-prone DNA repair "fixes" mistakes in the DNA prior to fertilization.
Flavonoids do not act as UV-B shields in pollen. Using genotypes that differ in pollen pigmentation (white pollen = no flavonoids, yellow pollen = flavonoids, red pollen = flavonoids including red anthocyanins), we tested whether modulating the amount of UV-B absorbing flavonoid pigments contributed to DNA shielding in pollen. Using the antibody detection methods outlined above, we determined that pollen flavonoid pigments provide only a small component of shielding. This was a surprising result. We propose that flavonoid distribution within pollen may be punctate, allowing most UV-B to pass through to the DNA. In these experiments, we also verified that reciprocity holds in DNA damage induction in pollen?doubling dosage results in equivalent damage in half the time as a comparison dose. Reciprocity was verified at several fluence rates.
Technical Limitations of the Work
The pollen assays are difficult to perform, because corn pollen is viable for 15 minutes to 2 hours after shedding. This provides only a short window of time for treatments; this was the primary reason that we did not implement additional experiments asking if photoreactivating radiation provided during UV-B treatment, or immediately following UV-B treatment, would result in lower damage levels. With every passing minute in the pollen manipulations, total pollen viability decreases based on pollen germination assays. The advantages to using corn include the ability to collect very large (>107 grains) pollen samples quickly, and the fact that cross-pollination is so easy. In other species, anther emasculation is typically required to prevent self-pollination of flowers. Despite the added technical difficulty of emasculation to achieve crosses, we recommend that future studies of UV-B impact on pollen be conducted with species in which pollen viability is higher. For example, tomato pollen can be stored for up to 2 years under desiccation, and it is viable for several days under laboratory conditions with no special precautions. The added flexibility in handing pollen samples would be a significant advantage.
- Test for Reactivation of Cryptic Mu Transposable Elements
Background to the Hypothesis
Transposable elements were discovered in corn by Barbara McClintock; such elements were subsequently discovered in all organisms examined. When these elements insert into genes, function is usually disrupted. Consequently, transposable elements are an endogenous mutagen in an organism or species. In most individuals, however, the endogenous transposable elements are immobile or silent. In bacteria, such silent elements can be reactivated by chemical and radiation treatments. Similarly, Walbot (1986, 1992) demonstrated that highly energetic UV-C and gamma irradiation of corn could reactivate maize Mutator transposable elements. These harsh treatments are never found in a terrestrial ecosystem. The purpose of the study was to determine whether the conditions established as causing DNA damage in maize leaves and pollen were sufficient to activate silent Mutator transposable elements.
Verifying lines with completely silent Mutator transposons. When the Mutator system is active, the Mu elements disrupting the function of Bz2 cause kernels to be complete bronze; in contrast, when the Mutator system is active, the Mu elements excise restoring red pigmentation in small sectors in all somatic tissues that normally synthesize anthocyanin. bz2::Mu1 and bz2::MuDR reporter alleles were used in this study. Lines homozygous for one of the two reporter alleles were self-pollinated, and bronze kernels selected. We verified that the Mutator system was off and that the reporter allele itself was still intact and potentially mutable, but crossing plants grown from bronze kernels to an active bz2 Mutator tester. The progeny had red spots. The test plant also was self-pollinated. This protocol was followed for seven generations to develop lines that had silenced Mu elements for one to seven generations. At each generation the frequency of spontaneous reactivation was measured; we determined that such spontaneous events are very rare by the fourth generation and exceedingly rare by the seventh generation.
Pollen irradiation of silent Mutator lines can restore mutability. Using the lines described above, pollen was irradiated using the protocols developed for DNA damage assessment with antibodies. In these experiments, we measured restoration of red spots in progeny kernels as evidence for the reactivation of Mutator activity. Tens of thousands of kernels were examined in the course of these experiments. The experiments demonstrated reactivation that could be as efficient as crossing to an active Mutator plant, although in most experiments, reactivation was modest. Reactivation was confirmed by demonstrating that reactivated individuals had RNA transcripts encoded by MuDR, the "master" element programming Mutator activities, whereas the silent population lacked these transcripts by RNA blot hybridization assays.
Because maize pollen contains two sperm, we also could ask if reactivation was a physiological effect impacting both sperm, which exist in a single cytoplasm, or a nuclear-specific event that would affect one sperm but not the other in the endosperm. This test is called a concordance assay because it determines whether the two sperm in one pollen grain produce the same phenotype in the endosperm and embryo. The results indicate conclusively that Mutator reactivation is non-concordant, and must, therefore, occur in individual sperm. Collectively, the results indicate the potential for modest increases in UV-B to reactivate cryptic transposable elements in maize. Once active, the transposons can cause new mutations to appear for many generations.
- Additional Studies
Because of our expertise in DNA damage detection in plant tissues, we were asked to collaborate in an analysis of the defect in the uvr2 mutation in Arabidopsis. Our contribution verified that plants defective in this gene function are unable to repair pyrimidine dimers. The gene encodes a photolyase (based on function in vitro and by sequence homology to other proteins with this activity). This project established that plants lacking this photolyase activity could be killed by low fluence UV-B.
We propose that a genomics approach to understanding UV-B impact in maize will be the next step in our research. Rapid progress in implementing new tools for surveying gene expression patterns and for identifying mutations in specific genes provides an excellent opportunity for broadening the definition of the impact of UV-B on maize. Microarrays of tens of thousands of expressed sequence tags (ESTs) are under construction for maize, based on the sequenced cDNAs from the Maize Gene Discovery Project (National Science Foundation funded plant genomics project, the principal investigator is V. Walbot). These arrays will contain approximately 20,000 gene tags, allowing surveys of changes in gene expression for about 40 percent of maize genes. For example, it would be highly instructive to compare the range of gene expression in leaves grown with UV-B removed by filters, normal fluence sunlight, and sunlight supplements with UV-B. By comparing the gene expression status of genetically identical individuals, genes whose expression increases or decreases with treatment can be identified. This subset of genes?perhaps 100 to 200?can be analyzed for common features, such as participation in particular pathways. Indeed, we expect up-regulation of the anthocyanin biosynthetic pathway to produce additional sunscreen and would expect to see more abundant transcripts for the seven genes in this pathway after supplemental UV-B exposure. Genes for repair of DNA, protein, and membrane damage caused by radiation also would be expected to be up-regulated in parallel with increasing dosage of non-lethal radiation. In addition, genes heretofore unexpected to participate in the responses to UV-B also could be identified, resulting in new insights into damage consequences, damage repair, and physiological/developmental responses to radiation changes.
Microarray technology will revolutionize the ability of molecular biologists to determine which genes are coordinately regulated in response to an environmental challenge. As maize is a large plant and it is easy to maintain and construct lines of known genetic status, it should be possible to obtain RNA samples from virtually any tissue and any genotype for analysis of gene expression changes resulting from UV-B exposure. These studies would greatly extend the now proven ability of UV-B to activate cryptic transposable elements in maize.
A second path to understanding the role of specific genes in UV-B tolerance and damage repair is to exploit gene "knockout" technology. Here, the sequences of genes identified as expressed under specific UV-B regimes are used to screen a large collection of RescueMu transposon insertions into maize genes. A collection of approximately 150,000 such "knockout" mutations is being assembled and sequenced by the Maize Gene Discovery Project (http://zmdb.iastate.edu) and is summarized in: Walbot, V. Saturation mutagenesis using maize transposons. Current Opinion in Plant Biology, 2000 (in press). Eliminating the function of individual genes provides genetically defined material to ask if a particular gene is lethal if mutant or if a visible phenotype occurs during UV-B exposure. Furthermore, hidden phenotypes can be detected by microarray analysis on RNA samples from individual mutants compared to the normal parental line.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
|Other project views:||All 5 publications||3 publications in selected types||All 3 journal articles|
||Landry LG, Stapleton AE, Lim J, Hoffman P, Hays JB, Walbot V, Last RL. An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation. Proceedings of the National Academy of Sciences, USA 1997;94(1):328-332.||
||Stapleton AE, Thornber CS, Walbot V. UV-B component of sunlight causes measurable damage in field-grown maize (Zea mays L.): Developmental and cellular heterogeneity of damage and repair. Plant, Cell and Environment 1997;20(3):279-290.||
||Walbot V, Stapleton A. Reactivation potential of epigenetically inactive Mu transposable elements of Zea mays L. decreases in successive generations. Maydica 1998;43(3):183-193.||