A maize MuDR transposon promoter shows limited autoregulation.

Transgenic maize expressing luciferase under the control of the mudrB terminal inverted repeat promoter (TIRB) of the MuDR transposon was assayed for transgene expression in active and inactive Mutator lines. We find that active MuDR elements increase TIRB-luciferase expression by 2- to 10-fold, relative to nonMuDR or silenced MuDR lines, in embryonic leaves in 75% of plants tested. However, this increase does not persist in juvenile and adult leaves. In pollen, TIRB-luciferase expression is up to 100-fold higher than in leaves but is unaffected by the presence or absence of active MuDR. Because the MuRA transposase binds to a motif within TIRB, we hypothesize that MURA may act as a weak transcriptional activator of TIRB or may partly inhibit host-induced silencing of TIRB in active Mutator lines during the early stages of somatic growth. Our results contrast with those for the maize transposon Spm, in which the TNPA transposase acts as a repressor of the Spm promoter in active Spm lines.

Expression and post-transcriptional regulation of maize transposable element MuDR and its derivatives.

The transposition of Mu elements underlying Mutator activity in maize requires a transcriptionally active MuDR element. Despite variation in MuDR copy number and RNA levels in Mutator lines, transposition events are consistently late in plant development, and Mu excision frequencies are similar. Here, we report previously unsuspected and ubiquitous MuDR homologs that produce both RNA and protein. MuDR transcript levels are proportional to MuDR copy number, and homolog transcript levels increase in active Mutator lines. A subset of homologs exhibits constitutive transcription in MuDR(-) and epigenetically silenced MuDR lines, suggesting independent transcriptional regulation. Surprisingly, immunodetection demonstrated nearly invariant levels of MuDR and homolog protein products in all tested Mutator and non-Mutator stocks. These results suggest a strict control over protein production, which might explain the uniform excision frequency of Mu elements. Moreover, the nonfunctional proteins encoded by homologs may negatively regulate Mutator activity and represent part of the host defense against this transposon family.

The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs.

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near-identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20-fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell-cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.

Vacuolar uptake of the phytoalexin medicarpin by the glutathione conjugate pump.

We have studied the uptake of [3H]-medicarpin and its glutathione conjugate(s) into vacuolar membrane vesicles from etiolated hypocotyls of mung bean (Vigna radiata). Unconjugated medicarpin is taken up at a low rate in the presence or absence of MgATP. However, [3H]-medicarpin-glutathione conjugate(s), prepared by incubation of medicarpin with a total maize glutathione S-transferase preparation, is taken up more than four-fold faster than medicarpin in the presence of MgATP, and this uptake is MgATP-dependent. Uptake of medicarpin-glutathione was not significantly inhibited by the ionophore gramicidin-D, but was strongly inhibited by vanadate and the alternative transport substrate S-(2,4-dinitrophenyl) glutathione. Our results demonstrate, in a model system, the potential utilization of the high affinity, high capacity, uncoupler-insensitive glutathione conjugate pump for the vacuolar transport of an isoflavonoid phytoalexin.

An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation.

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.

Reactivation of Mutator transposable elements of maize by ultraviolet light.

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 DNA damage or immediate physiological stress in the treated pollen.

Developmental and genetic aspects of Mutator excision in maize.

The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.