Cloning of the Arabidopsis WIGGUM gene identifies a role for farnesylation in meristem development.

Control of cellular proliferation in plant meristems is important for maintaining the correct number and position of developing organs. One of the genes identified in the control of floral and apical meristem size and floral organ number in Arabidopsis thaliana is WIGGUM. In wiggum mutants, one of the most striking phenotypes is an increase in floral organ number, particularly in the sepals and petals, correlating with an increase in the width of young floral meristems. Additional phenotypes include reduced and delayed germination, delayed flowering, maturation, and senescence, decreased internode elongation, shortened roots, aberrant phyllotaxy of flowers, aberrant sepal development, floral buds that open precociously, and occasional apical meristem fasciation. As a first step in determining a molecular function for WIGGUM, we used positional cloning to identify the gene. DNA sequencing revealed that WIGGUM is identical to ERA1 (enhanced response to abscisic acid), a previously identified farnesyltransferase beta-subunit gene of Arabidopsis. This finding provides a link between protein modification by farnesylation and the control of meristem size. Using in situ hybridization, we examined the expression of ERA1 throughout development and found it to be nearly ubiquitous. This extensive expression domain is consistent with the pleiotropic nature of wiggum mutants and highlights a broad utility for farnesylation in plant growth and development.

Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro.

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

In vitro analysis of a constitutively active mutant form of the Escherichia coli global transcription factor FNR.

The Escherichia coli transcription factor FNR regulates expression of genes required for the metabolic switch between aerobic and anaerobic respiration. In order to investigate how FNR controls transcription of its target operons, DNA binding was examined for both the wild-type (WT) FNR protein and an altered function FNR* protein (DA154) that exhibits enhanced activity in the presence of oxygen both in vivo and in vitro, apparently due to the fact that DA154 is able to dimerize to a greater extent than WT FNR. Electrophoretic mobility shift assays, using a consensus symmetrical FNR target site, revealed that both DA154 and WT FNR gave rise to protein-DNA complexes of indistinguishable electrophoretic mobilities. In addition, an estimate of the molecular weight from the mobility of the DA154-DNA complex indicated that both mutant and WT FNR were dimeric when bound to DNA. Under the same binding conditions, DA154 showed an observed constant of approximately 3 x 10(8) M-1 for the consensus symmetrical target site. In addition, the results of DNA binding competition assays provided evidence that DA154 was a site-specific DNA binding protein, since this mutant protein bound to the consensus symmetrical target site with approximately 40-fold and 250-fold higher affinity than a natural target site from the nar promoter or a non-specific DNA target, respectively. Electrophoretic mobility shift DNA bending assays demonstrated protein-induced DNA bending by both DA154 and WT FNR. In addition, in vitro transcription assays using an FNR-dependent variant of the lac P1 promoter demonstrated levels of transcription activation by DA154 comparable to those observed in vivo. These results provide several new insights into how FNR functions to activate transcription of target genes.