Search

The promoter of the nuclear gene, GAPB, which encodes the B subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GADPH) of Arabidopsis thaliana, was previously shown to contain four direct repeats (Gap boxes, located between -237 and -181) that were necessary but not sufficient for light-activated gene transcription. To identify additional elements located between the Gap boxes and TATA box, various GAPB promoter fragments driving the beta-glucuronidase (GUS) reporter gene were constructed in transgenic Arabidopsis. We found a 23 bp element (the XXIII element), centered at -119, that is essential for promoter activity. Mutations in the XXIII element abolished transcription of GAPB completely. Furthermore, we have identified three additional elements, PI, Tboxes, and PII that serve as positive modulators in the light-activated transcription of GAPB. Mutations in any of these three elements resulted in the reduction in light inducibility of the GAPB gene. The PI, XXIII, Tboxes and PII sequences are novel cis-acting elements that are not present in the closely related GAPA promoter or other promoters that are similarly regulated by light. In our current study, we found that transgenic Arabidopsis containing a GAPB promoter::GUS construct with all four Gap boxes deleted exhibited significant GUS expression albeit reduced to 42% of the optimal expression level. In contrast, in previous studies on transgenic tobacco, total abolishment of GUS expression was seen when the Gap boxes were deleted. This suggests that different trans-acting factors present in heterologous systems may result in variability of the expression of the transgene.

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is known to be induced by environmental stresses and regulated developmentally. We used a negative-selection approach to isolate mutants that were defective in regulating the expression of the ADH gene during seed germination; we then characterized three recessive mutants, aar1-1, aar1-2, and aar2-1, which belong to two complementation groups. In addition to their defects during seed germination, mutations in the AAR1 and AAR2 genes also affected anoxic and hypoxic induction of ADH and other glycolytic genes in mature plants. The aar1 and aar2 mutants were also defective in responding to cold and osmotic stress. The two allelic mutants aar1-1and aar1-2 exhibited different phenotypes under cold and osmotic stresses. Based on our results we propose that these mutants are defective in a late step of the signaling pathways that lead to increased expression of the ADH gene and glycolytic genes.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.

We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.

We have characterized the effects of different light spectra on expression of the nuclear genes (GapA and GapB) encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis thaliana. Steady-state mRNA levels for both genes in etiolated seedlings increased after a short exposure to red or blue light. However, these increases could not be reversed by immediate far-red light following the initial light treatment. In mature plants, a short light pulse, regardless of its spectrum, had no apparent effect on GapA or GapB mRNA levels in dark-adapted plants. In contrast, continuous exposure to red, blue, or white light resulted in increases of GapA and GapB mRNA levels, with blue and white light being far more efficient than red light. Similarly, continuous exposure of etiolated seedlings to red, blue, or white light also resulted in increased GapA and GapB mRNA levels. In addition, we show that illumination of red light-saturated Arabidopsis plants with continuous blue light results in further increases of GapA and GapB mRNA levels. Based on these results, we conclude that both blue light photoreceptor- and phytochrome-mediated pathways are involved in light regulation of GapA and GapB genes in Arabidopsis, with blue light acting as the dominant regulator.

We report here effects of three environmental conditions, heat shock, anaerobic treatment, and carbon source supply, on expression of nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana. The steady-state mRNA level of the GapC increased when Arabidopsis plants were transferred from normal growth condition to heat-shock, anaerobiosis, or increased sucrose supply conditions. In contrast, the steady-state mRNA levels for GapA and GapB genes were unaffected or decreased transiently under the same treatments. To identify the cis-acting regulatory elements, transgenic tobacco plants containing a 820-bp GapC 5'-flanking DNA fragment and beta-glucuronidase (Gus) fusion were constructed. Analyses of these transgenic plants indicate that this 820-bp DNA fragment is sufficient to confer both heat-shock and anaerobic responses. These results suggest that transcriptional level control is involved in regulation of GapC expression under these stress conditions. Histochemical analysis of Gus activity indicates that expression of the GapC is cell-type specific and is probably linked to the metabolic activity of the cells.