Characterization of cis-acting elements in light regulation of the nuclear gene encoding the A subunit of chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.

Abstract

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.

Identification of a Light-Responsive Region of the Nuclear Gene Encoding the B Subunit of Chloroplast Glyceraldehyde 3-Phosphate Dehydrogenase from Arabidopsis thaliana

“…Analysis of the GapA gene, which is coordinately regulated with the GapB gene under several different growth conditions that we have examined (Shih and Goodman, 1988;Dewdney et al, 1993), reveals that three copies of similar repeats with the consensus sequence 5'-CAAATGAA(G/ A)A-3' exist in its promoter region (Conley et al, 1994). Deletion of one copy of these repeats from the GapA promoter reduces light induction by 3-fold, whereas deletion of all three repeats from the GapA upstream region abolishes light induction completely (Conley et al, 1994). Taken together these results suggest that the promoter regions containing these repeats (Gap boxes) are essential for light regulation of the GapA and GapB genes in A. thaliana.…”

“…The binding sites for GAPF are the Gap boxes within these two promoter fragments as shown using DNA fragments consisting only of a 32-bp Gap box dimer (AID) or a 16-bp Gap box monomer (AIM) as the competitor ( Fig. 10; Conley et al, 1994). In vitro binding of the GapA promoter fragment (Fig.…”

“…We have previously identified three direct repeats with the consensus sequence 5'-CAAATGAA(G/A)A-3' located within an upstream region of the Gap A gene that is essential for light responsiveness (Conley et al, 1994). Examination of the GapB upstream sequence indicated that there are four similar repeats located between -236 and -179 ( Fig.…”

Light Induces Peroxisome Proliferation in Arabidopsis Seedlings through the Photoreceptor Phytochrome A, the Transcription Factor HY5 HOMOLOG, and the Peroxisomal Protein PEROXIN11b

“…We found a total number of 19 putative LREs (Supplemental Fig. S3), including seven GATA boxes (Lam and Chua, 1989), six GT1 boxes (Villain et al, 1996), three I boxes (Giuliano et al, 1988), one AE box (Park et al, 1996), one ATCT motif (Conley et al, 1994), and one GGATAA element (Tepperman et al, 2001). GATA, GT1, and I boxes are cis-elements commonly existing in light-regulated promoters and shown to be essential for light-controlled transcriptional activity (Tobin and Kehoe, 1994;Terzaghi and Cashmore, 1995;Millar and Kay, 1996).…”

DORNRÖSCHENis a direct target of the auxin response factor MONOPTEROS in theArabidopsisembryo

“…For crosslinking, nuclei were prepared from immature siliques (5g), essentially as described in (Conley et al, 1994), isolated nuclei were resuspended in nuclear isolation buffer (NIB: 50 mM Hepes pH 7.4, 5 mM MgCl 2 , 25 mM NaCl, 5% sucrose, 30% glycerol, 0.1% β-mercaptoethanol) supplemented with 20 μg/ml plant protease inhibitor cocktail (PPIC; Sigma P9599), 1 mM Na-orthovanadate and 1 mM NaF (NIBA). Crosslinking was performed with 1% formaldehyde for 5 minutes at room temperature in NIBA and stopped by pelleting and resuspending nuclei in NIBA containing 0.1 M glycine.…”