Differential expression of the Arabidopsis cytochrome c genes Cytc-1 and Cytc-2. Evidence for the involvement of TCP-domain protein-binding elements in anther- and meristem-specific expression of the Cytc-1 gene

Abstract

The promoters of the Arabidopsis (Arabidopsis thaliana) cytochrome c genes, Cytc-1 and Cytc-2, were analyzed using plants transformed with fusions to the beta-glucuronidase coding sequence. Histochemical staining of plants indicated that the Cytc-1 promoter directs preferential expression in root and shoot meristems and in anthers. In turn, plants transformed with the Cytc-2 promoter fusions showed preferential expression in vascular tissues of cotyledons, leaves, roots, and hypocotyls, and also in anthers. Quantitative measurements in extracts prepared from different organs suggested that expression of Cytc-1 is higher in flowers, while that of Cytc-2 is higher in leaves. The analysis of a set of deletions and site-directed mutants of the Cytc-1 promoter indicated that a segment located between -147 and -156 from the translation start site is required for expression and that site II elements (TGGGCC/T) located in this region, coupled with a downstream internal telomeric repeat (AAACCCTAA), are responsible for the expression pattern of this gene. Proteins present in cauliflower nuclear extracts, as well as a recombinant protein from the TCP-domain family, were able to specifically bind to the region required for expression. We propose that expression of the Cytc-1 gene is linked to cell proliferation through the elements described above. The fact that closely located site II motifs are present in similar locations in several genes encoding proteins involved in cytochrome c-dependent respiration suggests that these elements may be the target of factors that coordinate the expression of nuclear genes encoding components of this part of the mitochondrial respiratory chain.

Figure 1.

Histochemical localization of GUS activity in Arabidopsis plants transformed with either the Cytc-1 (A–L) or Cytc-2 (M–X) promoters fused to the gus reporter gene. A, Three-day-old seedling showing staining in the root tip (RT). B, Upper portion of a 7-d-old seedling showing staining in cotyledon tips (CT) and apical meristem (AM). C and D, Upper portion of 12- and 15-d-old seedlings showing staining in nascent leaves (NL) and apical meristem (AM). E and F, Cotyledon and leaf of a 20-d-old plant. G and H, Primary root showing staining at the tip (RT) and nascent secondary root (NSR). I, Silique. J, Anther. K, Flower. L, Lateral root showing staining at the base (LRB). M to P, Seedlings at different developmental stages. Q and R, Cotyledon and leaf of a 20-d-old plant. S and T, Primary root and nascent secondary root showing staining in the vascular cylinder (VC). U and V, Siliques showing staining in the septum (S) and the funiculus (F). W, Flower. X, Lateral root.

Figure 2.

Analysis of GUS activity in protein extracts from different organs of plants transformed with either the Cytc-1 or the Cytc-2 promoter-gus fusions. Specific GUS activity was determined using protein extracts prepared from different organs (as indicated) of plants carrying either the Cytc-1 or the Cytc-2 promoters fused to gus. Activity was also measured in extracts from plants transformed with a promoterless gus gene (pBI101). Bars indicate the mean activity values obtained with organs from three independent transformants for each construct. Error bars represent se. Similar results were obtained in other experiments in which different lines were used.

Figure 3.

Analysis of GUS activity driven by truncated forms of the Cytc-1 promoter. A, Constructs used for the analysis of the Cytc-1 promoter. Numbers indicate the upstream end of the promoter fragment present in each construct, with respect to the translation start site (TS); the downstream end was at +54 for all constructs. Construct Δ (147–218) carries a deletion comprising the indicated nucleotides within the context of the −369 promoter fragment. Plants transformed with the promoterless gus gene (pBI101) were also used. B, GUS expression levels in organs of plants carrying the different constructs. The results indicate the mean (± se) of three independent lines. Numbers below the bars indicate the respective construct. Similar results were obtained in other experiments in which different lines were used.

Figure 4.

Mutagenic analysis of the Cytc-1 promoter region required for expression. A, Sequence of the Cytc-1 promoter regions that were analyzed by mutagenesis. Introduced mutations are specified below the wild-type sequence; asterisks indicate identical nucleotides. Numbers indicate the position relative to the translation start site. Site II elements and the telo-box are underlined. B, Specific GUS activity was determined using protein extracts prepared from different organs (as indicated) of plants carrying mutagenized promoter fragments fused to gus. All constructs contain the region-spanning nucleotides −218 to +54 of the Cytc-1 gene. Activity was also measured in extracts from plants transformed with the −146 to +54 fragment (−146) and with the promoterless gus gene (pBI101). The bars represent mean (± se) values for three independent lines carrying each construct. Similar results were obtained in other experiments in which different lines were used.

Figure 5.

Histochemical analysis of GUS activity in Arabidopsis plants transformed with mutagenized promoters fused to the gus reporter gene. Seedlings (A–F), cotyledons and nascent leaves (G–L), flowers (M–R), or roots (S–X) from plants transformed with either the wild-type (−218) promoter or the same promoter with mutations (as described in Fig. 4) in the telo-box, the site II elements, the pollenQ motif, or the regions from −147 to −156 or from −187 to −196 were analyzed by histochemical staining.

Figure 6.

Induction of the Cytc-1 gene by Suc and cytokinins. GUS activity was measured using the fluorogenic substrate MUG and protein extracts prepared from 7-d-old seedlings grown in Murashige and Skoog (MS) medium alone or supplemented with either 3% Suc or 50 μm BAP, as indicated. The plants used for the analysis were a mix of four independent lines for each construct, as indicated. Activity was also measured in extracts from nontransformed plants (wt). Similar results were obtained in other experiments in which different lines were used.

Figure 7.

Nuclear proteins from cauliflower inflorescences and recombinant proteins AtTCP20 and AtPuralpha specifically bind to the Cytc-1 promoter. A, Nuclear extracts (3 μg) from cauliflower inflorescences were analyzed by an electrophoretic mobility shift assay for the presence of proteins that bind to labeled DNA spanning nucleotides −21 to −218 (lane 1), −21 to −146 (lane 2), or −126 to −218 (lane 3). In lane 4, a fragment spanning nucleotides −126 to −218 with mutations in its site II elements (as described in Fig. 4) was used. B, The binding of nuclear proteins to labeled fragment −126 to −218 was analyzed in the presence of only unspecific competitor (lane 1) or with the addition of a 10- or a 50-fold molar excess of unlabeled wild-type (lanes 2 and 3) or mutated forms (lanes 4 and 5) of the same fragment. In lanes 6 to 10, a similar experiment was carried out using the mutated form of the fragment as labeled probe. C, Binding of recombinant protein AtTCP20 to the Cytc-1 promoter. An aliquot (200 ng) of recombinant protein AtTCP20 was analyzed by an electrophoretic mobility shift assay for binding to a Cytc-1 promoter fragment spanning nucleotides −126 to −218 (lane 1) or to the same fragment with mutations introduced in the regions −187/196 (lane 2), −177/−186 (lane 3), or in the site II elements (lane 4). D, Binding of recombinant protein AtPuralpha (200 ng) to Cytc-1 promoter fragments spanning nucleotides −21 to −218 (lane 1) or −21 to −146 (lane 2), or to a similar fragment with mutations within the telo-box (lane 3) as described in Figure 4.