Isolation of a gene encoding a copper chaperone for the copper/zinc superoxide dismutase and characterization of its promoter in potato

Abstract

Gene expression during the potato (Solanum tuberosum) tuber lifecycle was monitored by cDNA-amplified fragment-length polymorphism, and several differentially expressed transcript-derived fragments were isolated. One fragment, named TDFL431, showed high homology to a copper (Cu) chaperone for Cu/zinc superoxide dismutase (CCS). The Ccs protein is responsible for the delivery of Cu to the Cu/zinc superoxide dismutase enzyme. The potato CCS (StCCS) full-length gene was isolated, and its sequence was compared with CCSs from other species. The promoter region of this gene was isolated, fused to the firefly luciferase coding sequence, and used for transformation of potato plants. The highest level of StCCS-luciferase expression was detected in the cortex of stem (like) tissues, such as stem nodes, stolons, and tubers; lower levels were detected in roots and flowers. The StCCS promoter contains regions highly homologous to several plant cis-acting elements. Three of them are related to auxin response, whereas four others are related to response to various stresses. Induction of the StCCS promoter was analyzed on 18 media, differing in hormone, sugar, and Cu content. StCCS expression was induced by auxin, gibberellins (GA4 + 7), fructose, sucrose, and glucose and was inhibited by relatively high concentrations of Cu.

Figure 1.

cDNA AFLP analysis of TDFL431 expression during the tuber life cycle. The expression profile during the 10 d of the in vitro tuberization system (A) and in the 12 time points of dormancy template (B). The numbers indicate days in A and weeks in B; *, 1 week after sprouting; #, 3 weeks after sprouting. C, The expression in 12 different plant tissues: young leaves (Ly), old leaves (Lo), petioles (P), stem nodes (N), stem internodes (IN), roots (R), young stolons (Sy), swelling stolons (Sw), young tubers (Ty), dormant tubers (To), tubers after sprouting (Tas), and sprouts (Sp). The black dotted line indicates the band corresponding to the TDFL31.

Figure 2.

Genomic organization of potato CCS gene(s) revealed by Southern-blot analysis. Genomic DNA from potato cv Bintje was digested with the following restriction enzymes: XhoI (Xo), XbaI (Xa), HindIII (H), EcoRV (EV), BamHI (B), and EcoRI (EI). The immobilized fragments were hybridized with the TDFL431 fragment.

Figure 3.

StCCS gene and its promoter. A, Organization of the five introns (In1, 290 bp; In2, 679 bp; In3, 113 bp; In4, 266 bp; and In5, 521 bp) and six exons (E1, 255 bp; E2, 27 bp; E3, 73 bp; E4, 135 bp; E5, 200 bp; and E6, 243 bp) in the StCCS genomic fragment of 2,802 bp encoding a protein of 311 amino acids. The TIS is represented with an arrow and the poly(A) tail is represented with AAA. B, StCCS promoter sequence. Putative promoter cis-elements are labeled with boxes (Box A–H). Both the TATA box and CAAT box are built-in bold lines. The TIS is labeled with an arrow, and the translation start site with M.

Figure 4.

The amino acid sequence of the potato StCcsp (accession no. AY196210) is shown in alignment with the sequences from other species available in GenBank. These include tomato (AF117707.2), Arabidopsis (AF179371.1), soybean (AF329816.1), human (AF002210.1), and yeast Lys7p (AAC49068.1). The amino acids different from the consensus are shaded. Vertical bars separate the three protein domains identified in human and mouse. The asterisks represent the localization of the introns in potato CCS, and the black horizontal line represents the TDFL431. The conserved motifs are in bold lettering, and the consensus sequence is indicated underneath. The circles indicate the Cys residues conserved in human and mouse.

Figure 5.

Expression of the firefly luciferase gene under the control of the potato StCCS promoter. On the left side of this figure, there is a schematic drawing illustrating the organs where luciferase expression has been measured. The second panel includes 16 pictures of the organ(s) measured in the luminometer (left side) and the results of the luciferase measurements (right side). Both pictures of the luciferase activity and corresponding plant organs are displayed with the same amplification. The colors resulting from the luciferase activity range from blue (low activity) to red (high activity) as represented in the lower left corner of the figure. A and B illustrate LUC⁺ expression in the aerial part, whereas C shows the underground part of a 2.5-month-old potato plant. The part of the plant where measurements A, B, and C were performed is indicated in the drawing next to these pictures. D shows expression in a tuber sprout, E in a flower, F and G in a sectioned stem node, and H in a sectioned tuber. The white bars in A through D correspond to 3.5 cm and in E through H to 0.35 cm. The white arrow in C points to the tuber. Rt, Relative time of luciferase activity measurements. Underneath D and H, an overview of the relative expression in different organs is given (0 corresponds to no activity and +++++ to the highest activity).

Figure 6.

Northern-blot analysis of StCCS transcript. A indicates the results of the hybridization with the StCCS gene and B with the 25S ribosomal probe.

Figure 7.

Activity of StCCS promoter driving luciferase gene in transgenic plants grown for 8 d on media supplemented with 18 different sugars, hormones, and Cu. Cu, Cu sulfate; JA, jasmonic acid; GA, gibberellins (GA_{4 + 7}); ABA, abscisic acid; 2,4-D is a synthetic auxin; BAP, benzylaminopurine; CCC, 2-chloroethyltrimethylammonium chloride. In each sample, the luciferase expression was measured for 1 h, and the results are shown in relative light units per minute (RLU/min).