Figure 1
Comparison of the deduced amino acid sequence of CrDHS1 with known plant nuclear coded chloroplast-specific DHS proteins. Sequences were aligned using ClustalW program located at ExPasy site http://www.expasy.ch. Sequence descriptions and Genbank accession numbers are: StSHKB, S. tuberosum deoxy arabino heptulosonate phosphate synthase (M95201); LeDHS, L. esculentum deoxy arabino heptulosonate phosphate synthase (Z21792); AtDHS, A. thaliana deoxy arabino heptulosonate phosphate synthase (M74820); StDHS, S. tuberosum deoxy arabino heptulosonate phosphate synthase (AB061254), and CrDHS1, C. roseus deoxy arabino heptulosonate phosphate synthase. Numbers of amino acids are indicated on the right. Dashes indicate gaps introduced in order to optimize the alignment. Asterisks (*) indicate conserved residues in all DHS sequences. Single or double dots represent similar amino acids.
Figure 2
Expression patterns of CrDHS1 in response to UV-B irradiation in C. roseus cultured cells. Six-day-old cultured cells were irradiated for 5 min with UV-B light or left untreated, harvested at the indicated time points, and total RNA was isolated and used for RT-PCR as described in materials and methods. Equal cDNA amounts were ensured by amplification of constitutively expressed Rps9 gene as positive control. Quantification of CrDHS1 and CrRPS9 expression was performed. The mean ± S.D. values of three independent experiments using Image Guage software (version 2.54) are shown in an inset. Each figure shows visual (gel plots) and graphical representation of the expression
Figure 3
Effect of suramin on UV-B-induced expression of CrDHS1. Six-day-old cultured cells were treated for 10 min with 0.1 or 1 mM suramin, and were then irradiated with UV-B for 5 min. As control, one set of cells were irradiated with UV-B alone or left untreated. Other details are as in the legend to Fig. 2.
Figure 4
Effect of EGTA and verapamil on UV-B-induced expression of CrDHS1. Six-day-old cultured cells was treated for 10 min with 0.2 or 2 mM of EGTA (A) or 0.5 or 5 μM of verapamil (B), followed by 5 min of UV-B irradiation. Other details are as in the legend to Fig. 2.
Figure 5
Effect of protein kinase inhibitors on UV-B-induced expression of CrDHS1. Six-day-old cultured cells were treated for 10 min with 10 or 100 nM staurosporine (A), 70 nM SB203580 (P38 inhibitor) (B), 40 nM SB600125 (JNK inhibitor) (B) or 5 μM PD98059 (ERKK inhibitor) (C), followed by 5 min of UV-B irradiation. Other details are as in the legend to Fig. 2.
Figure 6
Effect of NAC, NaF and orthovanadate on UV-B-induced expression of CrDHS1. Six-day-old cultured cells were treated for 10 min with 1 and 10 mM of N-acetyl cysteine (A), 1 and 10 mM of NaF (B), or 1 and 10 mM of orthovanadate (C), followed by 5 min of UV-B irradiation. Other details are as in the legend to Fig. 2.
Figure 7
Model for UV-B-induced transcript accumulation of CrDHS1.