UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

Abstract

The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase.

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.