Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseus

Abstract

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Figure 1

PE and octadecanoids induce the TIA biosynthetic genes Tdc and Str. C. roseus cells were exposed to DMSO as a control (C) or PE, linoleic acid (LE), α-linolenic acid (α-LA), or MeJA for 6 h, after which time cells were harvested and total RNA was isolated. Northern blots were hybridized with Tdc, Str, and Rps9 cDNAs. The micromolar concentrations of the octadecanoids are given at the top.

Figure 2

YE elicitor induces JA biosynthesis. A, Amount of JA in C. roseus cells at different times after the addition of water as a control (□) or PE (▴) or crude (•) YE. DW, Dry weight. B, Str, Tdc, and Rps9 mRNA levels at different times after the addition of water as a control (C) or of PE or crude (CE) YE. RNA was extracted from the same sample of tissue used to extract JA. The times in hours are indicated (top).

Figure 3

A, Effect of DIECA on TIA biosynthetic gene expression. C. roseus cell suspensions were incubated with DIECA for 10 min prior to the addition of water as a control (C), PE, or 50 μm MeJA, and cells were harvested 6 h later. The micromolar concentrations of inhibitors are indicated at the top. B, Effect of octadecanoid inhibitor on PE-induced JA accumulation. C. roseus cells were incubated with water or PE for 3 h. DIECA was added 10 min before the addition of PE. Bars represent se (n = 3).

Figure 4

Protein kinases act upstream and downstream of JA. A, Effect of inhibitors on TIA biosynthetic gene expression. C. roseus cell suspensions were incubated with DSMO as a control (C), K-252a, calyculin A (CalA), or OA for 10 min before the addition of either PE or 50 μm MeJA, and cells were harvested 6 h later. The micromolar concentrations of the inhibitors are indicated at the top. B, Inhibition of PE-induced JA accumulation by a protein kinase inhibitor. C. roseus cells were incubated with 1 μm K-252a for 10 min before the addition of PE, and cells were harvested 3 h later. Bars represent the se (n = 3).

Figure 5

The Str promoter responds to elicitor and MeJA mRNA levels of gusA, Str and Rps9 in independent transgenic cell lines containing the BH construct (BH#1, BH#2, BH#3, and BH#4). Cells were incubated for 6 h with DMSO as a control (C) or with PE or MeJA. RNA (20 μg) was loaded per lane.

Figure 6

Model for elicitor signal transduction leading to TIA biosynthetic gene expression. The model shows the involvement of protein phosphorylation and the octadecanoid pathway in elicitor-induced TIA biosynthetic gene expression. Figure was adapted from Farmer and Ryan (1992). PK, Protein kinase; PP, protein phosphatase; TAF, transcription-activating factor.