SUMMARYJasmonates are plant signalling molecules that play key roles in defence against insects and certain pathogens, among others by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the AP2/ERF-domain transcription factor ORCA3 controls the jasmonate-responsive expression of several genes encoding enzymes involved in terpenoid indole alkaloid biosynthesis. ORCA3 gene expression is itself induced by jasmonate. The ORCA3 promoter contains an autonomous jasmonate-responsive element (JRE) composed of a quantitative sequence responsible for the high level of expression and a qualitative sequence that acts as an on/off switch in response to methyl-jasmonate (MeJA). Here, we identify the basic helix-loop-helix (bHLH) transcription factor CrMYC2 as the major activator of MeJA-responsive ORCA3 gene expression. The CrMYC2 gene is an immediate-early jasmonate-responsive gene. CrMYC2 binds to the qualitative sequence in the ORCA3 JRE in vitro, and transactivates reporter gene expression via this sequence in transient assays. Knock-down of the CrMYC2 expression level via RNA interference caused a strong reduction in the level of MeJA-responsive ORCA3 mRNA accumulation. In addition, MeJA-responsive expression of the related transcription factor gene ORCA2 was significantly reduced. Our results show that MeJA-responsive expression of alkaloid biosynthesis genes in C. roseus is controlled by a transcription factor cascade consisting of the bHLH protein CrMYC2 regulating ORCA gene expression, and the AP2/ERF-domain transcription factors ORCA2 and ORCA3, which in turn regulate a subset of alkaloid biosynthesis genes.

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys 2 /His 2 -type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene responsefactor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.

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Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.

A cDNA encoding a bHLH transcription factor was isolated by the yeast one-hybrid system from a Catharanthus roseus cDNA library using the G-box element of the Strictosidine synthase gene promoter as bait. The corresponding protein (named CrMYC1) was shown to bind specifically to the G-box in yeast. In C. roseus suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate suggesting that CrMYC1 may be involved in the regulation of gene expression in response to these signals.