Copper-controllable gene expression system for whole plants.

Abstract

We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast acel (activating gopper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the 3-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 ,M CuS04 to the nutrient solution or after application of 0.5 ,uM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal ofcopper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

Characterization of a Novel Plant Promoter Specifically Induced by Heavy Metal and Identification of the Promoter Regions Conferring Heavy Metal Responsiveness

“…The use of a heavy metal-inducible promoter to control the target gene expression could avoid potential harmful effects due to overexpression of the target gene under control of constitutive promoters in transgenic plants. For example, the yeast copper-inducible promoter system has been well characterized in transgenic tobacco plants for controlling GUS (Mett et al, 1993) and cytokinin synthase (ipt) genes (McKenzie et al, 1998). Several promoters have failed to induce gene expression in response to cadmium in transgenic plants.…”

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

“…In order to achieve an optimal expression of transgenes with minimum undesirable effects, it is highly desirable to regulate the expression of transgenes in a controllable fashion. A number of chemical inducible gene regulation systems, or gene switches, have been developed based on a diverse collection of non-plant regulatory elements that respond to a variety of chemicals (Gatz et al 1992;Wilde et al 1992;Williams et al 1992;Mett et al 1993;Rieping et al 1994;Weinmann et al 1994;Aoyama and Chua 1997;Caddick et al 1998;Bohner et al 1999;Martinez et al 1999a, b;Bruce et al 2000;Padidam et al 2003). A chemical inducible gene regulation system that specifically regulates transgene expression in response to an exogenous inducer at a particular stage of plant development or in a specific organ is very valuable when using transgenes whose constitutive over expression is detrimental or lethal to the plant.…”

“…In an effort to find an optimal gene switch, several approaches have been tried (Ainley and Key 1990;Schena et al 1991;Gatz et al 1992;Mett et al 1993;Lloyd et al 1994;Weinmann et al 1994;Aoyama and Chua 1997;Bruce et al 2000;. However, most of the gene switches developed to date for use in plants are not suitable for field applications because of the impractical or incompatible characteristics of the chemical ligands.…”

Gene silencing using a heat-inducible RNAi system in Arabidopsis

“…Among the inducible expression systems used in plants, several promoters responding to chemical inducers have been reported (for review, see Gatz and Lenk, 1998). Those promoters are controlled by various external inducers such as tetracycline (Gatz et al 1992), copper (Mett et al, 1993), ethanol (Ait-Ali et al, 2003, and steroid (Zuo et al, 2000). Application of chemical inducers is generally simple; they are effective at a low concentration, and in dose dependent manner.…”