Characterisation of the ethanol‐induciblealcgene expression system for transgenic plants

Abstract

SummaryWe describe a chemically induced gene control mechanism for plants based on the ALCR transcription factor and alcA promoter of Aspergillus nidulans, which we have called the alc system. Ethanol, the chemical inducer, is not toxic at levels required for induction, and can be applied to the plants by spraying, root drenching and addition to liquid growth media. The alc system is very sensitive to ethanol and the induction is rapid; 0.01% (1.7 mM) ethanol in liquid growth media initiates chloramphenicol acetyl transferase (CAT) reporter gene expression within 4 h, with maximal expression occurring after 4 days. In the complete absence of ethanol, there is no detectable expression of CAT, nor do we observe induction in plants subjected to wound, cold or drought stress, or following treatment with either salicylic acid or methyl jasmonate. However, extreme anoxia resulting in elevated levels of alcohol dehydrogenase activity in both roots and leaves gave substantial induction of CAT in leaves but not in roots. We believe that the alc system will have broad utility in the exogenous control of plant gene expression in pure science and that it also has considerable potential in agriculture.

Export of Salicylic Acid from the Chloroplast Requires the Multidrug and Toxin Extrusion-Like Transporter EDS5

“…Therefore, we searched for an alternative strategy that allowed us to increase SA levels in the chloroplasts and bypass the negative feedback regulation of SA accumulation. To that end, a genetic strategy was designed whereby a chloroplast-targeted, ethanol-inducible gene encoding a chloroplast-targeted bifunctional chimeric protein referred to as SA synthase (pSAS) was engineered into eds5 mutants and EDS5-overexpressing plants (Salter et al, 1998). pSAS consists of isochorismate synthase fused to isochorismate pyruvate lyase from Pseudomonas aeruginosa (Mauch et al, 2001).…”

“…The alc gene expression system (Salter et al, 1998) was used to express the plastid-targeted pSAS encoding a chimeric salicylate synthase (Mauch et al, 2001) under the control of an ethanol-inducible promoter. The alc gene expression system is composed of two elements: the alcR encoding the transcription factor (ALCR) and a promoter derived from alcA (alcohol dehydrogenase I) from Aspergillus nidulans.…”

Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS

“…Two of these lines were used for further functional characterization: CpNifS-RNAi-6 and CpNifS-RNAi-9 (denoted as CpNifS-6 and CpNifS-9, respectively). To induce the RNAi construct, plants grown in soil were sprayed and soil-drenched every 4 days with a 2% ethanol solution, a concentration that was reported not to induce stress (26,27); untreated control plants were sprayed with water. For RNAi induction on agar medium, plants were germinated on 0.5 strength Murashige and Skoog medium ϩ 1% sucrose (28) solidified with 0.4% Agargel (Sigma) in 15-cm-diameter Petri dishes with 50 l of 100% ethanol (or water for controls) placed in the center at the time of germination.…”

Synthetic Botany

“…Among proven orthogonal systems for modulation of gene expression are transactivation systems like the aforementioned GAL4 activator, a tTA tetracycline inducible system based on the Escherichia coli tetR repressor (Weinmann et al 1994), and an IPTG-inducible pOp/LhG4 system based on the E. coli lac operon (Moore et al 1998). The various inducible expression systems implemented in plants to date include the dexamethasone responsive rat glucocorticoid (GR) ligand-binding domain (Aoyama and Chua 1997;Craft et al 2005;Samalova et al 2005), ethanol/acetaldehyde-inducible ALCR transcription factor and alcA promoter Salter et al 1998;Roslan et al 2001), DNA-binding domain of the lexA oestrogen receptor (Bruce et al 2000;Zuo et al 2000), copper-inducible ace1 promoter (Mett et al 1993), and ecdysone receptor (EcR) ligand binding-domain inducible by insecticide methoxyfenozide (Martinez et al 1999;Padidam et al 2003;Koo et al 2004). Other useful circuits comprise the Cre-Lox recombinase system that can be used for induction of transcription (Hoff et al 2001) or transgene excision (Chakraborti et al 2008), and a catalytically inactive version of Cas9 fused to regulatory domains.…”