Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes  

Abstract

To monitor the expression of T-DNA-tagged plant genes in vivo, a collection of 20,261 transgenic lines of Arabidopsis (Columbia-0) were generated with the promoter trap vector pTluc, which carries a promoterless firefly luc (luciferase) reporter gene linked to the right T-DNA border. By detection of bioluminescence in 3-week-old seedlings, 753 lines were identified showing constitutive, organ-specific, and stress-responsive luciferase expression patterns. To facilitate the identification of well-defined luciferase expression patterns, a pooled seed stock was established. Several lines showed sugar, salt, and abscisic acid (ABA)-inducible luciferase activity. Segregation analysis of 215 promoter trap lines indicated that about 50% of plants contained single insertions, whereas 40% carried two and 10% carried three or more T-DNA tags. Sequencing the T-DNA insert junctions isolated from 17 luciferase-expressing lines identified T-DNA tags in 5Ј-and 3Ј-transcribed domains and translational gene fusions generated by T-DNA insertions in exons and introns of Arabidopsis genes. Tissue specific expression of eight wild-type Arabidopsis genes was confirmed to be similar to the luminescence patterns observed in the corresponding luciferase-tagged lines. Here, we describe the characterization of a transcriptional luc reporter gene fusion with the WBC-type ABC transporter gene At1g17840. Expression of wild-type and luciferase-tagged At1g17840 alleles revealed similar induction by salt, glucose, and ABA treatments and gibberellin-mediated down-regulation of ABA-induced expression. These results illustrate that luciferase gene traps are well suited for monitoring the expression of stress-responsive Arabidopsis genes in vivo.

The ArabidopsisDESPERADO/AtWBC11Transporter Is Required for Cutin and Wax Secretion

“…Supportive evidence for the importance of DSO function in response to stress, particularly salt stress, was provided by experiments showing that dso-1 seedlings are highly sensitive to salt treatment. Preliminary characterization of a gene trap line corresponding to DSO also showed that it is also induced by multiple stresses, including ABA, high salt, and Glc (Alvarado et al, 2004). Like DSO, the expression of CER6 encoding a VLCFA-condensing enzyme was enhanced by osmotic stress and the presence of ABA (Hooker et al, 2002).…”

Functional Identification of Arabidopsis Stress Regulatory Genes Using the Controlled cDNA Overexpression System

“…To test the applicability of COS technology in screening for factors that confer trans-activation of a stress-regulated promoter, we have constructed an ADH1-LUC luciferase reporter gene, the activation of which can be monitored using nondestructive lowlight imaging (Alvarado et al, 2004). Transcription of the Arabidopsis ADH1 gene (At1g77120) is induced by both ABA-dependent (dehydration, high salinity) and independent (cold and anoxia) regulatory pathways (de Bruxelles et al, 1996).…”

“…The resulting ADH1-LUC reporter construct was introduced into Arabidopsis (Col-0 ecotype) by Agrobacteriummediated gene transfer. Twenty independent transformants were selected and tested for segregation of the kanamycin resistance marker in the T 2 generation, as well as for the activity of the ADH1-LUC reporter using bioluminescence imaging (Alvarado et al, 2004). Induction of ADH1-LUC by ABA was performed by spraying seedlings with 50 mM ABA solution or transferring seedlings on culture medium supplemented by 200 mM NaCl, 400 mM Suc, or 10 mM H 2 O 2 and measuring bioluminescence in 30-min intervals for 18 h. Luminescence values were analyzed with the Metaview software (Universal Imaging).…”

“…Screening for enhanced luminescence of a pathogen responsive PR1-luciferase reporter gene was thus used for tagging of the Arabidopsis ADR1 pathogen resistance gene (Grant et al, 2003). Analogous genetic screens are facilitated by the gene trapping systems that offer a collection of promoter trap insertions in stress-responsive genes that control the expression of GFP, GUS, or luciferase reporters (Alvarado et al, 2004).…”

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

“…By detection of bioluminescence in three week-old seedlings, 753 lines were identified showing constitutive, organ-specific, and stress-responsive luciferase expression patterns. Several lines showed sugar, salt, and abscisic acid (ABA)-inducible luciferase activity (Alvarado et al 2004). Moreover the technique has now been adopted for the ongoing intensive functional genomics efforts in rice demonstrated by the trapping of two cold inducible genes from screening of T-DNA tagged rice lines for cold responsive GUS expression (Lee et al 2004).…”