Color profile Generic CMYK printer profile Composite Default screen Plant Molecular Biology Reporter 18 143a 143t 2000 2000 International Society for Plant Molecular Biology Printed in Canada Publish by Abstract The Use of the Luciferase Reporter System for in Planta Gene Expression Studies WESSEL VAN LEEUWEN1 MARC J M HAGENDOORN1 TOM RUTTINK2 REMCO VAN POECKE3 LINUS H W VAN DER PLAS1 and ALEXANDER R VAN DER KROL1 1 Laboratory of Plant Physiology Wageningen University Arboretumlaan 4 6703 BD Wageningen The Netherlands 2Laboratory of Molecular Biology Wageningen University Dreijenlaan 3 6703 HA Wageningen The Netherlands 3Laboratory of Entomology Wageningen University Binnenhaven 7 6709 PD Wageningen The Netherlands Abstract The properties of the firefly luciferase LUC make it a very good non destructive reporter to quantify and image transgene promoter activity in plants The short half life of the LUC mRNA and protein and the very limited regeneration of the LUC protein after reacting with luciferin enables monitoring of changes in gene activity with a high time resolution However the ease at which luciferase activity is measured in planta using a light sensitive camera system 2D luminometer contrasts sharply with the complications that arise from interpreting the results A variegated pattern of luciferase activity that is often observed in in planta measurements might either be caused by differences in influx availability of the substrates luciferin oxygen ATP or by local differences in reporter gene activity Here we tested the possible contribution of differences in the availability of each substrate to the variegated in planta luciferase activity and we show when in planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene Furthermore we demonstrate the effects of protein stability apparent half life of luciferase activity regeneration of luciferase and pH on the in vivo and in vitro luciferase measurements The combined results give the prerequisites for the correct utilisation of the luciferase reporter system especially for in vivo gene expression studies in plant research Key words Cauliflower Mosaic Virus 35S promoter luciferase reporter system Petunia hybrida reporter gene transgene expression variegation Abbreviations FW fresh weight LUC luciferase rlu relative light units Luciferase Introduction for gene expression studies Van Leeuwen et al The luciferase gene from the North American firefly Photinus pyralis has emerged as a popular choice for in vitro and in vivo reporting of transcriptional activity in eukaryotic cells Since the cloning of the cDNA encoding the enzyme Author for correspondence e mail Wessel vanLeeuwen Algem PF WAU NL fax 31 317 484740 ph 31 317 482822 J PMBR Pmbr18 02 R00 022 vp Thursday September 07 2000 1 49 47 PM Color profile Generic CMYK printer profile Composite Default screen 143b Van Leeuwen et al 1 LUC luciferin Mg ATP LUC luciferin AMP MgPPI 2 LUC luciferin AMP O2 LUC oxyluciferin AMP CO 2 3 LUC oxyluciferin AMP LUC oxyluciferin AMP hv 4 LUC oxyluciferin AMP LUC oxyluciferin AMP Figure 1 The luciferase reaction Aflalo 1991 Brackets and bullets indicate the complexes formed Step 1 is a fast equilibrium reaction Step 2 is the oxidative decarboxylation in which the oxyluciferin is excited Step 3 is the fast photon emission at 562 nm Step 4 is the very slow release of product from the active site of the LUC protein luciferase LUC by DeWet et al in 1985 the luciferase gene has been expressed in tobacco and carrot plants Ow et al 1986 mammalian cells DeWet et al 1987 and zebrafish Mayerhofer et al 1995 and Drosophila Brandes et al 1996 In firefly the LUC protein is targeted to the peroxisomes and the C terminal peroxisome import signal was shown to function in plants as well For enhanced expression in mammalian cells and plants the luciferase coding sequence was modified and the peroxisomal import sequence luc Promega was removed Sherf and Wood 1994 Luciferase catalyses the oxidative decarboxylation of the substrate firefly luciferin Figure 1 The reaction causes the release of a photon at 562 nm in 90 of the catalytic cycles with the substrates luciferin Mg2 ATP and oxygen DeLuca et al 1974 Aflalo 1991 The luciferase enzyme is only slowly regenerated after reacting with the substrate because the end product of the reaction oxyluciferin is only slowly released from the Luciferase Oxyluciferin complex Figure 1 step 4 Denburg et al 1969 In vitro in the presence of high ATP concentrations Coenzyme A enhances the light production through removal of oxyluciferin from luciferase resulting in a nearly constant production of light Ford et al 1995 We will discuss whether the enhanced regeneration of luciferase by the presence of Coenzyme A occurs in vivo This slow regeneration in combination with the short half life of luciferase Nguyen et al 1989 Thompson et al 1991 implies that in the presence of all substrates each luciferase molecule can only react once and emit one photon In the presence of all substrates the LUC protein will therefore not accumulate in vivo Luciferase as a reporter gene thus represents gene expression as the flux of protein molecules LUC made in the cell LUC sec while more stable reporter genes only show the accumulation of protein molecules as an indication of gene expression total amount of reporter protein in the cell at any given time point Therefore luciferase can be used as a non invasive reporter in plants to accurately mark changes in transgene expression After the plant tissue has been provided with luciferin the only substrate that is not naturally present in the plant cell in planta luciferase activity can be monitored with a 2D luminometer However in order to relate the changes in luciferase activity to changes in transgene expression the availability of each of the substrates luciferin oxygen and ATP should remain constant during the period J PMBR Pmbr18 02 R00 022 vp Thursday September 07 2000 1 49 50 PM Color profile Generic CMYK printer profile Composite Default screen Luciferase for gene expression studies 143c over which the luciferase activity is monitored In order to relate differences in luciferase activity within a tissue to local differences in transgene expression the availability of each of the substrates should also be similar in different parts of the tissue When we used the luciferase reporter system to measure gene