Photochemistry and Photobiology, 2003, 77(6): 668–674 Analysis of Hydrogen Peroxide–independent Expression of the High-light–inducible ELIP2 Gene with the Aid of the ELIP2 Promoter–Luciferase Fusion{ Mitsuhiro Kimura1,2,3, Katsushi Manabe3, Tomoko Abe2, Shigeo Yoshida2, Minami Matsui1 and Yoshiharu Y. Yamamoto*1,2 1Plant Function Exploration Team, RIKEN Genomic Sciences Center, Tsurumi-Ku, Yokohama, Kanagawa, Japan; 2Plant Functions Laboratory, RIKEN, Hirosawa, Wako, Saitama, Japan and 3Graduate School of Integrated Science, Yokohama City University, Kanazawa-Ku, Yokohama, Kanagawa, Japan Received 4 December 2002; accepted 20 March 2003 ABSTRACT However, an intense and unmanageable amount of light (high light) spells danger for the plant. Under high-light conditions, Intense and excessive light triggers the evolution of reactive electrons leak from excited chlorophylls as well as from the oxygen species in chloroplasts, and these have the potential to photosynthetic electron transport system (ETS), resulting in the cause damage. However, plants are able to respond to light generation of oxygen and lipid radicals. These damage proteins, stress and protect the chloroplasts by various means, including lipids, pigments, DNA and all other components of the chloroplast transcriptional regulation at the nucleus. Activation of light (1). However, higher plants have developed several strategies to stress–responsive genes is mediated via hydrogen peroxide– protect the chloroplasts from high light. These include reduction of dependent and –independent pathways. In this study, we antenna size, downregulation of Photosystem II, photorespiration, characterized the Early-Light–Inducible Protein 2 (ELIP2) development of radical scavengers and induction of nonphoto- promoter–luciferase gene fusion (ELIP2::LUC), which re- chemical quenching to reduce the excitation energy transferred sponds only to the hydrogen peroxide–independent pathway. from the antenna to the reaction centers (1–3). Some of these high- Our results show that ELIP2::LUC is expressed under light responses are controlled at the level of gene expression. nonstressful conditions in green tissue containing juvenile Several nuclear genes have been reported to be activated by high and developing chloroplasts. Upon light stress, expression was light, including Early-Light–Inducible Protein (ELIP) (4), genes activated in leaves with mature as well as developing encoding active oxygen scavengers (5), actin, LEA, metallothio- chloroplasts. In contrast to another high-light–inducible gene, nein (6), a putative transcription factor (7) and others with APX2, which responds to the hydrogen peroxide–dependent unknown functions (6,8). Recently, microarray analysis has pathway, the activation of ELIP2::LUC was cell autonomous. revealed about 100 high-light–inducible genes of Arabidopsis The activation was suppressed by application of 3-(3,4)- (9,10). The Arabidopsis APX2 gene that encodes cytosolic dichlorophenyl-1,1-dimethylurea, an inhibitor of the reduction ascorbate peroxidase has been most frequently used to study the of plastoquinone, whereas 2,5-dibromo-3-methyl-6-isopropyl- molecular mechanism of high-light activation of nuclear gene p-benzoquinone, an inhibitor of the oxidation of plastoquinone, expression. The high-light activation of APX2 is mediated by gave the contrasting effect, which may suggest that the redox hydrogen peroxide, which accumulates through excess light- state of the plastoquinone plays an important role in triggering dependent generation of oxygen radicals (11). Furthermore, it has the hydrogen peroxide–independent light stress signaling. been shown that the high-light signal for activation of APX2 is mediated from cell to cell (11). In addition, reduction of the plastoquinone pool has been suggested to be necessary for INTRODUCTION activation of APX2. However, many things need to be revealed For plants, sunlight is the one and only source of energy for to understand the whole signaling pathway from high-light photosynthesis and thus is indispensable for the growth of plants. perception to gene expression, including the relationship between reduction of the plastoquinone pool and generation of hydrogen peroxide, its perception and the downstream events after hydrogen {Posted on the website on 7 April 2003. peroxide accumulation. Furthermore, it is not known if all the high- *To whom correspondence should be addressed at: Plant Functions light–regulated genes are controlled by the same mechanism as Laboratory, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan. Fax: 8148-462-4674; e-mail: [email protected] APX2. Abbreviations: APX2::LUC, APX2 promoter–luciferase fusion; CCD, The ELIP was first identified from the pea as a transcript that charge-coupled device; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p- was rapidly induced by light during greening (12). Subsequent benzoquinone; DCMU, 3-(3,4)-dichlorophenyl-1,1-dimethylurea; ELIP, analyses revealed that ELIP belongs to the CAB superfamily and early-light–inducible protein; ELIP2::LUC, ELIP2 promoter–luciferase fusion; ETS, electron transport system; mRNA, messenger RNA; RNase, associates with Photosystem II at the thylakoid membrane (4,13). ribonuclease; RT-PCR, reverse transcriptase–polymerse chain reaction. Because its expression is induced by light stress in many species, it Ó 2003 American Society for Photobiology 0031-8655/03 $5.00þ0.00 has been speculated that ELIP is an antistress component for the 668 Photochemistry and Photobiology, 2003, 77(6) 669 MATERIALS AND METHODS Plant material and light treatment. Preparation of transgenic Arabi- dopsis containing ELIP2::LUC (17), APX2 promoter–luciferase fusion (APX2::LUC) (11), 35S::LUC and plastocyanin promoter–luciferase fusion (PC::LUC) (18) is described elsewhere. Seeds of wild-type and transgenic Arabidopsis were surface sterilized, sown on GM medium supplemented with 0.8% Bactoagar (Difco, Detroit, MI) and 1% sucrose, cold treated for 2–4 days and grown under low-light conditions (6 W mÿ2, equivalent to ca 30 lmol mÿ2 sÿ1)at228C (17). Eight day-old seedlings were subjected to high-light treatment with a fluence rate of 150 W mÿ2 (equivalent to ca 800 lmol mÿ2 sÿ1) for 3 h, as described previously (17). For the experiments shown in Fig. 5, leaves grown in a greenhouse were detached, kept on GM medium for 1 day under low light (6 W mÿ2)at228C and locally irradiated with high light (150 W mÿ2) for 3 h at 228C with the aid of stainless sheets cut to remove either a straight slit or a Y-shaped slit. For 3-(3,4)- dichlorophenyl-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6- isopropyl-p-benzoquinone (DBMIB) treatments, 2 mL of the inhibitor solutions containing 1.8% (vol/vol) ethanol were sprayed per circular culture dish (10 cm diameter) 3 h before high-light treatments and sprayed again just before high-light treatment only for the DBMIB treatment (19). For controls, the corresponding amount of the ethanol solution without DCMU or DBMIB was sprayed. Luciferase assay. In vitro luciferase assays and in vivo assays using a high-performance charge-coupled device (CCD) camera were performed as described previously (17). For in vivo temporal analysis as shown in Fig. 3, seeds were sown in 96-cell black plates containing GM medium supplemented with 0.8% Bactoagar, 1% sucrose and 1 mM luciferin. They were cold treated for 2–4 days. The plates were sealed with clear adhesive sheets that had a tiny hole for each well and put into an automated scintillation counter (Topcount, Packard Japan, Tokyo, Japan). The plates were kept in the dark assay chamber for 5 min before starting the counting to allow the delayed chlorophyll fluorescence to fade. An assay was done Figure 1. Observation of ELIP2::LUC activation by high light. A and B: ÿ2 Induction of luciferase activity by high light in ELIP2::LUC transgenic for a week at 228C under dark or light (6 W m ) conditions. The data from plants observed with a high-performance CCD camera. A: Photograph of 8 an assay were logged into a text file and subsequently analyzed using Excel day old transgenic seedlings. B: Pseudocolor image of bioluminescence by software (Microsoft Japan, Tokyo, Japan). Typically, an assay for a week luciferase activity. Left, seedlings treated without high light (ÿHL). Right, was composed of about 400 data points. seedlings treated with high light (þHL, 150 W mÿ2 for 3 h). The color scale RNA analysis. Eight day-old seedlings were treated with or without high on the right shows the luminescence intensity from dark blue (lowest) to light for 3 h, and the aerial part of the seedlings was harvested for RNA white (highest). C: Photograph of a 2 week old seedling grown under low extraction (20). The ELIP2 messenger RNA (mRNA) was detected by light (6 W mÿ2). D: The corresponding bioluminescence image representing quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) luciferase activity. The picture in (D) is a synthetic image created by according to a previous report (17). As a negative control, a RNA prep was overwriting the bioluminescence image (color) onto the black-and-white treated with ribonuclease (RNase) A for 6 h at 378C and used as a template image of the seedling. Both images in (D) were captured by the same CCD for RT-PCR. camera. Color bar indicates the scale of the color. protection of Photosystem II, although the biochemical function of RESULTS ELIP remains to be elucidated (4). Expression of ELIP2::LUC under nonstressful conditions Arabidopsis has two ELIP genes,