Analysis of hydrogen peroxide-independent expression of the high-light-inducible ELIP2 gene with the aid of the ELIP2 promoter-luciferase fusion[para]

Photochemistry and Photobiology, Jun 2003 by Kimura, Mitsuhiro, Manabe, Katsushi, Abe, Tomoko, Yoshida, Shigeo, Et al

ABSTRACT

Intense and excessive light triggers the evolution of reactive oxygen species in chloroplasts, and these have the potential to cause damage. However, plants are able to respond to light stress and protect the chloroplasts by various means, including transcriptional regulation at the nucleus. Activation of light stress-responsive genes is mediated via hydrogen peroxide-dependent and -independent pathways. In this study, we characterized the Early-Light-Inducible Protein 2 (ELIP2) promoter-luciferase gene fusion (ELIP2[four dots above]LUC), which responds only to the hydrogen peroxide-independent pathway. Our results show that ELIP2[four dots above]LUC is expressed under nonstressful conditions in green tissue containing juvenile and developing chloroplasts. Upon light stress, expression was activated in leaves with mature as well as developing chloroplasts. In contrast to another high-light-inducible gene, APX2, which responds to the hydrogen peroxide-dependent pathway, the activation of ELIP2[four dots above]LUC was cell autonomous. The activation was suppressed by application of 3-(3,4)-dichlorophenyl-1,1-dimethylurea, an inhibitor of the reduction of plastoquinone, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of the oxidation of plastoquinone, gave the contrasting effect, which may suggest that the redox state of the plastoquinone plays an important role in triggering the hydrogen peroxide-independent light stress signaling.

INTRODUCTION

For plants, sunlight is the one and only source of energy for photosynthesis and thus is indispensable for the growth of plants. However, an intense and unmanageable amount of light (high light) spells danger for the plant. Under high-light conditions, electrons leak from excited chlorophylls as well as from the photosynthetic electron transport system (ETS), resulting in the generation of oxygen and lipid radicals. These damage proteins, lipids, pigments, DNA and all other components of the chloroplast (1). However, higher plants have developed several strategies to protect the chloroplasts from high light. These include reduction of antenna size, downregulation of Photosystem II, photorespiration, development of radical scavengers and induction of nonphoto-chemical quenching to reduce the excitation energy transferred from the antenna to the reaction centers (1-3). Some of these highlight responses are controlled at the level of gene expression.

Several nuclear genes have been reported to be activated by high light, including Early-Light-Inducible Protein (ELIP) (4), genes encoding active oxygen scavengers (5), actin, LEA, metallothionein (6), a putative transcription factor (7) and others with unknown functions (6,8). Recently, microarray analysis has revealed about 100 high-light-inducible genes of Arabidopsis (9,10). The Arabidopsis APX2 gene that encodes cytosolic ascorbate peroxidase has been most frequently used to study the molecular mechanism of high-light activation of nuclear gene expression. The high-light activation of APX2 is mediated by hydrogen peroxide, which accumulates through excess light-dependent generation of oxygen radicals (11). Furthermore, it has 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 activation of APX2. However, many things need to be revealed to understand the whole signaling pathway from high-light 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 peroxide accumulation. Furthermore, it is not known if all the highlight-regulated genes are controlled by the same mechanism as APX2.

The ELIP was first identified from the pea as a transcript that was rapidly induced by light during greening (12). Subsequent analyses revealed that ELIP belongs to the CAB superfamily and associates with Photosystem II at the thylakoid membrane (4,13). Because its expression is induced by light stress in many species, it has been speculated that ELIP is an antistress component for the protection of Photosystem II, although the biochemical function of ELIP remains to be elucidated (4).

Arabidopsis has two ELIP genes, ELIP1 (MIPS protein code At3g22840) (14) and ELIP2 (At4g14690) (15,16). Both genes are activated by light stress (15), but their differential dependence on HY5 was also observed in expression during deetiolation (16). Recent analyses of the Arabidopsis ELIP2 gene have revealed that its expression is also activated by high-light treatment, and the activation is mediated by hydrogen peroxide (15,17). Furthermore, using an ELIP2 promoter-luciferase fusion (ELIP2[four dots above]LUC), it was shown that transcriptional activation induced by high light driven by the ELIP2 promoter was achieved independent of hydrogen peroxide (17). Therefore, high-light induction of ELIP2 gene expression is achieved by both hydrogen peroxide-dependent and -independent signaling pathways.