Plants respond to light as an environmental factor to optimize development and regulate other physiological phenomena. Phytochrome and blue light receptors, such as cryptochrome and phototropin (phot), are the main photoreceptors for plant photomorphogenesis. The goal of our research is to elucidate the photoperception and the signal transduction pathways of photo-morphogenesis.

I. Chloroplast relocation movement

One of our major subjects is chloroplast photo-relocation movement, which is thought to be one of the simplest phenomena in this field. We use the fern Adiantum capillus-veneris and the moss Physcomitrella patens as model plants for our cell biological approach since the gametophytes are very sensitive to light and the organization of the cells is very simple. We also use Arabidopsis mutants to identify the genes regulating chloroplast photo-relocation movement.

1- 1 Arabidopsis

Chloroplasts accumulate at the cell surface under weak light and escape from the cell surface to the anticlinal wall under strong light to optimize photosynthesis. We identified the photoreceptors in Arabidopsis. Phototropin2 (phot2) mediates the avoidance response under strong light and phot1 and phot2 mediate, redundantly, the accumulation response under weak light. However, components of signal transduction pathways still remain to be identified. A mutant called chup1 is deficient in chloroplast movement and consequently chloroplasts gather at the bottom of cells. CHUP1 is a novel gene that has a hydrophobic region in the N-terminus and an actin binding domain, a prolin-rich region, and two leucin zippers. It was confirmed that the actin-binding domain binds F-actin. When fusion proteins of the N-terminal, hydrophobic region of CHUP1 with GFP were expressed transiently in chup1 mutant cells, fluorescence of the fusion proteins were found in the outer part of chloroplasts, likely on the outer membrane of chloroplasts. CHUP1 is proposed to play an important role on chloroplasts movement of both accumulation and avoidance responses.

1- 2 Adiantum phy3

Adiantum phytochrome3 (PHY3) is a unique chimeric protein with phytochrome structure in the N-terminal half and phototropin structure in the C-terminal half. Transient expression of PHY3 or modified PHY3 genes in rap (red light-induced aphototropic) mutants of Adiantum reveal that phy3 is the photoreceptor of red light-induced chloroplast photorelocation movement, and that the phy3 signal might be transferred through the C-terminal, phototropin region. The phot region of phy3 has not yet been shown to function as a blue light photoreceptor, but the above method of transient expression of modified PHY3 in rap and phot mutants may provide experimental evidence of this possibility soon.

II. Gene targeting and gene silencing

In order to elucidate the role of genes in Adiantum and rice, we have tried to establish new methods for gene targeting in these organisms.

2- 1 Miniature transposable element

Transposable elements constitute a large portion of eukaryotic genomes and contribute to their evolution and diversification. Miniature inverted-repeat transposable elements (MITE) constitute one of the main groups of transposable elements. MITEs have been found in wide range of organisms but active MITEs have not been identified. We found a new class of MITEs in rice and named them miniature Ping (mPing). mPing was identified as the first active MITE from any organism and the first active DNA transposon from rice. mPing is a short 430 base pairs element with 15 base pair terminal inverted repeats that lacks a transposase. mPing elements are activated in calli derived from anther culture and excise efficiently from original sites to reinsert into new loci. The mPing-associated Ping element which has a putative transposase sequence was also found and shown to transpose within the rice genome.

Rice is the most agriculturally important crop in the world. mPing/Ping transposon system is a useful molecular tool for gene isolation and gene knockout in rice.

2- 2 Expression sequence tag in Adiantum

To understand the genetic information of a fern, Adiantum capillus-veneris, a normalized cDNA library was constructed from prothallia grown under white light and analysis of expressed sequence tags (EST) were carried out. Approximately 10,000 clones were sequenced and clustering of these obtained sequences was performed. As a result, 7,132 non-redundant groups were generated. These groups were subjected to similarity searches to identify putative function. Approximately 1,600 EST groups were found to be similar to sequences of genes registered in the public database. About 1,100 EST groups showed similarity to sequences of unknown function. The remaining EST groups showed no significant similarity and were classified as novel sequences.

Figure Localization of N-CHUP-GFP in Arabidopsis palisade mesophyll cells as confocal microscopic images of the optical sections transiently expressing GFP (A-C), pt-sGFP (D-F), AtOEP7-GFP (E-G) and N-CHUP-GFP (H-J). The green and red colors show fluorescence from GFP and autofluorescence from chloroplasts, respectively. Images of green and red channel were merged (C, F, I and L). Scale bar: 10 µm. OEP is a membrane protein known to distribute at the chloroplast outer membranes (Kind gift of Dr. Yasuo Niwa).