NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY

(Adjunct)

Professor (Adjunct):
Masamitsu Wada
Research Associate:
Tomohiro Kiyosue
Postdoctoral Fellow:
Takatoshi Kagawa
Graduate Students:
Takato Imaizumi (Tokyo Metropolitan University)
Kazusato Oikawa (Tokyo Metropolitan University)


Plants use light as an environmental factor which controls their development as well as their other physiological phenomena. Phytochrome and blue-light receptors, such as cryptochrome and phototropin (NPH1), are main photoreceptors for plant photomorphogenesis. The goal of our research is to clarify the signal transduction pathways of photomorphogenesis. We are focusing our study on chloroplast photorelocation movement which is thought to be one of the simplest phenomena in this field. We use the fern Adiantum gametophytes as a model plant not only because the gametophytes are very sensitive to light, but also because the organization of the cells is very simple and easy to manipulate. We also use Arabidopsis for mutant screening to clarify the genes regulating chloroplast photorelocation.


I. Cloning and characterization of blue-light photoreceptors in the fern Adiantum capillus-veneris

1-1 Cryptochromes

WWe have described many blue-light-induced photomorphological responses in gametophytes of the fern Adiantum capillus-veneris. As the first step in understanding the molecular mechanisms of these various blue-light responses in Adiantum, we isolated blue-light photoreceptor-related genes as homologues of Arabidopsis cryptochrome1.

We cloned five different genes, which are designated as Adiantum cryptochrome1, 2, 3, 4, and 5 (CRY1, 2, 3, 4, and 5), from a genomic DNA library. The N-termini of the five genes showed remarkable homology to Arabidopsis cryptochrome1, as well as to other cryptochromes which have already been identified. However, these genes have every distinct C-termini from each other.

To analyze the correlation between blue-light-induced responses and cryptochromes, we investigated the expression pattern of these genes in various developmental stages and also in different light conditions using RNA blot analysis and competitive PCR analysis.

The results showed CRY1, CRY2 and CRY3 to have similar expression patterns. Transcripts of these genes were elevated after spores imbibed water in the dark, but after that the level of these transcripts did not vary much. The character of this expression pattern is very similar to those of previously characterized plant cryptochromes. CRY4 and CRY5 showed unique light-regulated expression patterns. CRY4 was down regulated by light throughout the life cycle of Adiantum. The cDNA expression level was reduced by approximately 100 times when dark imbibed spores were irradiated with red light, which caused spore germination. CRY5 expression was induced rapidly after light exposure and kept increasing for 12 hours. The expression level was reduced for the next 12 hours and after that it did not vary much.

The major change in CRY4 and CRY5 expression level occurred before spore germination, so transcripts of these two genes might act somewhat in the process of light dependent germination of Adiantum.

1-2 Phototropin

N PH1 (phototropin) is another blue-light photoreceptor recently isolated in higher plants, which is a flavin binding protein with a light sensitive protein kinase activity. By RT-PCR with primers designed from highly conserved sequence motifs in NPH1 photoreceptors, we cloned a cDNA fragment from Adiantum. The RT-PCR experiment suggested that the accumulation of mRNA for the Adiantum NPH1 is down regulated by light. The structural and biochemical characterization of Adiantum NPH1 is in progress.


II. Chloroplast relocation

2-1 Arabidopsis

Chloroplasts accumulate at cell surface under weak light and escape from strong light to optimize photosynthesis. Which photoreceptors are active in chloroplast relocation, however, is not known.

We are studying light-induced chloroplast relocation in Arabidopsis leaves. In a light-adapted mesophyll cell, chloroplasts spread over the cell surface, but in a dark-adapted cell they escape from the cell surface. When a cell is partly irradiated with blue light at a strength of 30Wm-2, chloroplasts move out from the irradiated spot. In comparison, chloroplasts accumulate in a spot irradiated with blue light at a strength of 3Wm-2. Simultaneous irradiation with red light increased the sensitivity to the blue light, although the red light irradiation alone did not induce chloroplast relocation.

Photoreceptor mutants, such as cry1, cry2, cry1cry2 double mutant and nph1, were demonstrated to be normal for blue-light-induced chloroplast relocation for both weak and strong blue light, meaning thet CRY1, CRY2 and NPH1 are not the photoreceptors for chloroplast relocation. To find the photoreceptor for blue-light-induced chloroplast relocation, we are screening mutants from T-DNA tagging lines. Identification of the mutated genes of Arabidopsis mutants is now in progress.

2-2 Adiantum

Mutants of chloroplast relocation movement were screened and analyzed is Adiantum gametophytes. Mutants which were deficient either in high fluence response or in low fluence response were obtained independently, meaning that the signal transduction pathways of both responses are different.

Figure 1.
Serial photographs of chloroplast relocation in a dark-adapted (for 2 days) prothallial cell induced by one min illumination with a red microbeam (10 mm in diameter) of 30 Wm-2. Chloroplast moved toward the beam after the beam was switched off over 70 min and then dispersed. Bar, 20 mm.

Figure 2.
Serial photographs of chloroplast relocation in a dark-adapted prothallial cell induced by continuous blue microbeam irradiation (10 Wm-2, 27 mm in diameter). The illumination spot is shown only time 0 but the light was given from time 0 to 90 min. The numbers shown in the photographs are the time after onset of blue light irradiation. Chloroplasts move toward the beam, but they can not get into the beam irradiated area until the microbeam was switched off. Bar, 20 mm.


Publication
Nozue, K., Kanegae, T., Imaizumi, T., Fukada, S., Okamoto, H., Yeh, K.C., Lagarias, J.C. and Wada, M. (1998) A phytochrome from the fern Adiantum with features of the putative photoreceptor NPH1. Proc. Natl. Acad. Sci. USA, 95, 15826-15830.

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Last Modified: 12:00, May 28, 1999