Analysis of the light signalling pathway for the degradation of the circadian clock protein ROC15 in the green alga Chlamydomonas reinhardtii

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Analysis of the light signalling pathway for the
degradation of the circadian clock protein ROC15 in the
green alga Chlamydomonas reinhardtii（緑藻クラミドモ
ナスの時計タンパク質 ROC15 の分解を誘導する光シグナ
ル伝達経路の解析）

氏

名

GURURAJ Malavika

論 文 内 容 の 要 旨
Circadian rhythms are the rhythms with a period of ~24 hours
observed in phenomenon such as leaf movement of plants, the human
sleep/wake cycle and phototaxis in protists like Chlamydomonas
reinhardtii. These rhythms persist in constant environmental conditions
and are generated by a molecular circadian clock. The clock comprises of an
input, oscillator and output. Signals such as light and temperature reach
the oscillator component via the input, the oscillator is a gene-protein
system that generates and maintains the rhythm and the output involves
genes that are part of metabolic processes that are play a role in the
afore-mentioned phenomenon. The input to the clock ensures that the
rhythm generated is synchronized to the external environment i.e. it resets
the clock. Light plays a major role in resetting the circadian clock, allowing
the organism to synchronize with the environmental day and night cycle.
In C. reinhardtii the light-induced degradation of the circadian clock
protein, RHYTHM OF CHLOROPLAST 15 (ROC15), is considered

学位関係

one of the key events in resetting the circadian clock. Red/violet and
blue light signals have been shown to reach the clock via different
molecular pathways; however, many of the participating components of
these pathways are yet to be elucidated.
In my research, I followed a forward genetics approach and a
reverse genetics approach using a reporter strain that expresses a
ROC15-luciferase fusion protein. As result of the forward genetics
approach, I isolated a mutant that showed impaired ROC15
degradation in response to a wide range of visible wavelengths and
impaired light-induced phosphorylation of ROC15. These results
suggest that the effects of different wavelengths converge before acting
on ROC15 or at ROC15 phosphorylation. Furthermore, the mutant
showed a weakened phase resetting in response to light, but its
circadian rhythmicity remained largely unaffected under constant light
and constant dark conditions. Surprisingly, the gene disrupted in this
mutant was found to encode a protein that possessed a very weak
similarity to the Arabidopsis thaliana EARLY FLOWERING 3 (ELF3).
The results suggest that this protein is involved in the many different
light signaling pathways to the C. reinhardtii circadian clock. However,
it may not influence the transcriptional oscillator of C. reinhardtii to a
great extent. As a result of the reverse genetics approach, I
demonstrated that the mutant of the photoreceptor plant cryptochrome
(pCRY), also has an impaired ROC15 degradation in response to a wide
range of wavelengths, and an impaired ROC15 phosphorylation in
response to both blue and red light. These results suggest the
possibility that the red/violet and blue light signaling pathways are
integrated by pCRY before acting on ROC15. This study provides an
opportunity to further understand the mechanisms underlying
light-induced clock resetting and explore the evolution of the circadian
clock architecture in Viridiplantae.

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