부산대학교 도서관

식물의 광반응 운동은 빛의 조건에 따라 광합성 효율을 높이기 위해 발달된 생리적 반응이다. 식물은 광수용체를 통해 빛의 세기와 방향을 인지하고 광반응 운동을 조절한다. 각 광수용체는 특정 파장의 빛을 흡수할 수 있으며 서로 다른 신호전달 경로를 갖는다. 고등식물의 광반응 운동은 청색광 수용체 phototropin에 의해 조절된다. 조류를 포함하는 민꽃식물 (cryptogam)에서는 적색/원적색광 수용체 phytochrome과 neochrome도 광반응 운동에 관여한다. 녹조식물 해캄은 사상체가 서로 뭉치고 빛 방향으로 이동하는 복잡한 광반응 운동을 한다. 이전 연구에서 이와 같은 해캄의 운동은 각 단색광에 따라 다르게 반응하였다. 본 연구에서는 해캄 (Spirogyra varians)의 각 단색광에 따른 광반응 운동의 분석과 저해 실험을 수행하였으며 광수용체의 분리 및 분석을 통해 운동 조절기작을 규명하였다. 해캄의 사상체는 청색광에서 양성굴광성을 보인 반면 적색광에서는 불규칙적으로 구부러지는 운동을 보였다. 적색광에서 구부러진 사상체는 원적색광에서 빠르게 펴져 phytochrome의 가역적 조절이 예상되었다. Phototropin 신호 전달 요소로 알려진 phosphoinositide 3-kinase를 wortmannin과 LY294002를 통해 저해하였을 때 해캄의 청색광 운동은 저해되었지만 적색광 운동은 영향을 받지 않았다. 세포 내 칼슘 신호 저해제인 caffeine을 처리하였을 때에도 청색광 운동은 저해되었지만 적색광 운동은 영향을 받지 않았다. 세포골격에 대한 저해실험에서 microfilament 저해제인 cytochalasin D와 microtubule 저해제인 oryzalin을 동시에 처리하였을 때 청색광 운동이 저해되었다. 적색광 운동은 cytochalasin D에 영향을 받지 않았지만 oryzalin 처리를 통해 저해되었다. 원적색광에서 관찰된 사상체가 펴지는 운동은 mannitol과 sorbitol을 이용한 삼투압 조절을 통해 저해되었다. 해캄의 각 광반응 운동에 관여할 것으로 판단되는 phototropin과 phytochrome을 분리 및 분석하였다. 해캄의 phototropin은 2 개의 상동유전자를 분리하였으며 SvPHOTA와 SvPHOTB로 명명하였다. 각 phototropin은 모두 light–oxygen–voltage domain과 kinase domain으로 구성된 전형적인 phototropin 구조를 보였지만 분자계통분석에서 서로 다른 분기군에 속하였다. 또한 빛 조건에 따른 유전자 발현 분석과 애기장대를 이용한 형질전환 실험에서 유의한 차이를 보여 기능적 분화가 예상되었다. 해캄의 phytochrome은 canonical phytochrome인 SvPHY1과 SvPHY2 그리고 non-canonical phytochrome인 SvPHYX2을 분리하였으며 분자계통분석에서 윤조식물의 각 분기군에 속하였다. 해캄의 phytochrome은 별해캄목 조류에서 보고되지 않은 PHY1을 보유하고 PHYX1이 결핍된 특이한 구성을 보였다. 해캄 phytochrome의 신호 전달에 관여할 수 있는 FHY1, COP1, HY5, PIF-like가 해캄의 발현유전체에 보존되어 있었으며 적색광과 원적색광에 따른 발현 변화를 보였다. 이상의 결과는 해캄의 광수용체인 phototropin과 phytochrome이 서로 다른 신호 전달 경로와 기계적 반응기 (mechanical effector)를 이용하여 각 단색광 운동을 조절한다는 것을 의미하였다. 해캄 사상체에서 관찰되는 복잡한 광반응 운동은 광수용체들의 상보적인 운동 조절의 결과로 판단된다.
Plants and freshwater algae devoid of flagella evolved various photomovements to optimize their photosynthetic efficiency. The filaments of Spirogyra varians exhibit complex photomovement and form a compact mat which enables them to adjust their light exposure. In monochromatic lights, the S. varians filaments formed bundles and showed rapid undulating movement under blue light, while formed loose mat without distinct movement under red light. Photomovement of filament fragments (1–10 cells) was analyzed using various photoreceptor signaling component and cytoskeleton inhibitors under monochromatic light. Different patterns of movement were observed under blue and red light. The filaments showed positive phototropism under blue light. Under red light, the filaments bent to irregular shape, but rapidly became unbent by exposure to far-red light suggesting the involvement of phytochrome in this movement. The mechanical effector for the red-light response was microtubule; the movement was inhibited effectively by the microtubule inhibitor, oryzalin. The blue-light movement was partially inhibited by the single treatment of either cytochalasin D or oryzalin, but was completely blocked when both chemicals were applied together. Phototropin-signaling inhibitors, wortmannin and LY294002, reversibly inhibited the blue-light movement. Calcium signaling inhibitor, caffeine treatment reversibly stopped the blue-light movement, while the red-light movement was not affected by calcium inhibitors. To decipher the genetic control of blue light-induced photomovement, two phototropin homologues were isolated from S. varians, and named SvPHOTA and SvPHOTB. Both phototropins have similar molecular structure consisted of two light–oxygen–voltage domains (LOV1, LOV2) and a serine/threonine kinase domain. SvPHOTA and SvPHOTB had 48.7% sequence identity. Phylogenetic analysis showed SvPHOTA and SvPHOTB belong to different clades suggesting early divergence, possibly before the divergence of land plants from the Zygnematales. qPCR and northern blot analysis showed that SvPHOTA and SvPHOTB responded differently to red and blue light. SvPHOTA was consistently expressed in the dark and in blue light, while SvPHOTB was expressed only when the plants were exposed to light. When the filaments were exposed to red light, SvPHOTA was significantly downregulated whereas SvPHOTB was highly upregulated. When SvPHOTA was incorporated into the phot1-5 phot2-3 double mutant of Arabidopsis thaliana the phototropin-mediated responses including chloroplast accumulation and leaf flattening were restored, while SvPHOTB transgenic plant did not exhibit any significant restored responses.The red/far-red light reversible photomovement, which is expected to involvement of phytochrome, was observed using whole filaments of S. varians. The filaments slowly bent and aggregated to form a tangled mass in red light. In far-red light, the filaments unbent, stretched rapidly and separated from each other. Mannitol and sorbitol treatment significantly inhibited this far-red light movement suggesting that turgor pressure is the driving force of this movement. The bending and aggregating movement of filaments in red light were not affected by osmotic change. Three phytochrome homologues isolated from S. varians showed unique phylogenetic characteristics. Two canonical phytochromes were named as SvPHY1 and SvPHY2, and a non-canonical phytochrome as SvPHYX2. SvPHY1 is the first PHY1 family phytochrome reported in zygnematalean algae. The genes involved in the transport of phytochromes into the nucleus and following transcriptional regulation factors were characterized, and their expression was significantly changed in response to red and far-red light.These results suggest that the complex photomovement of S. varians is the results of a two-track control of mechanical effectors signaled by the combination of phototropin and phytochrome. Although the pattern of photomovement in S. varians is unique the downstream signaling process of each photoreceptor was similar to that of higher plant. The structure and function of photoreceptors were well conserved, and most genes involved in downstream signalling were also identified in S. varians.