植物學

為觀察不同波長光對植物光合作用要素「葉綠素含量」的影響。實驗材料甘藹(蕹菜種、台農57號、台農64號)組織培養苗。正常光照三週，使其具備基本之根、莖、葉，再移入不同顏色雙層玻璃紙(藍光、綠光、紅光)所構成不同光質之光照環境。除觀察生長狀況外，亦利用分光光度計測定葉綠素a、葉綠素b及兩者總含量。結果顯示藍光和紅光為促進葉綠素合成之主要光譜。藍光組合成的量為對照組的 80.5%;紅光組合成的量為對照組的82.5%。更進一步利用氧氣電極測定光合作用速率。結果顯示以紅光組的光合作用最為旺盛。紅光組光合作用速率為對照組的68%。This is study dealt with how light of different wavelengths make a difference on the content of chlorophyll. The material was tissue-cultured sweet potato(Weng-tsay native variety, Ipomoea batatasL cv. Tainung 57 and Ipomoea batatas L cv. Tainung 64). For first three weeks, we exposed them under normal sunlight so that they would possess basic structure such as root, stem and leaves. Then we moved them into different illumination environments (blue, green and red light) made by double-layered glass plates. Besides inspecting their growth, we also measured the quantity of total chlorophyll and chlorophyll a, b with spectrophotometer, Hitachi U2000. The results indicated that blue and red light was the main spectrum to accelerate the content of chlorophyll. The plant grown in blue environment had content 80.5% chlorophyll of the control group; while 82.5% in red environment. Furthermore, we used oxygen electrode to inspect their rate of photosynthesis. The result showed that the red light treatment highest rate of photosynthesis among treatments, which was 68% of the control group.

植物依靠向光性爭取最多的光線，以進行光合作用，製造食物供給所有生物。雖然在十九世紀時植物的向光性就已經被發現，並且參與植物向光性的主要荷爾蒙為植物生長素也已經熟知，但是主要是植物的哪一個組織接受光訊息以誘導向光性，以及細胞內的哪些分子參與訊息傳遞，則都不清楚。因此這個研究，以可以發射特殊波長的發光二極體為光源照射綠豆小苗以研究向光性，結果顯示藍光和綠光而不是紅和黃光可以誘導向光性。就向光性訊息傳導的組織層面的研究而言，將豆苗的葉、葉柄、生長點、子葉分別除去後，再側面照光，發現向光性要產生必須要有生長點或葉柄，並且發現莖可以誘導向光性，而葉子不能誘導向光性，因此莖是主要接受光訊息以誘導向光性的組織。就向光性訊息傳導的分子層面的研究而言，植物以鈣離子的螯合劑和鈣離子通道阻斷劑處理後發現，細胞質內鈣離子濃度的增加是藍光和綠光誘導的向光性所需要的過程，有趣的是藍光誘導向光性的訊息傳導過程中，除了經由細胞內的鈣離子濃度的增加外，還有其它鈣離子不參與的訊息傳導途徑。此外，以蛋白質磷酸?抑制劑和蛋白質去磷酸?抑制劑處理植物後發現，藍光和綠光所誘導的向光性訊息傳導，都包含蛋白質去磷酸?第1 和2a 型在細胞內的作用。因此植物的向光性需要有生長素才會表現，生長素由生長點製造後由生長點和葉柄儲存，在光刺激之下會誘導莖產生傳遞訊息，此訊息會傳遞到含有生長素的生長點和葉柄，使得生長素流向照光組織細胞，並且使得細胞內鈣離子濃度增加，活化蛋白質去磷酸?第1 和2a 型，進而造成植物的向光性。Phototropism allows plants to receive the most amount of light to perform photosynthesis, which produces food and energy for all organisms. The phenomenon of phototropism has been known since the 19th century, and auxin has been identified to be the main hormone involving in phototropism. However, the major plant tissue responsible for receiving light signal is not fully understood, and the signal transduction pathway within cells after light activation is not clear. Therefore, the phototropism of mungbean seedlings is examined by Light Emitting Diodes (LED) which produce the specific wavelength of light in this study. Results point out that blue and green lights rather than red and yellow lights induce phototropism of moonbeam. The phototropism of mungbean seedlings is further studied by plants whose leaves, petioles, apical meristem, or cotyledons were removed, showing that the presence of either apical meristem or petioles is needed for inducing phototropism. Also, stem ,not leaves, is the major tissue that receives light activation, and induces phototropism. The signal transduction of phototropism was further analyzed in the presence of calcium ion chelator and channel blockers. The signal transduction of phototropism induced by blue or green light contains the increasing concentration of calcium ion within cytosol. Interestingly, there is a calcium-independent tansduction pathway for blue light only to induce phototropism. Additionally, staurosporine (STA), a protein kinase inhibitor and okadaic acid (OKA), a protein phosphatase inhibitor, were used to study the signal transduction pathway of phototropism, and results indicated that protein phosphatase 1 and 2a is needed for both blue and green lights to induce phototropism. Conclusively, the phototropism is triggered by the reception of light by stem, and the light signal is transferred to apical meristem and petioles that reserve auxin produced from apical meristem. Auxin is then transferred to the cells that is illuminated, increases the concentration of calcium ion and activates protein phosphatase 1 and 2a in cells, and finally phototropism occurs.

隨著迅速的工業化，重金屬汙染已是嚴重的環境問題。在植物中，當植物體內累積過量的重金屬，對於植物根部、葉部等器官的生長與發育有嚴重的影響或傷害。銅離子為植物生長所必須之重金屬，但是過量銅離子會導致細胞死亡，生長受到抑制。本實驗以大豆( Glycine max )及田菁( Sesbania roxburghii )為植物材料，藉由Evans blue 染色法、螢光染色、西方墨點法、反轉錄聚合?鏈鎖反應等，觀察過量銅離子影響植物根部生長、細胞死亡和細胞訊息傳遞物質變化之情形，並探討過量銅離子影響大豆根部細胞死亡的訊息傳遞路徑。過量銅離子會限制植物根部的生長及造成根部細胞死亡。以螢光染色觀察根尖(ROS, reactive oxygen species)、Ca2+累積情形，根尖細胞Ca2+、ROS 累積隨處理銅濃度的增加而上升，可能影響細胞死亡程度。以Ca2+螯合劑EGTA 和W-7(CDPK(calcium-dependent protein kinase)、Calmodulin 抑制劑)前處理發現可以降低過量銅離子對大豆根部的細胞死亡程度，推測Ca2+、CDPK 參與銅引發大豆根部細胞死亡的途徑。為檢驗MAPK 參與根部細胞死亡的途徑，以西方墨點法偵測根部細胞MAPK 的TEY 或TDY 磷酸化，實驗結果發現，隨著過量銅離子濃度的升高，田菁、大豆根部42-kDa MAPK磷酸化情形有上升之趨勢。以RT-PCR 分析大豆MAPK1 及MAPK2 基因之表現量，發現在銅處理時大豆之MAPK1 和MAPK2 基因的轉錄情形增加。大豆( Glycine max )及田菁( Sesbania roxburghii )皆屬於豆科植物，可作為綠肥植物。探討銅影響大豆、田菁細胞死亡之訊息傳遞路徑，希望進而控制生物體所受的毒害情形及訊息傳遞途徑，加強生物體對重金屬的防禦機制，未來可以以基因轉殖等基因工程技術，轉入抗重金屬基因或增強植物體對抗重金屬的能力等，作為綠肥植物、抗重金屬植物吸 附重金屬來復育土地達綠色淨化等用途。 Many heavy metals are necessary for plants, but excessive quantities directly affect plant growth and survival of organisms, cause cell death, or even affect human life indirectly. Cu (copper ion) is a heavy metal, which is one of micronutrients essential for normal growth and development of plants. The purpose of this experiment is to study the effect of excessive copper on Glycine max and Sesbania roxburghii root tips. I conducted some experiments by means of Evans blue staining (analysis of cell death), western blot analyses, and fluorescence microscope in order to examine the way copper results in plant death. Measurement of root length and analysis of cell death showed that excessive copper could bring about the inhibition of plant growth as well as cell death. With fluorescence microscope, I found that excessive copper might increase the level of copper-caused reactive oxygen species (ROS) in both the root tips of Glycine max and Sesbania roxburghii. In addition, I used Oregon Green 488 BAPTA-1 so as to assess the accumulation of calcium ions in root tips and found that the exposure of root tips to excessive copper results in the accumulation of calcium ions. To investigate whether calcium ions and calcium-dependent protein kinase (CDPK) play a role in the cell death caused by excessive copper, I tested W-7, calmodulin and CDPK inhibitors, and EGTA, Ca2+ chelating agents, before copper treatment – immersing copper in CuCl2. In this way, plant cells would be effectively prevented from copper-caused death. Besides, to find out whether copper activates MAPKs in plant cells, I took advantage of western blot analysis with Phosphor-Map kinase Antibody and Map kinase Antibody. The results revealed that excessive copper might lead to TEY or TDY motif phosphorylation of approximate 42- and 64-kDa MAPKs in the cells of Glycine max root-tip and approximate 42-kDa MAPKs in the cells of Sesbania roxburghii root-tip. Furthermore, with RT-PCR, I found that the transcription of Glycine max MAPK1 and MAPK2 mRNA happens more frequently in root cells after copper treatment. In addition, this study suggested that the MAPK cascade CDPK pathway may function in the heavy-metal-signaling pathway in plant, and that calcium ions and ROS might get involved in the copper-caused death of plant cells. By studying signal transduction against heavy-metal toxicity in the plants, we can know how the organisms protect themselves. Sesbania roxburghiivv (or Glycine max), as green manure, could be used for metal-hyper-accumulator with the help of genetic engineering in the future.

水生食蟲植物絲葉狸藻 (Utricularia gibba) 是非常獨特的，它不但跟其他植物一樣能行光合作用，且具備捕蟲囊捕捉水中小生物，補充生長所必需的營養元素。捕蟲囊的構造精密卻不複雜，消化吸收主要靠囊內壁上的四爪腺毛，目前尚未有文獻實際以追蹤物質描述出整個共質體輸送路徑。我們是最先以螢光染劑 (carboxyfluorescein) 及共軛焦雷射掃描顯微鏡(confocal laser scanning microscope) 成功地描繪出捕蟲囊共質體運輸路徑。同時我們也以對細胞無害的食用色素，進行相同的實驗觀察。發現食用色素不但成本低，且較螢光染劑有更多的優點，如觀察時間較不受限制等，非常適合用來研究捕蟲囊吸收物質的路徑，因此，本實驗的模式可以應用在其他水生植物運輸路徑的研究。;The aquatic carnivorous plant Utricularia gibba is very unique. It has not only the ability to undertake photosynthesis just like other plants, but also can trap and obtain the nutrients from the freshwater zooplankton. Its trapping organ is very sophisticate but not complicate. The digestion and absorption process inside the trap are mainly accomplished by the quadrifids structure. According to our knowledge, we are the first to introduce the phloem-mobile, fluorescent probe carboxyfluorescein (CF) and confocal laser scanning microscope (CLSM) to the study of the symplastic transport in the Utricularia trap. In addition, we use edible food colorings as tracers for this transport study. Both approaches turn out to be very successful in delineating the symplastic transport of the trap. But CF quenches rapidly so the observation time is restricted. On the contrary, food colorings don’t have these disadvantages; it is inexpensive, easy to perform, and the transport process is not fast. As a result, the study is easily to be completed. These methods will be very helpful in the studies of symplastic transport in other plants.

槐葉蘋(Salvinia natnas)生長於台灣低海拔淡水濕地，目前已列為嚴重瀕臨滅絕的台灣原生物種，為不具有根的植物，是世界珍稀的漂浮型水生蕨類。本研究是探討槐葉蘋形態、生活史及生存環境因子，實驗發現可藉由成熟浮水葉外部形態特徵來區別槐葉蘋與外來種之人厭槐葉蘋(Salvinia molesta)；槐葉蘋成熟浮水葉呈橢圓形，葉上毛被物是叢生且分岔，人厭槐葉蘋成熟浮水葉呈雙耳形，葉上毛被物則像打蛋器。當兩物種共存於同一個環境空間時，人厭槐葉蘋以平均11.6 cm2/week 的生長率將槐葉蘋完全取而代之，顯示人厭槐葉蘋之入侵對槐葉蘋生存影響之深遠。經由兩年的槐葉蘋物候觀察，發現3~11 月為抽芽成長期、3~12 月為成熟繁殖期、12 月~隔年2 月為冬枯期及孢子囊果出現期，12 月~隔年5 月為孢子囊果成熟開裂期。其繁衍策略可分為無性繁殖（頂芽及側芽生長）及有性生殖（異配子體交配）。探討環境因子（光照度、氣溫、濕度、水質、水溫、pH 值）分析結果，適合槐葉蘋生存環境的條件為(1)陽光間接照射（半遮蔭，遮蔭度58.33%）、(2)乾淨未受污染的水質(pH 6.5~8)、(3)通風性良好。生長環境符合以上條件即可達到移地保育的目的。Salvinia natans, a floating fern without roots, grows in low elevation fresh water wetlands of Taiwan, and is a critically endangered precious Taiwanese native species. This research investigates the life form, life history, and living environment of Salvinia natans. Our experiments show that we can differentiate Salvinia natans and Salvinia molesta, two easily mixed up species. The shape of matured floating leaves of Salvinia natans is elliptical and smaller, while it is twin-ear shape and larger for Salvinia molesta. Also, they can be distinguished by their leaf hairs. The hairs of Salvinia natans are tufted and separated at the tips, while the hairs of Salvinia molesta form an ‘eggbeater’ shape at the tip. When these two species lived together, Salvinia molesta grew in a rate of 11.6 cm2/week and will replace all Salvinia natans eventually. This shows the profound impact of invasion of Salvinia molesta. From the data of 2-year phenology observation, we concluded that budding took place from Mar. to Nov., growing and reproducing from Mar. to Dec., decaying from Dec. to Feb. (sporocarps were born in this period), and sporocarps matured from Dec. to May. There are two reproduction strategies: sexual reproduction (intergametophytic mating), and asexual propagation (by terminal and axillary growth). After investigating the environment factors (illuminance, air temperature, water temperature, humidity, pH), we found that ex situ conservation for Salvinia natans requires 1) indirect sunshine, 2) unpolluted water (pH 6.5 ~8), and 3) good ventilation.

Lantana is a very common plant in our lives. It grows easily and it has a long florescence and various colors. The colors of particular types of lantana alter as the changing florescence. In this experiment, paper chromatography, high-performance liquid chromatography, SDS-gel electrophoresis, the measurement of petal cellular pH values, and the comparative study of forms of trachoma on the epidermal cells of petals are exerted in order to explore factors that change the colors of the lantanaThe findings are as follows:\r (1)Lantana’s colors have inseparable relationships with the compositions of anthocyanins and flavonoids, but not with the pH values of petal cells.(2)The anthocyanins of petal cells are cyanidm, with glycosides as well.(3)Beside the differences in the compositions of pigments, the forms of trachoma on the epidermis of the petal, cone-like or caniniform, can also be used to distinguish different types of lantana, because the trachoma can influence the reflections of light from the epidermis of the petals and also affect colors of the flowers.(4)The result of SDS-gel electrophoresis shows that the biochemical pathways of petal cells in all species of lantana are similar, so we assume that there is mutant in the series of synthesizing enzyme when the anthocyanins of petal cells are formed, and thus, there are no anthocyanins appearing in the yellow and white species of lantanaThe results above are helpful for the understanding and discovering of lantana’s biological mechanisms, and can be used to create new types of lantana and to make further study of the metabolism of lantana’s complete anthocyanin’s biochemical pathway馬纓丹(Lantana ssp.)是常見景觀植物，容易栽種、花期長、花色多，且有些品系花色會隨著花期而變化。本實驗利用濾紙色層分析、高效能液相層析、SDS-gel電泳、細胞pH值測定及花瓣表皮細胞之毛茸(trichoma)型態之比較等方法探討馬纓丹花色之不同及變化的原因。結果顯示: (1)馬纓丹的花色及花色變化與花青素(anthocyanins)和類黃素(flavonoids)之組成有密切關係，而與花瓣細胞內pH值無關。(2)花瓣中所含花青素為矢車菊色素(cyanidm)，並且具有配醣基(glycoside)。(3)花瓣表皮細胞之毛茸型態，如圓錐形或犬牙型，會影響光的反射，進而影響花色，所以毛茸型態可做為區分馬櫻丹品系之特徵。(4)SDS-gel電泳的結果顯示，馬櫻丹各品系的花瓣細胞生合成類似，推測花瓣細胞產生花青素的一系列酵素中，已有突變發生，而造成黃色、白色品系無花青素。以上結果有助於了解馬纓丹花色變化之機制，可將其應用於改良出新的馬櫻丹之品系，或更深入研究馬櫻丹花青素完整生成代謝路徑。

從研究抑制乙烯的實驗中碰巧得到的靈感，讓我們找到了水果中一種不可思議的變化，水果在撞擊之後乙烯量會增加，因而帶動水果的糖度上升，甜度增加!! 我們利用水果內的逆境機制，使得水果在外界刺激之下（如：撞擊），出現加速成熟的效果。我們經由多次的實驗，在各種水果的數據中，分析變甜的原因，及與乙烯量增加、pH 值下降的相互關係。並且找出除了搖動外，其他可以使水果糖度增加的方式。有了這些方法，我們可以在家中自行加工水果，使未成熟的水果快速成熟、使已經成熟的水果更甜，再也不會因非產季而妨礙到吃的興致!! We get an inspiration form the experiment for controlling ethylene. We find an unimaginably different change of fruits. After ramming, the amount of ethylene in the fruit will increase. This makes the sweet degree of the fruit increase, and it tastes more sweetly!! With adversity system of fruit, we make fruit ripe quickly by external excitement.（ex: ram）Through many experiments and the data of all kinds of fruits, we can assay the reason for fruits’ becoming sweeter, and interrelation between increasing ethylene and decreasing pH value. And find other ways except for shaking to make sugar degree rise. With these ways, we can process fruits by ourselves at home. We can make unripe fruit mature quickly, make ripe fruit sweeter, and we will no longer be obstructed to eat fruit even if it won’t be produced in that season.

含羞草會因為受到各種不同外界刺激(光照、外力等等)，而造成膨壓改變。外 觀上表現出葉片閉合或有葉柄下垂的現象。其中含羞草處在黑暗的環境下一段時 間後，會做出睡眠運動。當它重新暴露於光照之中，將會需要一段時間以恢復原 先葉片張開的樣子。這個實驗是研究不同類型的環境因子(主要是光照和溼度)在打 破睡眠運動之後，對其葉片復原時間所造成的影響。我們針對上述環境因子在不 同狀況時，進行我們的實驗上百次，進一步得到了多項的數據。也在實驗中，為 了精確了解整個恢復的程序，而將恢復程序程序做成書面說明。以下是我們所紀 錄與分析完數據後的結果，以及我們在實驗期間，進行一些不同的實驗嘗試，所 發現不同於表面所見的驚人事實。 The mimosa can accept plenty of stimulation (light, force, etc), which results in the change of turgor pressure, and on outward appearance, it shows the phenomenon that the leaves become closed or that the stalks get pendent. Among the stimulation, the mimosa will undergo nyctinasty when it is left in the dark for a period of time, in order to enable it to be exposed to the light once more, which requires certain time. This experiment is based on how different kinds of elements of the environment (primarily light and hydro level) effects the rehabilitation time after the nyctinasty is broken. A large number of data are gained after experimenting on it for hundreds of times. Steps of habilitation are also made into illustration in writing, so as to understand the whole steps accurately in the experiment. Below are our records and analysis based on the data, including a few special experimental tries during our working time, in which some surprising facts that were discovered are different from what are seen on the surface.

我們成功的用溶氣計偵測到了聚球藻 RF-1 在 28℃下光合作用的概日韻律。和傳統的研究方法比起來，這個方法具有連續偵測的優點，減少因不斷取樣所造成的影響，此實驗可觀察到聚球藻 RF-1 溶氣量之變化圖與一般藻類（如單殼縫藻）不同.在光 /暗條件下 RF-1 之溶氣量的增加與減少均呈週期性變化，而且此變化現象在進入連續照光後仍然可以維持兩個循環以上，這些結果顯示以溶氣計連續偵測聚球藻 RF -1之概日韻律是可行的，而且所得到的變化圖形遠比傳統方法（於不同時間取樣）所得者自然。本實驗同時發現含聚球藻之培養液的溫度，在進入黑暗週期時會有明顯的上升，由於其變化程度比其他藻類明顯，如加以探討應有助於對此藻以及其韻律特性之瞭解。We successfully detected the photosynthesis circadian rhythm of the prokaryote Synechococcus RF-1 under 28℃ by a DO (Dissolved-Oxygen) meter. The advantage of this method, comparing with the traditional methods, is that it can detect signals continuously, reduce the influence of discrete sampling. The DO curves of the Synechococcus RF-1 are different from that of other algae. Under Light/Dark conditions, the DO values of RF-i increased and decreased periodically. The periodic phenomena progressed over two cycles under constant lighting conditions. These results revealed the feasibility of using DO meter to continuous detect the circadian rhythm of the Synechococcus RF-1 The detected DO curves looked more natural than those obtained in the traditional discrete-sampling method. We also found that the temperature of the culture increased in dark cycle. Since the variation is clearer than that of other algae, further investigation will benefit the understanding of the Synechcoccus RF-i and its circadian rhythm.

微型RNA是最近發現的小RNA，調控生物體內的反應，包括生長、細胞分化、對抗病毒…等。植物利用RNA干擾 (RNAi) 或過敏反應 (HR) 對抗病毒感染。有趣的是，miR168可藉由降解mRNA或抑制轉譯，調控阿拉伯芥AGO1的表達，而AGO1是RNAi的一個重要元件。miR398則調控銅鋅超氧化物歧化? (CSD1, CSD2) 的表達，而CSD1, CSD2負責產生過氧化氫去引發細胞凋亡 (cell apoptosis)。帶有竹嵌紋病毒 (BaMV) 全長基因的轉殖菸草 (Nicotiana benthamiana) 品系27-17是我們的研究材料。27-17的幼葉不具病徵，隨著葉子的生長，病徵會漸漸變嚴重。我發現被病毒感染時，植物會提高AGO1的表達，使RNAi更有效率。然而，病毒藉提高miR168使AGO1的量無法上升。植物亦可提高CSD1, 2 mRNA的量，促進細胞凋亡。病毒卻會引發miR398降解CSD2 mRNA。在病毒力價高的葉子中，雖然CSD2 mRNA降低且miR398升高，植物仍可大量提高CSD2蛋白的量。CSD1 mRNA沒有被miR398負調控，詳細原因仍有待研究。