- 學位論文
蝴蝶蘭光合作用研究—生理試驗與生物晶片綜合分析
Meta Analysis of Photosynthetic Pathway in Phalaenopsis aphrodite Combining Physiological Approach and Gene Expression Profiling Studies
摘要
高等植物中，有7%的維管束植物行景天酸代謝 (Crassulacean acid metabolism, CAM)，以時間區隔光合作用之兩固碳模式，使CAM植物能於胞內濃縮二氧化碳，同時提高水分利用效率，演化出在逆境中存活的機制。蝴蝶蘭之光合作用型式為CAM。本試驗以台灣原生白花蝴蝶蘭 (Phalaenopsis aphrodite Rchb. f.) 為試驗材料，結合生理量測技術與生物晶片技術，同時從生理層面與基因表達層面，探討CAM代謝之作用機制。 光合作用量測之試驗環境為光周0700 HR到1900 HR、光度300 μmol•m-2•s-1、日夜溫30/25 oC、相對濕度50%到70%。台灣原生白花蝴蝶蘭之淨二氧化碳吸收速率呈四個階段之日變化，蘋果酸濃度具日韻律變化。蘋果酸酶 (malic enzyme)和蘋果酸脫氫酶 (malate dehydrogenase) 基因表現量呈白天下降夜晚上升之變化，皆屬生物時鐘基因。 Phosphoenolpyruvate carboxylase (PEPC) 活性呈現午後提高、夜晚下降之變化趨勢，在1800 HR達到最高峰278.2 μmol•mg Chl-1•hr-1，其基因表現量亦呈現白天上升夜晚下降之變化，其中以PATC152779最有可能是PEPC基因家族中，擔任固碳酵素之重要基因。Rubisco將胞內二氧化碳導入卡爾文循環 (Calvin cycle)，其初始活性於光期期間受光照影響，從2.5 μmol•mg Chl-1•hr-1提高到6.0 μmol•mg Chl-1•hr-1，唯其基因表現量除在2200 HR時略有上升，其餘時間變化不大。 部分的景天酸代謝日變化被認為與生物時鐘調節相關，可不受延長光周的處理影響，而維持其日韻律變化數日。本研究利用延長黑暗或光照的處理，篩選出生物時鐘基因，包括PEPC、PEPC kinase、Rubisco及Rubisco activase等，為直接參與光合作用暗反應者；另亦包括鉀離子、鈣離子與碳水化合物之轉運蛋白，可能間接調節有機酸進出液胞的運移和分配，顯示生物時鐘之調控因子包含暗反應酵素及轉運蛋白等。 蝴蝶蘭對乾旱之反應與其他作物相似，葉片於乾旱期間離層酸累積、葉片相對含水量下降且淨二氧化碳吸收受抑制，唯這些反應之發生較一般C3植物慢。短期乾旱誘導幼葉之PEPC活性提高，在PEPC基因家族中，PATC224556和PATC235744之基因表現量受乾旱誘導而提升，其他PEPC 基因表現量則受乾旱抑制，而Rubisco初始活性及基因表現量皆受乾旱抑制。另外，篩選出1758個基因受短期乾旱誘導，1654個基因短期乾旱而抑制，推測其中包含許多與CAM植物耐旱相關之基因，可供未來研究參考用。
並列摘要
Among higher plants, about 7% of vascular plants perform crassulacean acid metabolism (CAM). Those plants separate two carboxylation reactions by time. This strategy not only elevates intercellular CO2 concentration but also increases water usage efficiency, enabling CAM plants to survive under stresses. Current knowledge defines Phalaenopsis exhibiting CAM photosynthesis. Using Phalaenopsis aphrodite Rchb. f. as plant material, combining physiological and microarray techniques, we are expecting to reveal the strategy of CAM from the two points of view. Photosynthesis measurement was done with a 12-h photoperiod from 0700 HR to1900 HR, a photosynthetic flux density of 300 μmol•m-2•s-1, and with 50%-70% relative humidity. Malate concentration in Phalaenopsis leaves exhibited diurnal fluctuation. Net CO2 uptake rate performed typical four CAM phases of diurnal changes. Expression levels of malic enzyme and malate dehydrogenase (MDH) decreased during day and increased during night. Both malic enzyme and MDH are circadian genes. The activity of phosphoenolpyruvate carboxylase (PEPC) increased after noon and decreased at night. It reached its highest value, 278.2 μmol•mg Chl-1•hr-1, at 1800 HR. The expression level of PEPC changed similarily to the trend of enzyme activities. Among all PEPC genes, PATC152779 is the most likely one as a functional phosphoenolpyruvate carboxylase. Another carboxylase of CAM plants is Rubisco. Initial activity of Rubisco in Phalaenopsis increased from 2.5 μmol•mg Chl-1•hr-1 to 6.0 μmol•mg Chl-1•hr-1 during daytime. However, the expression level of Rubisco did not change much during a day, although it increased slightly at 2200 HR. Some diurnal changes in CAM were considered to be related to circadian rhythm. In spite of elongation of photoperiod, these physiological changes remain its rhythm for several days. In our study, we treated Phalaenopsis plants with a 12-h extended dark and a 12-h extended light after 24-h normal photoperiod, respectively. We selected ‘circadian genes’ by using GeneSpring® software. Among the genes identified, some genes were directly involved in carbon fixation in photosynthesis, such as PEPC, PEPC kinase, Rubisco, and Rubisco activase; others included the genes which may regulate malate translocation and distribution between vacuole and cytoplasm, such as transporters of potassium, calcium, and carbohydrates. It is speculated that circadian rhythm is regulated by enzymes of carbon reaction and several transporters. Phalaenopsis showed similar responses to drought as other plants. Abscisic acid was accumulated, leaf relative water content was decreased, and net CO2 uptake was inhibited after drought treatment. The difference between Phalaenopsis and C3 plants is the slow rate of those responses. Short-term drought induced PEPC activities in young leaf. Among all PEPC genes of Phalaenopsis, expression levels of PATC224556 and PATC 235744 were induced by drought. Expression levels of other PEPC genes were inhibited by drought. However, the initial activity and the level of gene expression of Rubisco were inhibited under drought stress. There are 1758 and 1654 genes which were induced or inhibited by short-term drought, respectively. We speculate that lots of these genes are related to the ability of drought tolerance in CAM plants. The information could provide references for future research.