臺灣蝴蝶蘭各發育階段之景天酸代謝

Abstract

成熟的蝴蝶蘭是一種典型的CAM植物，會於夜晚打開氣孔，利用PEPC固碳成有機酸儲存，具可滴定酸日韻律變化。然而，根據前人研究指出，CAM蘭花在原球體時期沒有顯著的可滴定酸變化，因而推測其光合作用型態可能為C3。為此本研究目的在證明CAM蘭花光合型態會隨著發育過程而從C3轉變成CAM，試驗以白花原生種的臺灣蝴蝶蘭為材料，依組織型態將臺灣蝴蝶蘭從種子至成熟大苗分為七個發育階段，藉由分析CO2吸收率、蘋果酸含量、PEPC活性以及PEPC、PPCK的基因表達來探究蝴蝶蘭在發育階段中的光合作用型態轉變。 從種子發芽至未長葉的原球體階段，CO2於白天吸收、夜晚釋放的特性以及蘋果酸與PEPC活性沒有日夜變化，顯示原球體時期的光合作用型態不具CAM特性而傾向C3。當原球體長出第一片葉後，葉片中的蘋果酸含量與PEPC活性開始出現CAM的日夜韻律變化，但仍維持白天吸收CO2的特性；隨著葉片發育越成熟，CAM生理特徵越明顯，準備出瓶的瓶苗葉片已展現絕對性CAM的日夜變化。觀察PEPC基因PPC的表現量，在各發育時期及各器官中都有表現，且無明顯日夜變化，顯示PEPC在CAM個體發育中不扮演重要調控角色。本研究更進一步選殖出調控PEPC活性的PPCK基因PaPPCK，PaPPCK在各發育階段的表現量與上述各發育時期的CAM特徵相吻合。 因此我們結論蝴蝶蘭從原球體發育至瓶苗的過程中，光合型態會由C3轉變為CAM。在轉變的過程中，PaPPCK是CAM光合型態展現的主要關鍵。原球體長出第一片葉是轉變過程的關鍵時期，表現CAM-cycling光合型態，此時期清楚區隔原球體之前的時期和成熟植株。

Mature Phalaenopsis is a typical CAM plant, which is characterized by opening stomata during the night and fixing CO2 by PEPC and thus showing a diurnal fluctuation of titratable acids. However, no remarkable day/night titratable acidity rhythm was detected during the protocorm stages, which suggested that the protocorm stages of Phalaenopsis may not undergo the CAM photosynthesis pathway but C3 pathway. Therefore, the objective of this study is to prove the existence of a photosynthetic transition from C3 to CAM coping with the ontogenetic development in the CAM orchids. Phalaenopsis aphrodite subsp. formosana were prepared according to the seven developmental stages we defined, and the CO2 uptake, malate accumulation, PEPC activity, PEPC and PEPC kinase were investigated. Stages before generation of the primary leaf from protocorm, no net CO2 uptake during the night but the day, and no significant daily change in malate accumulation and PEPC activity indicated that the stages was without CAM characters and behaved nearly-C3. As the protocorm developed the first leaf, the day/night malate change and PEPC activity were first observed; however, the gas exchange remained like the C3 pattern. The CAM characters were getting much more typical along with the leaf maturation, and the seedling ready to transfer out from the flask had turned into an obligate CAM plant just as the mature plant. The expression of PPC, encoded for PEPC, revealed a constitutional pattern in all organs at all developmental stages indicated that the PEPC did not play an important role in the CAM ontogenetic development. Moreover, the expression patterns of PaPPCK, encoded for PPCK (PEPC kinase) were in agreement with the physiological characters just mentioned above. Therefore, we conclude that the photosynthesis shift from C3 to CAM during the ontogenetic development in Phalaenopsis and PaPPCK played a key role in transition. CAM-cycling occurred in the critical stage, protocorm with the primary leaf, which spaced the protocorm and mature plant.