摘要: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。但根據前人研究指出,CAM蘭花在原球體時期沒有顯著的可滴定酸變化,推測其光合作用型態可能為C3,因此我們假設蝴蝶蘭從原球體發育成小苗的過程中,光合型態會從C3轉變成CAM。臺灣蝴蝶蘭是臺灣很重要的白花原生種,本研究以臺灣蝴蝶蘭為材料,依組織型態將臺灣蝴蝶蘭從種子至成熟大苗分為stage 0至stage 6七個發育階段,藉由分析CO2吸收率、蘋果酸含量、PEPC活性以及PEPC、PEPC kinase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(stage 0)至原球體的發育階段(stage 3),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是C3。當發育階段進入原球體長出第一片葉和第一條根的stage 4時,葉片中的蘋果酸含量與PEPC活性即有CAM的日夜韻律變化,但仍維持在白天吸收CO2的特性,故stage 4的葉片是進行CAM-cycling。隨著葉片發育越成熟,stage 5瓶苗的CO2吸收率、蘋果酸含量與PEPC活性皆有絕對性CAM的日夜變化。因此從原球體發育至瓶苗的過程中,光合型態會經歷C3、CAM-cycling與CAM的轉變,此種轉變是一種不受環境影響的個體發生過程。
在CAM途徑中的關鍵固碳酵素是PEPC,其基因PPC不論白天或夜晚,在各發育時期及各器官中都有表現。PPC的持續性表現表示光合型態轉變的關鍵不是PPC的表達,因此本研究進一步釣出了調控PEPC活性的PPCK基因PaPPCK,並分析PaPPCK在各發育階段的表現。PaPPCK在stage 0至stage 2無日夜表現的差異性,而當PEPC活性於stage 3開始出現日夜差異時,stage 3的PaPPCK夜晚的表達量開始高於白天的表達量。在各發育階段以及各器官中可以觀察到PaPPCK夜晚的表達量高於白天,尤其在葉片中的差異表現最為明顯,且隨著葉片的成熟,PaPPCK的日夜表達差異也越明顯。PaPPCK表達的日韻律影響PEPC活性與蘋果酸的日韻律,顯示PaPPCK才是CAM光合型態展現的主要關鍵。
Abstract: Mature Phalaenopsis is a typical CAM plant, which is characterized by opening stomata during the night, fixing CO2 via PEPC and 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 undergo the C3 photosynthesis pathway and lead to the hypothesis that a photosynthetic transition from C3 to CAM at certain developing stages may exist. The material used is Phalaenopsis aphrodite subsp. formosana, one of the important indigenous orchid species in Taiwan. We defined seven developmental stages from mature seed (stage 0) to adult plant (stage 6) according to the morphology, and analyzed CO2 uptake, malate accumulation, PEPC activity and the gene expression of PEPC and PEPC kinase to support this hypothesis of photosynthesis transition.
From stage 0 to stage 3, no significant daily change in malate accumulation and PEPC activity were detected, and apparent net CO2 uptake during the day indicated that at the protocorm stage it may undergo C3 photosynthesis. At stage 4, when the protocorm developed the first leaf, the day/night malate content and PEPC activity were first observed to show the typical CAM diurnal pattern in the leaf, except the gas exchange remained the C3 pattern. The character in the stage 4 leaf was CAM-cycling. With further leaf development, CO2 uptake, malate fluctuation and PEPC activity appeared obligate CAM rhythm since stage 5. Consequently, photosynthesis shifted from C3 to CAM-cycling to CAM from the protocorm to seedling, which was an ontogenetic development independent of environmental signals.
In CAM pathway, nocturnal CO2 is fixed by PEPC. The expression of PPC, encoded for PEPC, revealed a constitutional pattern in all organs at all developmental stages without diary rhythm. The results suggested that PPC expression was not the key factor controlling the transformation of photosynthesis type. We cloned PEPC kinase gene PaPPCK, which encoded PEPC kinase to regulate PEPC phosphorylation, and analyzed the expression of PaPPCK during different developmental stages. PaPPCK had no different expression from stage 0 to stage 2. At stage 3, PaPPCK expression started to exhibit day/night difference as PEPC activity was slightly higher at night. PaPPCK mRNA abundance was higher at night in every organ after stage 3, especially in the leaf. Furthermore, the day/night expression difference of PaPPCK was related to leaf age. PaPPCK transcripts showed a diurnal expression in the leaf, which in turn affected the rhythm of PEPC activity and malate. Therefore, PaPPCK expression is a major factor to control CAM expression and photosynthesis transition.