斑葉蝴蝶蘭之生理與生化特性

Physiological and biochemical characteristics of variegated orchids

台灣白花蝴蝶蘭（Phalaenopsis aphrodite subsp. formosana）及小花品種P. Sogo Vivien ‘F858’的斑葉突變株葉片上分布黃綠不均之斑紋。本試驗比較上述兩斑葉突變株與正常株組織、生理及分生之差異。在P. aphrodite斑葉突變株之黃色區域，葉綠體僅存在於靠近表皮細胞之葉肉細胞內；而P. Sogo Vivien斑葉突變株之黃色區域僅於近表皮之葉肉細胞有較少數葉綠體。利用穿透式電子顯微鏡觀察兩斑葉突變株之葉綠體發育，在P. aphrodite黃色區域可觀察到發育不全的基粒堆疊（stacked grana）及質體球（plastoglobules），而在P. Sogo Vivien之黃色區域並未觀察到任何相關之構造。兩斑葉品種之綠色區域及野生型植株，其葉片的葉綠素含量及類胡蘿蔔素含量皆較黃色區域高。進一步分別由葉綠素代謝、類胡蘿蔔素生合成、葉綠體發育及光合作用相關之基因挑選並進行定量即時聚合酶鏈鎖反應（quantitative real-time PCR）。結果顯示，三個次單位之鎂螯合酵素（Mg-chelatase）在兩斑葉品種的黃色區域其表現量皆較低，相反地，葉綠素降解相關基因（CLH and RCCR）則大量表現。推測兩斑葉品種葉片黃色區域可能為葉綠素降解相關基因大量表現之緣故。另外，兩斑葉品種之黃色區域其PsbO表現量較綠色區域高，可能因而使基粒堆疊受到抑制，進而導致光系統II（photosystem II）之D1和D2蛋白合成受阻；推測當PsbO於黃色區域表現量提高時，會抑制葉綠體發育相關基因（GLK）及光系統II等相關基因之表現。
Leaf variegation in Phalaenopsis aphrodite subsp. formosana and P. Sogo Vivien 'F858' showing irregular yellow stripes were observed. This work investigated both the histological and physiological characters of the variegated mutants and their wild-type plants. The chloroplast is only present in the sub-epidermal mesophyll cells in the yellow sectors of variegated P. aphrodite, while fewer chloroplast was present in sub-epidermal mesophyll of the yellow sectors of P. Sogo Vivien 'F858' when observed under light microscope on leaf cross-sections. Observation under transmission electron microscopy (TEM) revealed the presence of some underdeveloped stacked grana and plastoglobules in the yellow sectors of P. aphrodite but not in the yellow sectors of P. Sogo Vivien 'F858'. Both the chlorophyll and carotenoid contents in yellow sectors were remarkably lower than those in wild-types and green sectors in both accessions. We further analyzed probable candidate genes related to chlorophyll metabolism, carotenoid biosynthesis, chloroplast development and photosynthesis by quantitative real-time PCR. The three subunits of chlorophyll biosynthesis-related Mg-chelatase were all down-regulated, while chlorophyll degradationrelated CLH and RCCR genes were up-regulated in the yellow sectors of both mutant leaves. The lower chlorophyll contents in the yellow sectors probably were due to the activation of chlorophyll degradation-related genes in both genotypes. The up-regulated PsbO activity may inhibit the grana stacking in the yellow sectors of both genotypes and further blocking the synthesis of the D1 and D2 proteins in the PSII complex as revealed by downregulation of chloroplast development-related gene GLK, and the PSII complex-related genes.